Paranodin, a glycoprotein of neuronal paranodal membranes.

Ranvier nodes are flanked by paranodal regions, at the level of which oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region.

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.

Postnatal development of GFAP, connexin43 and connexin30 in cat visual cortex.

In cat visual cortex, neurons acquire progressively mature functional properties during the first postnatal months. The aim of this study was to analyze the development of astrocytes during this period. The patterns of expression of the glial fibrillary acidic protein (GFAP) as well as of two gap junction proteins expressed in astrocytes, connexin43 (Cx43) and connexin30 (Cx30), were investigated by immunohistochemistry and optical density measurements, in visual cortical areas 17 and 18 at four different ages: 2 weeks (postnatal days 12 to 15, P12-15), 1 month (P27-31), 2 months (P60-62) and beyond 1 year. Since visual experience is a key factor for neural development, the patterns of expression of these three proteins were studied both in normally-reared and monocularly deprived animals. Interestingly, the distribution of GFAP, Cx43 and Cx30 was found to change dramatically but independently of visual experience, during postnatal development, even beyond P60. During the first postnatal month, GFAP and Cx43 were mainly localized in the white matter underlying the visual cortical areas 17 and 18. Then, their distributions evolved similarly with a progressive decrease of their density in the white matter associated with an increase in the cortex. Connexin30 expression appeared only from the second postnatal month, strictly in the cortex and with a laminar distribution which was similar to that of Cx43 at the same age. In adults, a specific laminar distribution was observed, that was identical for GFAP, Cx43 and Cx30: their density was higher in layers II/III and V than in the other cortical layers.

Expression of calbindin D28K in the dopaminergic mesotelencephalic system in embryonic and fetal human brain.

A subset of tyrosine-hydroxylase (TH) neurons of the substantia nigra (A9) containing calbindin D28K (CaBP) appeared to be less vulnerable to cell death induced by Parkinson's disease than the subset containing dopamine (DA) alone. Because grafting procedures of fetal human neurons are increasingly used in the therapy of Parkinson's disease, it is important to study the development of DA neurons coexpressing CaBP. In humans, the genesis of TH immunoreactivity of A9, of the ventral tegmental area (A10), and of the retrorubral area (A8) occurred during a 2-week period from the 4. 5th gestational week (g.w.) in the ventricular zone of the floor plate and the contiguous basal plate of the mesencephalon and diencephalon, i.e., the prosomeres p1-p3. Double-immunolabeled TH-CaBP neurons were detected from 5.5 g.w. on, in the first wave of DA neuron's migration, and were observed in their final residence in the dorsal A9 by 10.5 g.w. Calretinin immunoreactivity was expressed in TH-immunoreactive (IR) neurons from 10.5 g.w. on. Ascending TH-CaBP-IR axons were observed toward the telencephalon from 6-7 g.w. , reaching the anlage of the nucleus accumbens and amygdaloid complex at 10.5 g.w., but were not detected in the ganglionic eminence at this latter stage. Dopaminergic patches were detected at 13 g.w. in the anlage of the putamen, but no TH-CaBP-IR fibers were observed in the matrix at this stage. In conclusion, even if CaBP immunoreactivity was detected in TH-IR cell bodies during the embryonic period, the TH-CaBP-IR axonal terminal was observed earlier in some limbic-related areas than in the matrix compartment of the basal ganglia in humans.

ATP-induced inhibition of gap junctional communication is enhanced by interleukin-1 beta treatment in cultured astrocytes.

Nucleotides are signaling molecules involved in variety of interactions between neurons, between glial cells as well as between neurons and glial cells. In addition, ATP and other nucleotides are massively released following brain insults, including inflammation, and may thereby be involved in mechanisms of cerebral injury. Recent concepts have shown that in astrocytes intercellular communication through gap junctions may play an important role in neuroprotection. Therefore, we have studied the effects of nucleotides on gap junction communication in astrocytes. Based on measurement of intercellular dye coupling and recording of junctional currents, the present study shows that ATP (10-100 microM) induces a rapid and a concentration-dependent inhibition of gap junction communication in cultured cortical astrocytes from newborn mice. Effects of agonists and antagonists of purinergic receptors indicate that the inhibition of gap junctional communication by ATP mainly involves the stimulation of metabotropic purinergic 1 (P2Y(1)) receptors. Pretreatment with the pro-inflammatory cytokine interleukin-1beta (10 ng/ml, 24 h), which has no effect by itself on gap junctional communication, increases the inhibitory effect of ATP and astrocytes become sensitive to uridine 5'-triphosphate (UTP). As indicated by the enhanced expression of P2Y(2) receptor mRNA, P2Y(2) receptors are responsible for the increased responses evoked by ATP and UTP in interleukin-1beta-treated cells. In addition, the effect of endothelin-1, a well-known inhibitor of gap junctional communication in astrocytes was also exacerbated following interleukin-1beta treatment. We conclude that ATP decreases intercellular communication through gap junctions in astrocytes and that the increased sensitivity of gap junction channels to nucleotides and endothelin-1 is a characteristic feature of astrocytes exposed to pro-inflammatory treatments.

Ontogeny of Rxt1, a vesicular "orphan" Na(+)/Cl(-)-dependent transporter, in the rat.

The developmental expression of the orphan Na(+)/Cl(-)-dependent transporter, Rxt1, was studied in the rat using a specific [(35)S]complementary RNA probe and affinity purified antibodies. Western blotting experiments allowed the detection of Rxt1 in brain as early as on embryonic day 16. After birth, the brain levels of Rxt1 increased dramatically up to a maximum around postnatal day 30 and then decreased slightly to the adult value. In situ hybridization experiments allowed the earliest detection of Rxt1 messenger RNA in the brain and spinal cord at embryonic day 14. In embryonic day 18 embryos, Rxt1 messenger RNA was present not only in the nervous system but also in the pituitary, the thymus and the heart. Immunoautoradiograms of whole embryo at embryonic days 16 and 18 showed high amounts of the Rxt1 protein in the spinal cord and brain. Moreover, at embryonic day 18, the orphan transporter was expressed in the thymus, heart and liver. At these ages, Rxt1 immunolabeling was localized in neurons of the subplate and in the ventricular zone of the brain. During early postnatal stages, Rxt1 messenger RNA expression demonstrated dynamic and complex changes until postnatal day 13. In particular, this transcript was relatively abundant in the striatum at postnatal days 3 and 5 and then decreased to very low levels after postnatal day 10. At the same period, Rxt1 immunostaining in the hippocampus and the cerebral cortex was observed all over the gray matter, in cell bodies as well as in the neuropil. Finally, the adult pattern was reached around postnatal day 13 for Rxt1 messenger RNA, but only at postnatal day 20 for the Rxt1 protein. The presence of Rxt1 messenger RNA and protein at embryonic stages and the high expression of the protein during synaptogenesis suggest that this vesicular "orphan" transporter is involved in the brain maturation process.

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.

Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms.

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

Endothelins regulate astrocyte gap junctions in rat hippocampal slices.

Gap junctional communication (GJC) is a typical feature of astrocytes proposed to contribute to the role played by these glial cells in brain physiology and pathology. In acutely isolated hippocampal slices from rat (P11-P19), intercellular diffusion of biocytin through gap junction channels was shown to occur between hundreds of cells immuno-positive for astrocytic markers studied in the CA1/CA2 region. Single-cell RT-PCR demonstrated astrocytic mRNA expression of several connexin (Cx) subtypes, the molecular constituent of gap junction channels, whereas immunoblotting confirmed that Cx43 and Cx30 are the main gap junction proteins in hippocampal astrocytes. In the brain, astrocytes represent a major target for endothelins (Ets), a vasoactive family of peptides. Our results demonstrate that Ets decrease the expression of phosphorylated Cx43 forms and are potent inhibitors of GJC. The Et-induced effects were investigated using specific Et receptor agonists and antagonists, including Bosentan (Tracleer trade mark ), an EtA/B receptor antagonist, and using hippocampal slices and cultures from EtB-receptor-deficient rats. Interestingly, the pharmacological profile of Ets effects did not follow the classical profile established in cardiovascular systems. The present study therefore identifies Ets as potent endogenous inhibitory regulators of astrocyte networks. As such, the action of these peptides on astrocyte GJC might be involved in the contribution of astrocytes to neuroprotective processes and have a therapeutic potential in neuropathological situations.