The Carboxyl-terminal Domain of Connexin43 Is a Negative Modulator of Neuronal Differentiation

Abstract

Connexin43 (Cx43) is widely expressed in embryonic brain, and its expression becomes restricted mainly to astrocytes as the central nervous system matures. Recent studies have indicated that Cx43 plays important, nonchannel, roles during central nervous system development by affecting neuronal cell migration. Here, we evaluated the effects of Cx43 on neuronal differentiation. For that we used an in vitro model of neural cell development (neurospheres) to evaluate, through immunocytochemistry, electrophysiology, and molecular biology, the degree of neuronal maturation from neurospheres derived from wild-type (WT) and Cx43-null mice. Our results indicate that Cx43 is a negative modulator of neuronal differentiation. The percent neurospheres containing differentiated neurons and the number of cells displaying inward currents were significantly higher in Cx43-null than in WT littermate neurospheres. Knockdown of Cx43 with small interfering RNA increased the number of WT neurospheres generating differentiated neurons. Blockade of gap junctional communication with carbenoxolone did not induce neuronal differentiation in WT neurospheres. Transfection of Cx43-null neurospheres with Cx43 mutants revealed that Cx43 carboxyl terminus prevents neuronal maturation. In agreement with these in vitro data, in situ analysis of embryonic day 16 brains revealed increased ␤-III-tubulin expression in germinal zones of Cx43-null compared with that of WT littermates. These results indicate that Cx43, and specifically its carboxyl terminus, is crucial for signaling mechanisms preventing premature neuronal differentiation during embryonic brain development.Multiple distinct mechanisms contribute to neocortical development. Diffusible and nondiffusible molecules have been implicated in neural cell proliferation, migration, and differentiation during brain development (1-4). Connexins (Cx), 2 the proteins forming gap junctions, are prominent in neural progenitor cells and play important roles in neural cell proliferation, migration, and differentiation (5-9). Among the central nervous system gap junction proteins, Cx43 and Cx26 are the two most abundantly expressed in neural stem cells, although transcripts for other connexins (Cx29, Cx30, Cx30.2, Cx32, Cx36, and Cx47) are also present in embryonic states (10 -12). The expression pattern of connexin proteins dramatically changes as the central nervous system matures, becoming restricted with respect to cell type. For instance, following postnatal development, Cx43 together with Cx30 are mainly found in astrocytes, whereas Cx29, Cx32, and Cx47 form the oligodendrocyte group of gap junction proteins, and Cx36, Cx45, and Cx30.2 are expressed in neurons (for review, see Ref. 13).The dramatic decrease in Cx43 expression during central nervous system development, specifically during neuroblast maturation, has raised the possibility that concurrent loss of gap junctional communication may play an important role during neuronal differentiation. Indeed, initial studies (14, 15) indicating an inver...

Connexin 43 controls the multipolar phase of neuronal migration to the cerebral cortex

“…Recently, studies using Cx43cKO and Cx43k258stop mice have indicated that the C terminus is required for cortical neuronal migration (30). In addition, independent regulation of the gap junction channel or C-terminal domain in neurite outgrowth (31,32) and cellular motility (33)(34)(35)(36)(37) have been demonstrated in various cell types, indicating that they may affect the neocortical development (38).…”

Internal Ribosomal Entry Site (IRES) Activity Generates Endogenous Carboxyl-terminal Domains of Cx43 and Is Responsive to Hypoxic Conditions

“…Cx43, besides conveying channel forming properties, has also been described as being involved in the regulation of additional biological functions, such as control of cell growth, cell survival, and differentiation (5,6); migration of neural precursor and glioma cells (7)(8)(9); and neuroprotection after hypoxic stress (10). An inverse relationship exists between Cx43 expression and cell growth (11,12), and a carboxyl-terminal domain was found to be as effective as full-length Cx43 in suppressing cell growth in a variety of cell lines (13)(14)(15).…”

Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology?

“…Several independent studies have highlighted an important role for Cxs, Cx43 in particular, in regulating NPC proliferation as well as the migration and differentiation of young excitatory neurons in the embryonic cerebral cortex. In Cx43-null brains of mice, the thickness of the CP was found to be reduced and this was accompanied by an impairment in lamination of the neocortex and accumulation of neurons in the IZ sublayer (E18, E21) [93,164,165] or the VZ/SVZ (E16) [166]. These effects may be caused by a defective migration [93,164] or by the induction of premature neuronal differentiation [166].…”

“…In Cx43-null brains of mice, the thickness of the CP was found to be reduced and this was accompanied by an impairment in lamination of the neocortex and accumulation of neurons in the IZ sublayer (E18, E21) [93,164,165] or the VZ/SVZ (E16) [166]. These effects may be caused by a defective migration [93,164] or by the induction of premature neuronal differentiation [166]. The role of neuronal Cx43 in cortical lamination was recently confirmed by the use of neuron-specific, conditional Cx43 KO mice.…”