Abl2 Kinase Differentially Regulates iGluRs Current Activity and Synaptic Localization.

Abelson non-receptor tyrosine kinases (Abl1 and Abl2) are established cellular signaling proteins, implicated in cytoskeletal reorganization essential for modulation of cell morphology and motility. During development of the central nervous system, Abl kinases play fundamental roles in neurulation and neurite outgrowth, relaying information from axon guidance cues and growth factor receptors to promote cytoskeletal rearrangements. In mature neurons, Abl kinases localize to pre- and postsynaptic compartments and are involved in regulation of synaptic stability and plasticity. Although emerging evidence indicates interchangeability of these isoforms in managing of cellular functions, in healthy adult neurons, Abl1 contribution is less elucidated, while Abl2 is required for optimal synaptic functioning. Our previous study demonstrated compartmentalization of Abl1 to the presynapse and Abl2 to the postsynapse and characterized their modulatory effect on spontaneous excitatory synaptic transmission. Here, we further delineate the role of Abl2 on regulation of the postsynaptic component of miniature excitatory postsynaptic current (mEPSC). Our findings show that both acute and prolonged activation of Abl2, in line with reduction of mEPSC amplitude, also decrease AMPA and NMDA current amplitudes. In contrast with the current-detrimental effect, prolonged Abl2 activity stabilizes spines, particularly contributing to maintenance of active synapses at distal (perhaps apical) segments of dendrites. Hence, we propose that attenuation of ion currents via ionotropic glutamatergic receptors by Abl2 kinase derives from either reduction of the receptor sensitivity for glutamate or is due to alteration of channel gating mechanisms. Abl2 and excitatory postsynapses: Abl2 expression level affects active excitatory synapse density on distal dendrites, while Abl2 activity impacts current density through AMPA and NMDA receptors.

Abelson Kinases Mediate the Depression of Spontaneous Synaptic Activity Induced by Amyloid Beta 1-42 Peptides.

Amyloid beta (Aß) peptides represent one of the most studied etiological factors of Alzheimer's disease. Nevertheless, the effects elicited by different molecular forms of amyloid beta peptides widely vary between the studies, mostly depending on experimental conditions. Despite the enormous amount of accumulated evidences concerning the pathological effects of amyloid beta peptides, the exact identity of the amyloid beta species is still controversial, and even less is clear as regards to the downstream effectors that mediate the devastating impact of these peptides on synapses in the central nervous system. Recent publications indicate that some of the neurotoxic effects of amyloid beta peptides may be mediated via the activation of proteins belonging to the Abelson non-receptor tyrosine kinase (Abl) family, that are known to regulate actin cytoskeleton structure as well as phosphorylate microtubule-associated tau protein, a hallmark of Alzheimer's disease. By performing series of miniature excitatory postsynaptic currents (mEPSC) recordings in cultured hippocampal cells, we demonstrate that activation of Abl kinases by acute application of 42 amino acid-length monomeric amyloid beta (Aß1-42) peptides reduces spontaneous synaptic release, while this effect can be rescued by pharmacologic inhibition of Abl kinase activity, or by reduction of Abl expression with small interfering RNAs. Our electrophysiological data are further reinforced by a subsequent biochemical analysis, showing enhanced phosphorylation of Abl kinase substrate CT10 Regulator of Kinase-homolog-Like (Crkl) upon treatment of hippocampal neurons with Aß peptides. Thus, we conclude that Abl kinase activation may be involved in Aß-induced weakening of synaptic transmission.

Membrane-tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post-synaptic AMPA receptor level.

The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long-term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long-term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long-term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post-synaptic expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post-synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post-synaptic membrane. © 2016 Wiley Periodicals, Inc.

Motion, creation, and annihilation of disclinations in multidomain structured nematic liquid crystal cells.

We study dynamical processes in a multidomain (MD) structured nematic liquid crystal cell with a particular emphasis on the motion, creation, and annihilation of disclinations. In the MD cell right- and left-handed director helices alternate due to a special choice of the director pretilt angles at the surfaces. As a result, a net of twist disclinations occurs. We have implemented a numerical algorithm based on a pure rotational dynamics of the director field to monitor the motion of the defect lines during the switching process, i.e., when an electric voltage is applied to or removed from the cell. We demonstrate that the total light transmission vs time is not affected by the presence of the defects compared to a conventional twisted nematic cell. If the pretilt angles at the surfaces are sufficiently small, the twisting sense of one species of helices is reversed and a configuration free of defects occurs. On the other hand, for an applied voltage twist disclinations close to the surface have to exist. Therefore, defect lines are created or they annihilate during the switching process. We investigate these situations in detail and reveal the underlying mechanisms.

A new beta-lactoglobulin-based vector targets luciferase cDNA expression to the mammary gland of transgenic mice.

A beta-lactoglobulin (BLG)/luciferase gene vector (p907), composed of a luciferase intronless gene inserted between the second and sixth BLG exons was constructed. Stable transfections of CID-9 cells with this vector, as well as with a series of additional vectors, were performed to define regulatory regions within the BLG sequence, and the contribution of the SV40 polyadenylation (PA) site to luciferase expression. A relatively low level of luciferase activity was supported by vector p907. It was partially rescued by vector p906, in which the BLG 3' region, downstream of the luciferase cDNA, was replaced with the SV40 PA site. Flanking the SV40 region of vector p906, at its 3' end, with BLG sequences of exon 6/intron 6/exon 7 and the 3' region of the gene resulted in vector p904. This vector supported the highest luciferase activity, 10 times or 2.5 times higher than that measured in cells transfected with vectors p907 and p906, respectively. The induced activity supported by vector p904 is attributed to interaction between the SV40 PA site and elements of the distal part of the BLG 3' flanking sequences. The BLG 5' regulatory region of vector p904 encompasses a 3-kb promoter sequences. Deletion of 935 bp of its proximal end resulted in a 60% decrease in luciferase activity. Reduced activity was also seen with vector p915 lacking sequences of exon 1/intron 1/exon 2. This decrease could not be rescued with heterologous sequences of insulin intron 1, inserted upstream of the luciferase cDNA. Two sets of transgenic mice carrying vectors p907 and p904 were generated. Vector p907 supported only marginal luciferase activity in the mammary gland of all transgenic mice tested and luciferase RNA could not be detected by northern analysis. In contrast, 50% of the transgenic mice carrying vector p904 expressed luciferase RNA in the mammary gland and tissue-specific, hormonal-dependent activity was determined. However, the new p904 vector was not able to insulate the transgene from surrounding host DNA sequences, as reflected by its copy number-independent manner of expression. Nevertheless, vector p904 may represent a valuable tool for the expression of cDNAs in the mammary gland of transgenic animals.

A novel transgenic marker for migrating limb muscle precursors and for vascular smooth muscle cells.

A unique pattern of LacZ expression was found in a transgenic mouse line, likely due to regulatory elements at the site of integration. Two new genes flanking the transgene were identified. At early stages of development, the transgene is transiently expressed in ventro-lateral demomyotomal cells migrating from the somites into the limb buds. At late developmental stages and in the adult, lacZ staining marks vascular smooth muscle cells throughout the vascular bed, with the exception of the major elastic arteries, and in pericytes. No expression was detected in skeletal and smooth muscles. Different patterns of expression in vascular smooth muscles was observed at distinct levels of the vascular tree, in arteries as well as in veins. Vessel injury, resulting in stimulation of smooth muscle cells proliferation and migration, is associated with transgene down-regulation. After the formation of neointima thickening, it is reactivated. This transgenic insertion may therefore be used as a useful marker to identify novel physiological cues or genetic elements involved in the regulation of the vascular smooth muscle phenotype(s). It may also provide an experimental tool for studying vasculature and the involvement of pericytes in regulating microvascular homeostasis.