Identification of hub genes associated with cognition in the hippocampus of Alzheimer's Disease.

Alzheimer's Disease (AD) is a neurodegenerative disease featured by cognitive impairment. This bioinformatic analysis was used to identify hub genes related to cognitive dysfunction in AD. The gene expression profile GSE48350 in the hippocampus of AD patients aged >70 years was obtained from the Gene Expression Omnibus (GEO) database. A total of 96 differentially expressed genes (DEGs) were identified, and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses; a protein-protein interaction (PPI) network was constructed. The DEGs were enriched in synapse-related changes. A protein cluster was teased out of PPI. Furthermore, the cognition ranked the first among all the terms of biological process (BP). Next, 4 of 10 hub genes enriched in cognition were identified. The function of these genes was validated using APP/PS1 mice. Cognitive performance was validated by Morris Water Maze (MWM), and gene expression by RT-qPCR, Cholecystokinin (CCK), Tachykinin precursor 1 (TAC1), Calbindin 1 (CALB1) were downregulated in the hippocampus. These genes can provide new directions in the research of the molecular mechanism of AD.

Calbindin expression in adult vestibular epithelia.

The mammalian vestibular epithelia exhibit a remarkably stereotyped organization featuring cellular characteristics under planar cell polarity (PCP) control. PCP mechanisms are responsible for the organization of hair cell morphologic polarization vectors, and are thought to be responsible for the postsynaptic expression of the calcium-binding protein calretinin that defines the utricular striola and cristae central zone. However, recent analyses revealed that subtle differences in the topographic expression of oncomodulin, another calcium-binding protein, reflects heterogeneous factors driving the subtle variations in expression. Calbindin represents a third calcium-binding protein that has been previously described to be expressed in both hair cells and afferent calyces in proximity to the utricular striola and crista central zone. The objective of the present investigation was to determine calbindin's topographic pattern of expression to further elucidate the extent to which PCP mechanisms might exert control over the organization of vestibular neuroepithelia. The findings revealed that calbindin exhibited an expression pattern strikingly similar to oncomodulin. However, within calyces of the central zone calbindin was colocalized with calretinin. These results indicate that organizational features of vestibular epithelia are governed by a suite of factors that include PCP mechanisms as well others yet to be defined.

Electrophysiological properties of rat subfornical organ neurons expressing calbindin D28K.

The subfornical organ (SFO) is forebrain sensory circumventricular organ, characterized by lack of a blood-brain barrier. Neurons of the SFO can detect circulating molecules such as peptide hormones and communicate this information to regulatory centers behind the blood-brain barrier, thus playing a critical role in homeostatic processes including regulation of energy balance, hydromineral balance and cardiovascular control. The SFO contains two subregions defined by neuronal expression of molecular markers: the dorsolateral peripheral or shell SFO (sSFO) neurons express calretinin, and the ventromedial core (cSFO) neurons express calbindin D28K. Neurons from these two subregions project to different locations to subserve different roles in homeostatic regulation. It is unknown whether neurons from these two subregions exhibit unique or identifiable electrophysiological properties. This study used a gold nanoparticle-conjugated RNA fluorescent probe on dissociated SFO neuron cultures and patch clamp electrophysiology to characterize the intrinsic electrophysiological properties of cSFO and sSFO neurons. Our studies revealed that neurons originating from the core region exhibited significantly more action potential bursting, while neurons from non-core regions exhibited more tonic firing neurons, albeit at a higher overall frequency. The difference in activity is correlated with a more depolarized resting membrane potential and a higher density of voltage gated Na+ currents.

Downregulation of Calbindin 1 by miR-454-3p Suppresses Cell Proliferation in Nonsmall Cell Lung Cancer In Vitro.

BACKGROUND: Calbindin 1 (CALB1), a constituent Ca2+-binding protein, has been reported to prevent apoptotic death in tumor cells. However, the microRNA-mediated regulatory mechanism of CALB1 expression in nonsmall cell lung cancer (NSCLC) has not been reported so far. METHODS AND RESULTS: In this study, CALB1 was found to be overexpressed in NSCLC tissues through the immunohistochemistry assay. Higher CALB1 expression levels were significantly associated with the tumor-node-metastasis (TNM) stage. Moreover, higher expression of CALB1 predicts poor survival in NSCLC patients using the Kaplan-Meier plotter online analysis. In addition, miR-454-3p was predicted to target CALB1 using a software algorithm, validated by the luciferase assay, and analyzed by quantitative polymerase chain reaction and Western blot. The authors further found that miR-454-3p was downregulated in NSCLC tissues and cell lines. Downregulation of CALB1 or upregulation of miR-454-3p significantly suppressed NSCLC cell proliferation and induced cell apoptosis as shown by CCK-8 and flow cytometry analysis, respectively. CONCLUSIONS: Our results suggest that CALB1 is a direct target of miR-454-3p and downregulation of CALB1 by miR-454-3p suppressed NSCLC cell functions, which may shed light on its potential application in NSCLC therapy.

Calcipotriol inhibits α-synuclein aggregation in SH-SY5Y neuroblastoma cells by a Calbindin-D28k-dependent mechanism.

Many neurodegenerative diseases are characterized by the formation of microscopically visible intracellular protein aggregates. α-Synuclein is the key aggregating protein in Parkinson's disease which is characterized by neuronal cytoplasmic Lewy body inclusions. Previous studies have shown relative sparing of neurons in Parkinson's disease and dementia with Lewy bodies that are positive for the vitamin D-dependent calcium-buffering protein, calbindin-D28k, and that α-synuclein aggregates are excluded from calbindin-D28k-positive neurons. Recent cell culture studies have shown that α-synuclein aggregation can be induced by raised intracellular-free Ca(II) and demonstrated that raised intracellular calcium and oxidative stress can act synergistically to promote α-synuclein aggregation. We hypothesized that calcipotriol, a potent vitamin D analogue used pharmaceutically, may be able to suppress calcium-dependent α-synuclein aggregation by inducing calbindin-D28k expression. Immunofluorescence and western blot analysis showed that calcipotriol potently induced calbindin-D28k in a dose-dependent manner in SH-SY5Y human neuroblastoma cells. Calcipotriol significantly decreased the frequency of α-synuclein aggregate positive cells subjected to treatments that cause raised intracellular-free Ca(II) (potassium depolarization, KCl/H2 O2 combined treatment, and rotenone) in a dose-dependent manner and increased viability. Suppression of calbindin-D28k expression in calcipotriol-treated cells using calbindin-D28k-specific siRNA showed significantly higher α-synuclein aggregation levels, indicating that calcipotriol-mediated blocking of calcium-dependent α-synuclein aggregation was dependent on the induction of calbindin-D28k expression. These data indicate that targeting raised intraneuronal-free Ca(II) in the brain by promoting the expression of calbindin-D28k at the transcriptional level using calcipotriol could prevent α-synuclein aggregate formation and ameliorate Parkinson's disease pathogenesis.

The expression patterns of Tetratricopeptide repeat domain 36 (Ttc36).

Tetratricopeptide repeat domain 36 (Ttc36), whose coding protein belongs to tetratricopeptide repeat (TPR) motif family, has not been studied extensively. We for the first time showed that Ttc36 is evolutionarily conserved across mammals by bioinformatics. Rabbit anti-mouse Ttc36 polyclonal antibody was generated by injecting synthetic full-length peptides through "antigen intersection" strategy. Subsequently, we characterized Ttc36 expression profile in mouse, showing its expression in liver and kidney both from embryonic day 15.5 (E15.5) until adult, as well as in testis. Immunofluorescence staining showed that Ttc36 is diffusely expressed in liver, however, specifically in kidney cortex. Thus, we further compare Ttc36 with proximal tubules (PT) marker Lotus Tetragonolobus Lectin (LTL) and distal tubules (DT) marker Calbindin-D28k respectively by double immunofluorescence staining. Results showed the co-localization of Ttc36 with LTL rather than Calbindin-D28k. Collectively, on the basis of the expression pattern, Ttc36 is specifically expressed in proximal distal tubules.

Immunocytochemical localization of the calcium-binding proteins calbindin D28K, calretinin, and parvalbumin in bat visual cortex.

It is a common misconception that bats are blind, and various studies have suggested that bats have visual abilities. The purpose of this study was to investigate the cytoarchitecture of calbindin D28K (CB)-, calretinin (CR)-, and parvalbumin (PV)-immunoreactive (IR) neurons in the bat visual cortex using immunocytochemistry. The highest density of CB- and PV-IR neurons was located in layer IV of the visual cortex. The majority of CB- and PV-IR neurons were characterized by a stellate or round/oval shape. CR-IR neurons were predominantly located in layers II/III, and the cells were principally round/oval in shape. Two-color immunofluorescence revealed that 65.96%, 24.24%, and 77.00% of the CB-, CR-, and PV-IR neurons, respectively, contained gamma-aminobutyric acid (GABA). We observed calcium-binding protein (CBP)-IR neurons in specific layers of the bat visual cortex and in specific cell types. Many of the CBP-IR neurons were GABAergic interneurons. These data provide useful clues to aid in understanding the functional aspects of the bat visual system.

Calbindin-D28K is increased in the ventral horn of spinal cord by neuroprotective factors for motor neurons.

Slow glutamate-mediated neuronal degeneration is implicated in the pathophysiology of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The calcium-binding proteins calbindin-D28K and parvalbumin have been reported to protect neurons against excitotoxic insults. Expression of calbindin-D28K is low in adult human motor neurons, and vulnerable motor neurons additionally may lack parvalbumin. Thus, it has been speculated that the lack of calcium-binding proteins may, in part, be responsible for early degeneration of the population of motor neurons most vulnerable in ALS. Using a rat organotypic spinal cord slice system, we examined whether the most potent neuroprotective factors for motor neurons can increase the expression of calbindin-D28K or parvalbumin proteins in the postnatal spinal cord. After 4 weeks of incubation of spinal cord slices with 1) glial cell line-derived neurotrophic factor (GDNF), 2) neurturin, 3) insulin-like growth factor I (IGF-I), or 4) pigment epithelium-derived factor (PEDF), the number of calbindin-D28K -immunopositive large neurons (>20 µm) in the ventral horn was higher under the first three conditions, but not after PEDF, compared with untreated controls. Under the same conditions, parvalbumin was not upregulated by any neuroprotective factor. The same calbindin increase was true of IGF-I and GDNF in a parallel glutamate toxicity model of motor neuron degeneration. Taken together with our previous reports from the same model, which showed that all these neurotrophic factors can potently protect motor neurons from slow glutamate injury, the data here suggest that upregulation of calbindin-D28K by some of these factors may be one mechanism by which motor neurons can be protected from glutamate-induced, calcium-mediated excitotoxicity.

SUN11602-induced hyperexpression of calbindin D-28k is pivotal for the survival of hippocampal neurons under neurotoxic conditions.

Basic fibroblast growth factor (FGF-2/bFGF) possesses neuroprotective activity and promotes cell proliferation. In this study, the novel synthetic compound 4-({4-[[(4-amino-2,3,5,6-tetramethylanilino)acetyl](methyl)amino]-1-piperidinyl}methyl)benzamide (SUN11602) exhibited neuroprotective activities similar to those of FGF-2 without promoting cell proliferation. In primary cultures of hippocampal neurons, stimulation with SUN11602 or FGF-2 increased calbindin D-28k (CalB) gene expression and prevented glutamate-induced neuronal death. These effects were abolished by pretreatment with PD166866 (FGF receptor 1 [FGFR1] tyrosine kinase-specific inhibitor). This indicated that FGFR1 activation and increased CalB expression were involved in SUN11602-mediated neuroprotection. However, receptor-binding assays revealed that unlike FGF-2, SUN11602 did not alter the binding of (125)I-labeled FGF-2 to FGFR1. To investigate the possible proliferative activity of SUN11602, we utilized BHK21 and SKN cells expressing endogenous FGFR1. FGF-2 promoted cell proliferation whereas SUN11602 did not. In in vivo studies, wild-type (WT) and CalB-deficient (CalB(-/-)) mice were injected with aggregated Aß1-40 and ibotenate (NMDA receptor agonist) to severely damage the hippocampal tissue. Treatment with SUN11602 (orally) or FGF-2 (intraparenchymally) at the midpoint of Aß1-40 and ibotenate injections prevented the hippocampal damage in WT mice, however this effect was abolished in CalB(-/-) mice. Thus, SUN11602 exerted protective effects on hippocampal neurons through activation of FGFR1 and increased CalB expression. Moreover, the neuroprotective effects of SUN11602 depended upon the various biological activities of FGF-2.

Expression and colocalization patterns of calbindin-D28k, calretinin and parvalbumin in the rat hypothalamic arcuate nucleus.

Calcium binding proteins (CaBPs) form a diverse group of molecules that function as signal transducers or as intracellular buffers of Ca(2+) concentration. They have been extensively used to histochemically categorize cell types throughout the brain. One region which has not yet been characterized with regard to CaBP expression is the hypothalamic arcuate nucleus, which plays a vital role in neuroendocrine control and the central regulation of energy metabolism. Using in situ hybridization and immunofluorescence, we have investigated the cellular distribution of the three CaBPs, calbindin-D28k (CB), calretinin (CR) and parvalbumin (PV) in the rat arcuate nucleus. Both mRNA and immunoreactivity was detected in the arcuate nucleus for CB - located in the medial aspects - and CR - located ventrolaterally. No PV mRNA was detected in the arcuate nucleus. Immunofluorescence results for PV were ambiguous; while one antibody detected a group of cell somata, a different antibody failed to visualize any arcuate nucleus cell profiles. Using double-labeling, neither of the examined CaBPs were observed in cells immunoreactive for the signaling molecules agouti gene-related protein, tyrosine hydroxylase, neurotensin, growth hormone-releasing hormone, somatostatin, enkephalin, dynorphin or galanin. We did, however, observe CB- and CR-immunoreactivity, in two distinct populations of neurons immunoreactive for the melanocortin peptide α-melanocyte-stimulating hormone. These data identify distinct subpopulations of arcuate neurons defined by their expression of CaBPs and provide further support for differentiation between subpopulations of anorexigenic melanocortin neurons.

Estrogen deficiency-induced Ca balance impairment is associated with decrease in expression of epithelial Ca transport proteins in aged female rats.

AIMS: The study is designed to determine whether estrogen and vitamin D endocrine systems interact to regulate calcium (Ca) balance as well as changes in mRNA expression of epithelial Ca transport proteins involved in intestinal and renal Ca transport in aging animals in response to ovariectomy and low dietary Ca intake. MAIN METHODS: Eleven-month-old female sham or ovariectomized (OVX) rats were divided into four groups and fed with either a low-Ca (LCD; 0.1% Ca, 0.65% P) or a high-Ca (HCD; 1.2% Ca, 0.65% P) diet for 12weeks. Ca balance and mRNA expression of Ca transport proteins in the intestine and kidney from rats were systematically studied. KEY FINDINGS: OVX rats fed with LCD resulted in a negative Ca balance. LCD suppressed serum Ca in OVX but not sham rats, resulting in an induction of serum PTH and 1,25(OH)2D3 levels. The surge in serum 1,25(OH)2D3 levels in LCD-fed OVX rats was associated with an increase in mRNA expression of intestinal transient receptor potential cation channel (TRPV6) and calbindin D9k (CaBP9k) as well as renal vitamin D receptor (VDR), but such an induction was unable to restore Ca balance in vivo. In contrast, the negative Ca balance was associated with suppression of intestinal plasma membrane Ca pump (PMCA1b) and renal transient receptor potential cation channel (TRPV5), calbindin D28k (CaBP28k) and PMCA1b mRNA expression in aged OVX rats. SIGNIFICANCE: Negative Ca balance in aged female OVX rats is associated with estrogen-dependent and vitamin D-independent downregulation of epithelial Ca transport protein mRNA expression.

Calbindin-D28k and calretinin in chicken inner retina during postnatal development and neuroplasticity by dim red light.

Members of the family of calcium binding proteins (CBPs) are involved in the buffering of calcium (Ca2+) by regulating how Ca2+ can operate within synapses or more globally in the entire cytoplasm and they are present in a particular arrangement in all types of retinal neurons. Calbindin D28k and calretinin belong to the family of CBPs and they are mainly co-expressed with other CBPs. Calbindin D28k is expressed in doubles cones, bipolar cells and in a subpopulation of amacrine and ganglion neurons. Calretinin is present in horizontal cells as well as in a subpopulation of amacrine and ganglion neurons. Both proteins fill the soma at the inner nuclear layer and the neuronal projections at the inner plexiform layer. Moreover, calbindin D28k and calretinin have been associated with neuronal plasticity in the central nervous system. During pre and early postnatal visual development, the visual system shows high responsiveness to environmental influences. In this work we observed modifications in the pattern of stratification of calbindin immunoreactive neurons, as well as in the total amount of calbindin through the early postnatal development. In order to test whether or not calbindin is involved in retinal plasticity we analyzed phosphorylated p38 MAPK expression, which showed a decrease in p-p38 MAPK, concomitant to the observed decrease of calbindin D28k. Results showed in this study suggest that calbindin is a molecule related with neuroplasticity, and we suggest that calbindin D28k has significant roles in neuroplastic changes in the retina, when retinas are stimulated with different light conditions.