Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi

The neuronal calcium sensor-1 (NCS-1) possesses a consensus signal for N-terminal myristoylation and four EF-hand Ca(2+)-binding sites, and mediates the effects of cytosolic Ca(2+). Minute changes in free intracellular Ca(2+) are quickly transformed into changes in the activity of several kinases including calcium/calmodulin-dependent protein kinases (Ca(2+)/CaMKs) that are involved in regulating many eukaryotic cell functions. However, our current knowledge of NCS-1 and Ca(2+)/CaMKs comes mostly from studies of the mammalian enzymes. Thus far very few fungal homologues of NCS-1 and Ca(2+)/CaMKs have been characterized and little is known about their cellular roles. In this minireview, we describe the known sequences, interactions with target proteins and cellular roles of NCS-1 and Ca(2+)/CaMKs in fungi.     

Activity-dependent regulation of the subcellular localization of neuronal calcium sensor-1 in the avian cochlear nucleus

Neurons in the avian cochlear nucleus, nucleus magnocellularis (NM), are highly sensitive to manipulations of afferent input, and removal of afferent activity through cochlear ablation results in the death of approximately 20-40% of ipsilateral NM neurons. The intracellular cascades that determine whether an individual NM neuron will die or survive are not fully understood. One early event observed in NM following deafferentation is a rapid rise in intracellular calcium concentration. In most cellular systems, the activity of calcium-binding proteins is believed to accommodate calcium influx. The calcium-binding protein, neuronal calcium sensor-1 (NCS-1), is an intracellular neuronal calcium sensor belonging to the EF-hand superfamily. NCS-1 has been implicated in calcium-dependent regulation of signaling cascades. To evaluate NCS-1 action in NM neurons, the localization of NCS-1 protein was examined. Double-label immunofluorescence experiments revealed that NCS-1 expression is evident in both the presynaptic nerve terminal and postsynaptic NM neuron. The postsynaptic expression of NCS-1 typically appears to be closely associated with the cell membrane. This close proximity of NCS-1 to the postsynaptic membrane could allow NCS-1 to function as a modulator of postsynaptic signaling events. Following deafferentation, NM neurons were more likely to show diffuse cytoplasmic NCS-1 labeling. This increase in the number of cells showing diffuse cytoplasmic labeling was observed 12 and 24 h following cochlea ablation, but was not observed 4 days following surgery. This activity-dependent regulation of NCS-1 subcellular localization suggests it may be associated with, or influenced by, processes important for the survival of NM neurons.     

神经元钙传感蛋白R102Q突变体的动力学构象特征

背景：神经元钙传感蛋白参与多种生理功能,在大脑皮质不同脑区都有很高的分布。在自闭症患者基因测序中识别出神经元钙传感蛋白第102个氨基酸精氨酸ARG102突变成谷氨酰胺Glu102(R102Q)。实验研究显示,R102Q突变对神经元钙传感蛋白局部区域影响很大,发生本质性的构象改变。 目的：确定神经元钙传感蛋白单一氨基酸R102Q突变引起结构构象动力学变化的具体原因。 方法：采用计算机分子动力学模拟的方法,进行6个独立的、模拟时间是450 ns的全原子动力学模拟。 结果与结论：①神经元钙传感蛋白R102Q突变对蛋白整体结构影响不大,在整个模拟过程中都没有进行大的构象重组,但导致螺旋改变,结构更加稳定。②R102Q突变导致盐桥网络发生改变,一方面降低了L2的柔性,使其更加稳定;另一方面改变L3在疏水口袋中的位置,使其在疏水口袋中更加舒展。结果表明,螺旋在蛋白结构稳定中起到一定的作用,盐桥改变也是蛋白动力学变化的重要原因。这项研究可能从分子的层面和结构的视角,为与R102Q突变有关的蛋白质功能缺失提供理论参考。  中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Acute changes in short-term plasticity at synapses with elevated levels of neuronal calcium sensor-1

Short-term synaptic plasticity is a defining feature of neuronal activity, but the underlying molecular mechanisms are poorly understood. Depression of synaptic activity might be due to limited vesicle availability, whereas facilitation is thought to result from elevated calcium levels. However, it is unclear whether the strength and direction (facilitation versus depression) of plasticity at a given synapse result from preexisting synaptic strength or whether they are regulated by separate mechanisms. Here we show, in rat hippocampal cell cultures, that increases in the calcium binding protein neuronal calcium sensor-1 (NCS-1) can switch paired-pulse depression to facilitation without altering basal synaptic transmission or initial neurotransmitter release probability. Facilitation persisted during high-frequency trains of stimulation, indicating that NCS-1 can recruit 'dormant' vesicles. Our results suggest that NCS-1 acts as a calcium sensor for short-term plasticity by facilitating neurotransmitter output independent of initial release. We conclude that separate mechanisms are responsible for determining basal synaptic strength and short-term plasticity.     

Effect of metrazol on time course of membrane-bound calcium level in the cerebral cortex

I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR. S. M. Kirov Military Medical Academy, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR N. G. Ivanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, pp. 50–51, January, 1989.     

Regulation of neurite outgrowth mediated by neuronal calcium sensor-1 and inositol 1,4,5-trisphosphate receptor in nerve growth cones

Calcium acts as an important second messenger in the intracellular signal pathways in a variety of cell functions. Strictly controlled intracellular calcium is required for proper neurite outgrowth of developing neurons. However, the molecular mechanisms of this process are still largely unknown. Neuronal calcium sensor-1 (NCS-1) is a high-affinity and low-capacity calcium binding protein, which is specifically expressed in the nervous system. NCS-1 was distributed throughout the entire region of growth cones located at a distal tip of neurite in cultured chick dorsal root ganglion neurons. In the central domain of the growth cone, however, NCS-1 was distributed in a clustered specific pattern and co-localized with the type 1 inositol 1,4,5-trisphosphate receptor (InsP₃R1). The pharmacological inhibition of InsP₃ receptors decreased the clustered specific distribution of NCS-1 in the growth cones and inhibited neurite outgrowth but did not change the growth cone morphology. The acute and localized loss of NCS-1 function in the growth cone induced by chromophore-assisted laser inactivation (CALI) resulted in the growth arrest of neurites and lamellipodial and filopodial retractions. These findings suggest that NCS-1 is involved in the regulation of both neurite outgrowth and growth cone morphology. In addition, NCS-1 is functionally linked to InsP₃R1, which may play an important role in the regulation of neurite outgrowth.     

Fos expression and task-related neuronal activity in rat cerebral cortex after instrumental learning

Learning has been shown to induce changes in neuronal gene expression and to produce development of task-specific neuronal activity. The connection between these two features of neuronal plasticity remains of a great interest. To address this issue we compared distribution of c-Fos expressing and task-related neurons in the rat cerebral cortex following instrumental learning of appetitive lever-press task. The number of Fos-positive neurons was determined immunohistochemically in the retrosplenial and the motor cortex of naive ("control" group), newly trained ("acquisition" group) and overtrained ("performance" group) animals. A significant activation of c-Fos expression was observed in the neurons of the retrosplenial but not motor cortex in the "acquisition" group rats, as compared with the "control" and "performance" groups. In accordance with this c-Fos expression difference, the retrosplenial cortex of the trained animals contained significantly more neurons with lever-press-related activity than the motor cortex. Therefore, the two examined cortical areas showed a parallel between experience-dependent induction of c-Fos and development of task-related neuronal activity. These data support a notion that learning-induced activation of c-Fos is associated with long-term neurophysiological changes produced by training.     

Immunocytochemical localization of neuronal calcium sensor-1 in the hippocampus and cerebellum of the mouse,with special reference to presynaptic terminals

Neuronal calcium sensor-1 (NCS-1) is a member of the EF-hand calcium-binding protein superfamily, which is considered to modulate synaptic transmission and plasticity. In this work, we first examined the distribution patterns of NCS-1 in the hippocampus and cerebellum. The intense NCS-1-immunoreactive (IR) elements in the hippocampus were restricted to dendritic layers, while those in the cerebellum occurred in both dendritic and cellular layers. Then, we examined the exact localization of NCS-1 using immunofluorescent double labeling for NCS-1 and synaptophysin, a marker of presynaptic terminals. In the hippocampus, the mossy fiber systems (terminals and bundles) exhibited intense NCS-1 immunoreactivity. On the other hand, the presumed principal cell dendrites were also NCS-1-IR in the stratum lacunosum-moleculare of Ammon's horn and molecular layer of the dentate gyrus, where NCS-1-IR elements and synaptophysin-IR presynaptic terminals showed characteristic complementary distribution patterns. In the cerebellum, some of the basket cell axon terminals surrounding the somata of Purkinje cells exhibited NCS-1 immunoreactivity, while the pinceau showed consistent labeling for NCS-1. Higher magnification observations revealed that the NCS-1-IR presumed granule cell dendrites and synaptophysin-IR mossy fiber terminals in the glomeruli of the cerebellum showed characteristic complementary distribution patterns. Furthermore, we estimated quantitatively the relative amount of NCS-1 in the presynaptic terminals in individual layers, and confirmed that the mossy fiber terminals in the hippocampus contained comparatively high amounts of NCS-1. These results showed the diverse localization of NCS-1 in pre- and/or postsynaptic elements of the hippocampus and cerebellum, and suggest potential roles in specific synaptic transmission.     

p27kip1 independently promotes neuronal differentiation and migration in the cerebral cortex

The generation of neurons by progenitor cells involves the tight coordination of multiple cellular activities, including cell cycle exit, initiation of neuronal differentiation, and cell migration. The mechanisms that integrate these different events into a coherent developmental program are not well understood. Here we show that the cyclin-dependent kinase inhibitor p27(Kip1) plays an important role in neurogenesis in the mouse cerebral cortex by promoting the differentiation and radial migration of cortical projection neurons. Importantly, these two functions of p27(Kip1) involve distinct activities, which are independent of its role in cell cycle regulation. p27(Kip1) promotes neuronal differentiation by stabilizing Neurogenin2 protein, an activity carried by the N-terminal half of the protein. p27(Kip1) promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Thus, p27(Kip1) plays a key role in cortical development, acting as a modular protein that independently regulates and couples multiple cellular pathways contributing to neurogenesis.     

Chronic unpredictable stress promotes neuronal apoptosis in the cerebral cortex

Stress-mediated loss of synaptogenesis in the hippocampus appears to play a role in depressive and mood disorders. However, little is known about the effect of stress/depression on the plasticity and survival of cortical neurons. In this report, we have examined whether chronic stress increases the vulnerability of neurons in the rat cortex. We have used a chronic unpredictable mild stress (CMS) as a rat model of depression. CMS (5 weeks treatment) produced anedonia and increased corticosterone levels. These effects were accompanied by a detectable increase in caspase-3 positive neurons in the cerebral cortex, suggesting apoptosis. Desipramine (DMI), a well known antidepressant, reversed the pro-apoptotic effect of CMS. These results suggest that antidepressants may reduce the pathological changes seen in stress-induced depressive disorders.     

1Alpha,25-dihydroxyvitamin D3 treatment does not alter neuronal cyclooxygenase-2 expression in the cerebral cortex after stroke

The inducible prostaglandin synthase, cyclooxygenase-2, is upregulated in response to cerebral ischemia and contributes to potentiation of oxidative injury. Cyclooxygenase-2 expression is regulated by retinoic acid receptors, which form heterodimers with vitamin D receptors and vitamin D. In addition, vitamin D has been reported to have neuroprotective qualities. The aim of this study was to examine whether the biologically active vitamin D₃-metabolite 1α,25-dihydroxyvitamin D₃ (1,25-D₃), influences the expression of inducible cyclooxygenase-2 in photothrombotically lesioned brain or is part of an independent neuroprotective mechanism. We compared groups of nonlesioned control rats and infarcted animals, which were treated with either 1,25-D₃ or solvent at different times postlesion. In control animals, cyclooxygenase-2 immunoreactivity was readily evident in almost all cortical neurons of layers II/III as well as in a few pyramidal cells in layer V. Following photothrombotic infarction of the right cortical hindlimb area, there was a significant, but transient, increase in cyclooxygenase-2 labeling which was restricted to neurons of the injured hemisphere in both 1,25-D₃-treated and solvent-treated rats. Highest levels of cyclooxygenase-2 immunoreactivity were seen at 12 and 24 h postlesion, followed by a gradual decrease at later time points. However, no significant differences were detected between 1,25-D₃-treated and solvent-treated lesioned rats, indicating that postischemic neuronal cyclooxygenase-2 upregulation is not influenced by 1,25-D₃. It is concluded that the neuroprotective effect of 1,25-D₃ does not depend on modulations of neuronal COX-2 expression caused by postlesional hyperexcitation.     

Human slow-wave sleep and the cerebral cortex

SUMMARY  Recent hypotheses about the roles of human slow-wave sleep (hSWS—delta EEG activity) are appraised. The possible linkage between hSWS and the functions of the prefrontal cortex (PFC) are explored with respect to normal subjects and to disorders involving PFC deficits.     

Ultrastructural pathology of neuronal membranes in the oedematous human cerebral cortex

Surgical biopsies of frontal, parietal and temporal regions of thirty two patients with clinical diagnosis of congenital hydrocephalus, brain trauma, tumours, and vascular anomalies were examined with the transmission electron microscope. The main goal was to study the submicroscopic alterations of somatodendritic, axonal, and synaptic plasma membranes, cytomembranes, and the cytoskeleton. In both, moderate and severe oedema, fragmentation of plasma membrane, enlargement and focal necrosis of rough endoplasmic cisterns and nuclear envelope, detachment of membrane-bound ribosomes and reduction of polysome were observed. The degenerated myelinated axons exhibited discontinuities of the axolemma, disorganisation of multiple myelin lamellae, myelin sheath vacuolization, and formation of myelin ovoids. In severe oedema, synaptic disassembly was frequently found characterized by separate pre- and postsynaptic endings and loss of perisynaptic glial ensheathment. Fragmented and intact microtubules and actin-like filaments also were distinguished. The alterations of plasma membranes and cytomembranes are related with the anoxic-ischaemic conditions of brain parenchyma. The role of free radical and lipid peroxidation, disturbed energy metabolism, altered metabolic cascades, excitotoxicity, protein aggregation, and presence of extracellular oedema fluid is discussed in relation with the derangement of neuronal membranes.     

Anoxic changes in cAMP level in the cat cerebral cortex

Laboratory for Regulation of Functions of Brain Neurons, I. P. Pavlov Institute of Physiology, Academy of Sciences of Russia, St. Petersburg. (Presented by Academician of the Russian Academy of Medical Sciences A. N. Klimov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 114, No. 9, pp. 227–228, September, 1992.     

Area- and lamina-specific organization of a neuronal subpopulation defined by expression of latexin in the rat cerebral cortex

The aim of the present study was to investigate the density, laminar distribution, size, morphology, and neurotransmitter phenotype of rat cortical neurons expressing latexin, an inhibitor of carboxypeptidase A. Immunohistochemical analyses established that latexin-immunoreactive neurons are restricted essentially to the infragranular layers of lateral cortical areas in the rat. The overall density, laminar or sublaminar localization, and cell size distribution of latexin-positive neurons differed substantially across cytoarchitectonic areas within lateral cortex. Numerous latexin-positive neurons had the morphology of modified pyramidal cells especially of layer VI. The vast majority of latexin-positive neurons were glutamate-immunoreactive in the six lateral neocortical areas examined, while neurons immunoreactive for both latexin and GABA were virtually absent. Thus the majority of latexin-positive neurons are likely to be excitatory projection neurons. The area- and lamina-specific distribution of the latexin-expressing subpopulation of glutamate-immunoreactive neurons is a distinctive feature that may contribute to the functional specialization of the lateral cortical areas.     

Intramitochondrial crystalloid inclusions in neuronal processes of human cerebral cortex

Summary Orbital cortex specimens obtained as postmortem brain needle samples from 4 of 7 elderly subjects contained inclusion-carrying mitochondria in a small percentage of neuronal processes. Banded, lattice-like and hexagonal patterns were all seen in the inclusions. These patterns are thought to result from different planes of section through ordered aggregates of tubules. The crystalloid inclusions in mitochondria are tentatively regarded as a morphologic variant within the normal range, possibly brought about by some metabolic changes intervening, for instance, just before death.