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Single and double antigen localization procedures were used to study the distribution of the calcium-binding proteins calretinin, calbindin and parvalbumin in the cerebellum of the squirrel monkey (Saimiri sciureus). The immunostaining experiments have revealed that each of the three calcium-binding proteins occurred, either alone or in various combinations, in many neuronal types of the monkey cerebellum, including the Purkinje cells. Immunoreactivity for calbindin was detected in virtually all Purkinje cells, whereas immunoreactivity for calretinin and parvalbumin was encountered only in some subpopulations of Purkinje cells. In the vermal region, parvalbumin immunostaining appeared in the form of typical weak and strong alternating parasagittal bands. Calretinin immunoreactivity was found in virtually all neurons and fiber systems related to the granular layer, including the monodendritic cells, the granule cells and their parallel fibers, the Golgi and Lugaro cells and the mossy fibers. The Golgi cells also displayed calbindin and parvalbumin immunoreactivity. Parvalbumin was found to labeled both the climbing and mossy fibers, as well as the basket and stellate cells lying in the molecular layer. These results reveal that virtually all the different neuronal types in the primate cerebellum contain at least one of three calcium-binding proteins investigated in the present study. Furthermore, calretinin appears to be a particularly reliable molecular maker for all the neuronal elements associated with the granular layer in the primate cerebellum.
Ndr is a nuclear serine/threonine protein kinase that belongs to a subfamily of kinases identified as being critical for the regulation of cell division and cell morphology. The regulatory mechanisms that control Ndr activity have not been characterized previously. In this paper, we present evidence that Ndr is regulated by EF-hand calcium-binding proteins of the S100 family, in response to changes in the intracellular calcium concentration. In vitro, S100B binds directly to and activates Ndr in a Ca2+-dependent manner. Moreover, Ndr is recovered from cell lysates in anti-S100B immunoprecipitates. The region of Ndr responsible for interaction with Ca2+/S100B is a basic/hydrophobic motif within the N-terminal regulatory domain of Ndr, and activation of Ndr by Ca2+/S100B is inhibited by a synthetic peptide derived from this region. In cultured cells, Ndr is rapidly activated following treatment with Ca2+ ionophore, and this activation is dependent upon the identified Ca2+/S100B-binding domain. Finally, Ndr activity is inhibited by W-7 in melanoma cells overexpressing S100B, but is unaffected by W-7 in melanoma cells that lack S100B. These results suggest that Ndr is regulated at least in part by changes in the intracellular calcium concentration, through binding of S100 proteins to its N-terminal regulatory domain.
Motifs within proteins help us categorize their functions. Intrinsically disordered proteins (IDPs) are rich in short linear motifs, conferring them many different roles. IDPs are also frequently highly charged and, therefore, likely to interact with ions. Canonical calcium-binding motifs, such as the EF-hand, often rely on the formation of stabilizing flanking helices, which are a key characteristic of folded proteins, but are absent in IDPs. In this study, we probe the existence of a calcium-binding motif relevant to IDPs. Upon screening several carefully selected IDPs using NMR spectroscopy supplemented with affinity quantification by colorimetric assays, we found calcium-binding motifs in IDPs which could be categorized into at least two groups-an Excalibur-like motif, sequentially similar to the EF-hand loop, and a condensed-charge motif carrying repetitive negative charges. The motifs show an affinity for calcium typically in the ~100 μM range relevant to regulatory functions and, while calcium binding to the condensed-charge motif had little effect on the overall compaction of the IDP chain, calcium binding to Excalibur-like motifs resulted in changes in compaction. Thus, calcium binding to IDPs may serve various structural and functional roles that have previously been underreported.
Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.
A novel calcium-binding protein (EhCaBP) has been recently identified and characterized from the protozoan parasite Entamoeba histolytica. In order to decipher the function of this protein, a few basic properties were investigated and compared with the ubiquitous Ca(2+)-signal transducing protein calmodulin (CaM). Indirect immunofluorescence and immunoprecipitation analyses using specific antibodies against EhCaBP suggest that it is a soluble cytoplasmic protein with no major post-translational modification. EhCaBP did not stimulate cAMP-phosphodiesterase activity, differentiating it from all known CaMs. Affinity chromatography of [35S]methionine-labelled proteins of E. histolytica trophozoites using EhCaBP-sepharose column showed Ca(2+)-dependent binding of a group of proteins. Radiolabelled proteins from the same extract also bound to CaM-sepharose. However, the proteins bound to the two columns were different as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At least one of the EhCaBP-binding proteins became phosphorylated as revealed by in vivo phosphorylation analysis. The binding-proteins could not be detected in E. invadens (a species that is pathogenic in reptiles) and E. moshkovskii (which is found in the human gut but is not pathogenic), two species in which EhCaBP-like protein has not been found. Two distinct Ca(2+)-dependent protein kinases, which get activated by EhCaBP and CaM respectively, were detected in E. histolytica. These kinases require different levels of Ca2+ for their maximal activities. Affinity chromatography also showed the binding of protein kinase(s) to EhCaBP in a Ca(2+)-dependent manner. Our data suggest that there may be novel Ca(2+)-signal transduction pathway in E. histolytica mediated by EhCaBP.
Calcium-binding proteins regulate ion metabolism and vital signalling pathways in all living organisms. Our aim is to rationalize the molecular basis of their function by studying their evolution using computational biology techniques. Phylogenetic analysis is of primary importance for classifying cognate orthologs; profile hidden Markov models (HMM) of individual subfamilies discern functionally relevant sites by conservation probability analysis; and 3-dimensional structures display the integral protein in context. The major classifications of calcium-binding proteins, viz. EF-hand, C2 and ANX, exhibit structural diversity in their HMM fingerprints at the subfamily level, with functional consequences for protein conformation, exposure of receptor interaction sites and/or binding to membrane phospholipids. Calmodulin, S100 and annexin families were characterized in Petromyzon marinus (sea lamprey) to document genome duplication and gene creation events during the key evolutionary transition to primitive vertebrates. Novel annexins from diverse organisms revealed calcium-binding domains with accessory structural features that define their unique molecular fingerprints, protein interactivity and functional specificity. These include the first single-domain, bacterial annexin in Cytophaga hutchinsonii, the 21 tetrad annexins from the unicellular protist Giardia intestinalis, an ancestor to land plant annexins from the green alga Ostreococcus lucimarinus, invertebrate octad annexins and a critical polymorphism in human ANXA7. Receptor docking models supported the hypothesis of a potential interaction between annexin and C2 domains as a propitious mechanism for ensuring membrane translocation during signal transduction.
Calcium-binding proteins may endow tumor cells with properties related to their malignancy and metastatic phenotype. Chromatographic procedures and amino acid sequence analysis were used in this study to identify seven calcium-binding proteins, annexin VI, cap g, annexin V, calmodulin, S100A11, S100B and S100A6, associated with uveal melanoma, the primary ocular tumor of adults. This series of calcium-binding proteins was identified in both primary tumors and cell lines of uveal melanoma. Several of the proteins were shown by immunochemical methods to be differentially expressed between normal uveal melanocytes and malignant melanomas of the uvea. In addition, the expression of S100A6 may correlate with the malignant properties of the tumor.
Apoptosis is a highly ordered and orchestrated multiphase process controlled by the numerous cellular and extra-cellular signals, which executes the programmed cell death via release of cytochrome c alterations in calcium signaling, caspase-dependent limited proteolysis and DNA fragmentation. Besides the general modifiers of apoptosis, several tissue-specific regulators of this process were identified including HAX1 (HS-1 associated protein X-1) - an anti-apoptotic factor active in myeloid cells. Although HAX1 was the subject of various experimental studies, the mechanisms of its action and a functional link connected with the regulation of apoptosis still remains highly speculative.
Cisplatin (CDDP) is currently one of the most effective FDA-approved treatments for breast cancer. Previous studies have shown that CDDP-induced cell death in human breast cancer (MCF-7) cells is associated with disruption of calcium homeostasis. However, whether the sensitivity of breast cancer cells to cisplatin is associated with dysregulation of the expression of calcium-binding proteins (CaBPs) remains unknown. In this study, we evaluated the effect of the intracellular calcium chelator (BAPTA-AM) on viability of MCF-7 cells in the presence of toxic and sub-toxic doses of cisplatin. Furthermore, this study assessed the expression of CaBPs, calmodulin, S100A8, and S100A14 in MCF-7 cells treated with cisplatin. Cell viability was determined using MTT-based in vitro toxicity assay. Intracellular calcium imaging was done using Fluo-4 AM, a cell-permeant fluorescent calcium indicator. Expression of CaBPs was tested using real-time quantitative PCR. Exposure of cells to increasing amounts of CDDP correlated with increasing fluorescence of the intracellular calcium indicator, Fluo-4 AM. Conversely, treating cells with cisplatin significantly decreased mRNA levels of calmodulin, S100A8, and S100A14. Treatment of the cells with calcium chelator, BAPTA-AM, significantly enhanced the cytotoxic effects of sub-toxic dose of cisplatin. Our results indicated a statistically significant negative correlation between calmodulin, S100A8, and S100A14 expression and sensitivity of breast cancer cells to a sub-toxic dose of cisplatin. We propose that modulating the activity of calcium-binding proteins, calmodulin, S100A8, and S100A14, could be used to increase cisplatin efficacy, lowering its treatment dosage while maintaining its chemotherapeutic value.
The molecular repertoire of the "Ca2+-signaling toolkit" supports the specific kinetic requirements of Ca2+-dependent processes in different neuronal types. A well-known example is the unique expression pattern of calcium-binding proteins, such as parvalbumin, calbindin, and calretinin. These cytosolic Ca2+-buffers control presynaptic and somatodendritic processes in a cell-type-specific manner and have been used as neurochemical markers of GABAergic interneuron types for decades. Surprisingly, to date no typifying calcium-binding proteins have been found in CB1 cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons that represent a large population of GABAergic cells in cortical circuits. Because CB1/CCK-positive interneurons display disparate presynaptic and somatodendritic Ca2+-transients compared with other interneurons, we tested the hypothesis that they express alternative calcium-binding proteins. By in silico data mining in mouse single-cell RNA-seq databases, we identified high expression of Necab1 and Necab2 genes encoding N-terminal EF-hand calcium-binding proteins 1 and 2, respectively, in CB1/CCK-positive interneurons. Fluorescent in situ hybridization and immunostaining revealed cell-type-specific distribution of NECAB1 and NECAB2 throughout the isocortex, hippocampal formation, and basolateral amygdala complex. Combination of patch-clamp electrophysiology, confocal, and STORM super-resolution microscopy uncovered subcellular nanoscale differences indicating functional division of labor between the two calcium-binding proteins. These findings highlight NECAB1 and NECAB2 as predominant calcium-binding proteins in CB1/CCK-positive interneurons.
Motor neurons with different susceptibility to degeneration have been identified in amyotrophic lateral sclerosis (ALS). Increase of intracellular calcium has been proposed as a mediator, amplifying the damage through a positive feedback of the known pathological processes. Accordingly, the potential of motor neurons to limit calcium increases during injury might be proportional to their viability. A basic mechanism of reducing calcium amplitudes depends on the calcium-buffering capacity, determined by the calcium-binding protein content. In this study, oculomotor and hypoglossal neurons, prototypes of resistant and vulnerable motor neurons in ALS were examined in axotomy experiments. Total calcium-, parvalbumin-, and calbindin-D28k levels of motor neurons of adult mice were characterized by electron microscopic histochemistry and light microscopic immunostaining. In hypoglossal neurons, compared with oculomotor neurons, larger and more enduring increases of calcium were detected. The perikarya of hypoglossal neurons remained immunonegative for both parvalbumin and calbindin-D28k. Qualitatively, no major cell loss was noted after axotomy, but a decreased neuronal marker staining at days 1-14 suggested a reversible injury of hypoglossal neurons. Oculomotor neurons were not stained for calbindin-D28k but stained for parvalbumin in control conditions, staining which increased at postoperative days 7-14 before returning to baseline. Neuronal marker staining did not change in these cells during the observed period. The higher level of parvalbumin in resistant motor neurons and their ability to up-regulate parvalbumin after injury, paralleled by a smaller increase of intracellular calcium suggest that parvalbumin may have a protective effect in these cells.
Studying the distribution of Ca2+-binding proteins allows one to discover specific neuron chemotypes involved in the regulation of the activity of various neural elements. While extensive data exist on Ca2+-binding proteins in the nervous system, in particular, in the cerebellar cortex of terrestrial mammals, the localization of these proteins in the cerebellar cortex of marine mammals has not been studied. We studied the localization of calretinin, calbindin, and parvalbumin immunoreactivity in the cerebellar cortex of the bottlenose dolphin Tursiops truncates and harbour porpoise Phocoena phocoena. In both species, most Purkinje cells were calbindin-immunoreactive, while calretinin and parvalbumin were expressed in a small portion of Purkinje cells. In addition, calretinin-immunoreactive unipolar brush and granule cells and calbindin- and parvalbumin-immunoreactive basket, stellate, and Golgi cells were observed. Calretinin-immunoreactive corticopetal (mossy and climbing) fibers were found. Based on the length of the primary dendrite, short-, middle-, and long-dendrite unipolar brush cells could be distinguished. The validity of this classification was supported using cluster analysis suggesting the presence of several natural types of these cells. The distribution of Ca2+-binding proteins in the cerebellar cortex of the cetaceans studied was generally similar to that reported for terrestrial mammals, suggesting that this trait is evolutionarily conservative in mammals.
The development of therapeutic approaches to improve the life quality of people suffering from different types of body paralysis is a current major medical challenge. Brain-machine interface (BMI) can potentially help reestablishing lost sensory and motor functions, allowing patients to use their own brain activity to restore sensorimotor control of paralyzed body parts. Chronic implants of multielectrodes, employed to record neural activity directly from the brain parenchyma, constitute the fundamental component of a BMI. However, before this technique may be effectively available to human clinical trials, it is essential to characterize its long-term impact on the nervous tissue in animal models. In the present study we evaluated how chronic implanted tungsten microelectrode arrays impact the distribution and morphology of interneurons reactive to calcium-binding proteins calbindin (CB), calretinin (CR) and parvalbumin (PV) across the rat's motor cortex. Our results revealed that chronic microelectrode arrays were well tolerated by the nervous tissue, with recordings remaining viable for up to 6 months after implantation. Furthermore, neither the morphology nor the distribution of inhibitory neurons were broadly impacted. Moreover, restricted microglial activation was observed on the implanted sites. On the whole, our results confirm and expand the notion that tungsten multielectrodes can be deemed as a feasible candidate to future human BMI studies.
We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.
We have localized the spinocerebellar neuron groups in C57BL/6J mice by injecting the retrograde neuronal tracer Fluoro-Gold into the cerebellum and examined the distribution of SMI 32 and the calcium-binding proteins (CBPs), calbindin-D-28K (Cb), calretinin (Cr), and parvalbumin (Pv) in the spinal precerebellar nuclei. The spinal precerebellar neuron clusters identified were the dorsal nucleus, central cervical nucleus, lumbar border precerebellar nucleus, lumbar precerebellar nucleus, and sacral precerebellar nucleus. Some dispersed neurons in the deep dorsal horn and spinal laminae 6-8 also projected to the cerebellum. Cb, Cr, Pv, and SMI 32 were present in all major spinal precerebellar nuclei and Pv was the most commonly observed CBP. A number of genes expressed in hindbrain precerebellar nuclei are also expressed in spinal precerebellar groups, but there were some differences in gene expression profile between the different spinal precerebellar nuclei, pointing to functional diversity amongst them.
In Parkinson's disease oxidative stress and calcium-induced excitotoxicity have been considered important mechanisms leading to cell death for decades, but the factors that make some neurons vulnerable to neurodegeneration while others remain resistant are not fully understood. Studies of the disorder in animal models suggest that the voltage-gated calcium channel subtype Ca(V)1.3 has a role in making neurons susceptible to neurodegeneration and support earlier work in post-mortem human brain that suggested loss of calcium buffering capacity in neurons correlated with areas of neuronal loss in the substantia nigra of parkinsonian brain. This study examined expression of Ca(V)1 subtypes and the calcium-binding proteins calbindin, calmodulin and calreticulin in areas vulnerable and resistant to neurodegeneration in Parkinson's disease, in brain from neurologically normal individuals and patients with Parkinson's disease. In control brain the expression of a specific Ca(V)1 subtype or distribution of each calcium-binding protein did not associate with those regions prone to neurodegeneration in Parkinson's disease. Whereas, alterations in the amount of both Ca(V)1 subtypes and the calcium-binding proteins were found throughout the brain in Parkinson's disease. Some changes reflected the cell loss seen in Parkinson's disease, whereas others represented altered levels of cellular expression, which as they occurred in the absence of cell loss could not be explained as solely compensatory to the neurodegeneration. The finding of increased Ca(V)1.3 subtype expression in the cerebral cortex of early stage Parkinson's disease, before the appearance of pathological changes, supports the view that disturbed calcium homeostasis is an early feature of Parkinson's disease and not just a compensatory consequence to the neurodegenerative process. This interpretation is supported further by the finding that the ratio of Ca(V)1 subtypes differed throughout the brain in patients with Parkinson's disease compared with control subjects, in favour of an increased use of Ca(V)1.3, which would add to the metabolic burden for cells that rely on this Ca(V)1 subtype for electrical activity and could therefore render specific neuronal populations more vulnerable to neurodegeneration.
Neuronal networks in the spinal cord generate and execute all locomotor-related movements by transforming descending signals from supraspinal areas into appropriate rhythmic activity patterns. In these spinal networks, neurons that arise from the same progenitor domain share similar distribution patterns, neurotransmitter phenotypes, morphological and electrophysiological features. However, subgroups of them participate in different functionally distinct microcircuits to produce locomotion at different speeds and of different modalities. To better understand the nature of this network complexity, here we characterized the distribution of parvalbumin (PV), calbindin D-28 k (CB) and calretinin (CR) which are regulators of intracellular calcium levels and can serve as anatomical markers for morphologically and potential functionally distinct neuronal subpopulations. We observed wide expression of CBPs in the adult zebrafish, in several spinal and reticulospinal neuronal populations with a diverse neurotransmitter phenotype. We also found that several spinal motoneurons express CR and PV. However, only the motoneuron pools that are responsible for generation of fast locomotion were CR-positive. CR can thus be used as a marker for fast motoneurons and might potentially label the fast locomotor module. Moreover, CB was mainly observed in the neuronal progenitor cells that are distributed around the central canal. Thus, our results suggest that during development the spinal neurons utilize CB and as the neurons mature and establish a neurotransmitter phenotype they use CR or/and PV. The detailed characterization of CBPs expression, in the spinal cord and brainstem neurons, is a crucial step toward a better understanding of the development and functionality of neuronal locomotor networks.
Information about the position and movement of the head in space is coded by vestibular receptors and relayed to four nuclei that comprise the vestibular nuclear complex (VNC). Many additional brainstem nuclei are involved in the processing of vestibular information, receiving signals either directly from the eighth nerve or indirectly via projections from the VNC. In cats, squirrel monkeys, and macaque monkeys, we found neurochemically defined subdivisions within the medial vestibular nucleus (MVe) and within the functionally related nucleus prepositus hypoglossi (PrH). In humans, different studies disagree about the borders, sizes, and possible subdivisions of the vestibular brainstem. In an attempt to clarify this organization, we have begun an analysis of the neurochemical characteristics of the human using brains from the Witelson Normal Brain Collection and standard techniques for antigen retrieval and immunohistochemistry. Using antibodies to calbindin, calretinin, parvalbumin, and nitric oxide synthase, we find neurochemically defined subdivisions within the MVe similar to the subdivisions described in cats and monkeys. The neurochemical organization of PrH is different. We also find unique neurochemical profiles for several structures that suggest reclassification of nuclei. These data suggest both quantitative and qualitative differences among cats, monkeys, and humans in the organization of the vestibular brainstem. These results have important implications for the analysis of changes in that organization subsequent to aging, disease, or loss of input.
The pathogenesis of kidney stone formation includes multi-step processes involving complex interactions between mineral components and protein matrix. Calcium-binding proteins in kidney stones have great influences on the stone formation. The spatial distributions of these proteins in kidney stones are essential for evaluating the in vivo effects of proteins on the stone formation, although the actual distribution of these proteins is still unclear. We reveal micro-scale distributions of three different proteins, namely osteopontin (OPN), renal prothrombin fragment 1 (RPTF-1), and calgranulin A (Cal-A), in human kidney stones retaining original mineral phases and textures: calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD). OPN and RPTF-1 were distributed inside of both COM and COD crystals, whereas Cal-A was distributed outside of crystals. OPN and RPTF-1 showed homogeneous distributions in COM crystals with mosaic texture, and periodically distributions parallel to specific crystal faces in COD crystals. The unique distributions of these proteins enable us to interpret the different in vivo effects of each protein on CaOx crystal growth based on their physico-chemical properties and the complex physical environment changes of each protein. This method will further allow us to elucidate in vivo effects of different proteins on kidney stone formation.
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