Expression Of calbindin-D28k Research Articles

Effects of Dietary Vitamin D3 Over-Supplementation on Broiler Chickens' Health; Clinicopathological and Immunohistochemical Characteristics

Introduction: Vitamin D3 is used as a supplement in the feeds of livestock, pets, and human infants. However, the presence of excessive Vitamin D3 has been shown to cause toxicity in humans and animals. This study investigated the clinicopathological aspects of Vitamin D3 toxicity in broiler chickens. Materials and methods: The median lethal dose (LD50) of Vitamin D3 was estimated by the up-and-down method. To determine long term (21 days) toxic effects of oral Vitamin D3 supplementation, 90 (14-day-old) IBL-80 unsexed chicks were randomly divided into three groups as group A (control, received basal diet), B (basal diet + Vitamin D3 at 16.67 mg/kg body weight daily), and C (basal diet + Vitamin D3 at 33.33 mg/kg body weight daily). Results: The findings indicated that broiler chickens tolerated a single oral dose of Vitamin D3 up to 550 mg/kg body weight (22,000,000 IU/kg) without mortality. The results of long-term (21 days) oral supplementation of divided doses of Vitamin D3 in broiler chickens (groups B and C) showed progressive emaciation, elevated hemoglobin, hypercalcemia, hypophosphatemia, and increased alkaline phosphatase activity. At necropsy, pale liver and kidneys, congestion and hardening of lungs, mild congestion in the brain, and soft bones were observed in Vitamin D3 treated chicks (groups B and C). Microscopically, degeneration and metastatic calcification in lung parenchyma and peribronchiolar epithelium, coagulative necrosis and calcification in kidneys, and calcification with fibroplasia in proventriculus was detected. Lungs and kidneys showed a significant difference in calcification score between groups B and C. Broiler chickens from Vitamin D3 treated groups (B and C) showed strong immunohistochemical expression of Calbindin D28K in the intestine and kidneys but weak expression in the lungs. Conclusion: This study demonstrates that broiler chicks can tolerate very high levels of a single oral dose of Vitamin D3. Toxic effects of prolonged exposure to Vitamin D3 are due to over-expression of Calbindin D28k in the intestine and kidneys, disturbing the calcium and phosphorous homeostasis and leading to metastatic calcification of vital organs. This study supports that prolonged over-supplementation of Vitamin D3 causes toxic effects; hence, appropriate dietary Vitamin D3 supplementation limits should be set.

Effects of RNA M6A demethylase ALKBH5 gene deficiency on morphology and function of cerebellum in aged mice

Objective: To investigate the effects of RNA m6A demethylase ALKBH5 gene deficiency on cerebellar morphology and function in the aged mice, and to explore the role of ALKBH5 in cerebellar degeneration. Methods: Western blot was performed to detect the protein level of ALKBH5 in the cerebellum of wild-type mice of various ages. The expression of NeuN, Calbindin-D28K, MAP2, GFAP and other proteins in the cerebella of middle-aged (12-month-old) and aged (18-month-old) wild-type mice and ALKBH5-/- mice was examined using immunohistochemistry. The balance beam test and gait analysis were performed to test the balance ability and motor coordination of the mice. Results: With aging of the mice, the expression of ALKBH5 in the cerebellum increased gradually in an age-dependent manner. In the aged mice, but not middle-aged mice, the body weight, whole brain weight and cerebellum weight of ALKBH5-/- mice decreased by 15%, 10% and 21%, respectively (P<0.05). The expression of ALKBH5 in the Purkinje cells was much higher than that in other types of neural cells. Correspondingly, ALKBH5-deficiency caused 40% reduction in the number of Purkinje cells, as well as the length and density of neuronal dendrites in the aged mice (P<0.01). In addition, the time for the aged ALKBH5-/- mice to pass the balance beam was 70% longer than that of the wild type mice of the same age, with unstable gaits (P<0.01). Conclusions: Gene deficiency of RNA m6A demethylase ALKBH5 causes cerebellar atrophy, Purkinje neuron loss and damage in the aged mice. These changes eventually affect mice's motor coordination and balance ability. These results suggest that imbalanced RNA m6A methylation may lead to neurodegenerative lesions in the cerebellum of mice.

Rabies virus infection is associated with variations in calbindin D-28K and calretinin mRNA expression levels in mouse brain tissue.

Rabies virus (RABV) infection leads to a fatal neurological outcome in humans and animals and is associated with major alterations in cellular gene expression. In this study, we describe the effects of RABV infection on the mRNA expression levels of two genes, encoding the Ca2+-binding proteins (Ca-BPs) calbindin D-28K (Calb1) and calretinin (Calb2), in the brains of BALB/c mice. Sixty 4-week-old mice were divided into two test groups and one control group. Mice were inoculated intramuscularly with either a street rabies virus (SRV) strain or a challenge virus standard (CVS-11) strain and sacrificed at 3-day intervals up to day 18 postinfection. A direct fluorescent antibody test (DFAT) was used to verify the presence of RABV antigen in brain tissues, and real-time quantitative PCR (RT-PCR) was used to assess gene expression. Infection with both RABV strains resulted in significant (p < 0.05) increases in Calb1 and Calb2 expression in the test animals when compared with the controls at various time points in the study. Correlation analysis indicated very weak insignificant (p > 0.05) negative and positive relationships, respectively, between Calb1 expression (r = -0.04) and Calb2 expression (r = 0.08) with viral load (CVS-11 strain). Insignificant (p > 0.05) relationships were also observed Calb1 expression (r = -0.28) and Calb2 expression (r = 0.06) and viral load for the SRV strain.The observed alterations in Calb1 and Calb2 expression in this study indicate possible impairments in neuronal Ca2+ buffering and Ca2+ homeostasis as a result of RABV infection and, consequently, possible involvement of calbindin-D28K and calretinin in the neuropathogenesis of rabies.

Neuroprotective effects of quercetin on the cerebellum of zinc oxide nanoparticles (ZnoNps)-exposed rats

ABSTRACT Engineered nanomaterials induce hazardous effects at the cellular and molecular levels. We investigated different mechanisms underlying the neurotoxic potential of zinc oxide nanoparticles (ZnONPs) on cerebellar tissue and clarified the ameliorative role of Quercetin supplementation. Forty adult male albino rats were divided into control group (I), ZnONPs-exposed group (II), and ZnONPs and Quercetin group (III). Oxidative stress biomarkers (MDA & TOS), antioxidant biomarkers (SOD, GSH, GR, and TAC), serum interleukins (IL-1β, IL-6, IL-8), and tumor necrosis factor alpha (TNF-α) were measured. Serum micro-RNA (miRNA): miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-3p expression levels were quantified by real-time quantitative polymerase-chain reaction (RT-QPCR). Cerebellar tissue sections were stained with Hematoxylin & Eosin and Silver stains and examined microscopically. Expression levels of Calbindin D28k, GFAP, and BAX proteins in cerebellar tissue were detected by immunohistochemistry. Quercetin supplementation lowered oxidative stress biomarkers levels and ameliorated the antioxidant parameters that were decreased by ZnONPs. No significant differences in GR activity were detected between the study groups. ZnONPs significantly increased serum IL-1β, IL-6, IL-8, and TNF-α which were improved with Quercetin. Serum miRNA-21-5p, miRNA-122-5p, miRNA-125b-5p, and miRNA-155-p expression levels showed significant increase in ZnONPs group, while no significant difference was observed between Quercetin-treated group and control group. ZnONPs markedly impaired cerebellar tissue structure with decreased levels of calbindin D28k, increased BAX and GFAP expression. Quercetin supplementation ameliorated cerebellar tissue apoptosis, gliosis and improved calbindin levels. In conclusion: Quercetin supplementation ameliorated cerebellar neurotoxicity induced by ZnONPs at cellular and molecular basis by different studied mechanisms.Abbreviations: NPs: Nanoparticles, ROS: reactive oxygen species, ZnONPs: Zinc oxide nanoparticles, AgNPs: silver nanoparticles, BBB: blood–brain barrier, ncRNAs: Non-coding RNAs, miRNA: Micro RNA, DMSO: Dimethyl sulfoxide, LPO: lipid peroxidation, MDA: malondialdehyde, TBA: thiobarbituric acid, TOS: total oxidative status, ELISA: enzyme-linked immunosorbent assay, H2O2: hydrogen peroxide, SOD: superoxide dismutase, GR: glutathione reductase, TAC: total antioxidant capacity, IL-1: interleukin-1, TNF: tumor necrosis factor alpha, cDNA: complementary DNA, RT-QPCR: Real-time quantitative polymerase-chain reaction, ABC: Avidin biotin complex technique, DAB: 3′, 3-diaminobenzidine, SPSS: Statistical Package for Social Sciences, ANOVA: One way analysis of variance, Tukey’s HSD: Tukey’s Honestly Significant Difference, GFAP: glial fiberillar acitic protein, iNOS: Inducible nitric oxide synthase, NO: nitric oxide, HO-1: heme oxygenase-1, Nrf2: nuclear factor erythroid 2-related factor 2, NF-B: nuclear factor-B, SCI: spinal cord injury, CB: Calbindin

Expression of Calbindin-D28K in the Developing and Adult Mouse Cochlea.

Herein, we aimed to use double-labeling immunofluorescence to describe the expression pattern of Calbindin-D28K (CaBP28K) in the mouse cochlea from late embryonic (E) stages to the adulthood. CaBP28K was expressed in the inner hair cells (IHCs) and the greater epithelial ridge (GER) at E17. In addition, its expression was observed in the interdental cells. On postnatal day 1 (P1), CaBP28K immunoreactivity was observed in the IHCs and outer hair cells (OHCs) and was also specifically expressed in the nucleus and the cytoplasm of spiral ganglion neurons (SGNs). At P8, CaBP28K labeling disappeared from the interdental cells, and the CaBP28K-positive domain within the GER shifted from the entire cytoplasm to only the apical and basal regions. At P14, CaBP28K immunoreactivity was lost from the GER; however, its expression in the IHCs and OHCs, as well as the SGNs, persisted into adulthood. The identification of CaBP28K in the hair cells (HCs) and cuticular plates, as well as SGNs, was confirmed by its colocalization with several markers for Sox2, Myosin VIIa, Phalloidin, and Tuj1. We also detected colocalization with calmodulin in the cytoplasm of both HCs and SGNs. Western blot revealed an increase in CaBP28K postnatal expression in the mouse cochlea.

Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

ABSTRACTBranaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Hydrogen sulfide inhibits calcium and phosphorus loss after fracture by negatively regulating glucocorticoid/glucocorticoid receptor α

AimsPost-fracture calcium and phosphorus excretion is greater than influx, which might be caused by stress. Glucocorticoid is known to enhance calcium and phosphorous excretion, and hydrogen sulfide (H2S) has been shown to exert inhibitory effects on glucocorticoid. Therefore, this study explored whether H2S could inhibit calcium and phosphorus loss after fracture by regulating glucocorticoid and/or its receptor. Main methodsThe following properties were analyzed in rats with femur fractures: serum and urinary calcium and phosphorus (by colorimetry); bone turnover markers alkaline phosphatase, serum type 1 collagen amino terminal peptide, type 1 procollagen carboxy terminal peptide, and anti-tartaric acid phosphatase (by ELISA); factors related to calcium-phosphorus metabolism including glucocorticoid, parathyroid hormone, calcitonin, fibroblast growth factor 23, and 1,25(OH)2D3 (by ELISA); and sulfhydration of glucocorticoid receptor α in the kidney (by immunoprecipitation linked biotin-switch assay), after supplementing with mifepristone, the H2S donor GYY4137 or H2S generating enzyme inhibitors aminooxyacetic acid and propargylglycine. Key findingsSerum H2S decreased and glucocorticoid secretion increased in rats post-fracture. The glucocorticoid receptor inhibitor mifepristone partly blunted calcium and phosphorus loss. Furthermore, supplementation with GYY4137 reduced glucocorticoid secretion; inhibited glucocorticoid receptor α activity by sulfhydration; downregulated vitamin D 1α-hydroxylase expression; and upregulated 24-hydroxylase, calbindin-D28k, and sodium phosphate cotransporter 2a expression in the kidney; thereby inhibiting calcium and phosphorus loss induced by fracture. Moreover, inhibiting endogenous H2S generation showed opposite effects. SignificanceOur findings suggest that H2S antagonized calcium and phosphorus loss after fracture by reducing glucocorticoid secretion and inhibiting glucocorticoid receptor α activity by sulfhydration.

Methionine supplementing effects on intestine, liver and uterus morphology, and on positivity and expression of Calbindin-D28k and TRPV6 epithelial calcium carriers in laying quail in thermoneutral conditions and under thermal stress.

This study aimed to provide the performance, localization and expression of the epithelial calcium transporter channels Calbindin-D28k (Calb) and TRPV6, and of the morphology of the digestive and reproductive system of laying quail under heat stress (HS), and with methionine supplementation (MS). This study characterized the positivity (immunohistochemistry) and expression (real-time PCR) of calcium channels in the kidneys, intestine and uterus of 504 laying quails under different MS (100, 110 and 120%) and temperatures (20, 24, 28 and 32°C). The animals under HS (32°C) had lower villus height, villus:crypt ratio, and goblet cell index in the duodenum and jejunum, fewer secondary and tertiary uterine folds, smaller hepatic steatosis, and increased number of distal convoluted renal tubules (CT) positive to Calb, and increased positivity in proximal CTs. Deleterious effects of HS were minimized with MS for: duodenal crypts, number of goblet cells of the jejunum, number of uterine folds, decreased Calb positivity in intestines and kidney, increased positivity of Calb in the uterus and increased TRPV6 gene expression in the kidney (P≤0.05). Epithelial calcium transporters were altered due to less need for calcium absorption and reabsorption due to more calcium available with the MS, increasing egg production in HS and quality in termoneutrality (P≤0.05). MS further increased intestinal villus absorption area and height, increased steatosis, decreased Calb positivity in the intestine and kidney, increased uterine positivity of Calb, and increase Calb and TRPV6 expression in the kidney (P≤0.001) under thermoneutrality. It was concluded that the use of MS (120%) is justifiable in order to partially reverse the deleterious effects of HS on the production, in the epithelial calcium carriers, and in the digestory and reproductive morphology of laying quail.

Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage.

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood–brain barrier, which triggers gliosis after ischemic insults.

Circadian zinc feeding regime in laying hens related to laying performance, oxidation status, and interaction of zinc and calcium

This study investigated that circadian zinc (Zn) feeding regime affected laying performance, Zn and calcium (Ca) status, antioxidant capacity and gene expression of circadian clock, and Ca and Zn transporter in laying hens. In total, 162 of 21-wk Hyline Sophie laying hens were assigned randomly into 3 groups including CON group (Control Zn, basal diets supplemented 60 mg/kg Zn), HL group (high-low Zn, basal diets supplemented 120 mg/kg Zn—basal diets), and LH group (low-high Zn, basal diets—basal diets supplemented 120 mg/kg Zn), which were fed at 0,530 h and 1,530 h, respectively. Blood, tibia, duodenum, and eggshell gland samples were collected at 8 h intervals with starting at 0,000 h in 1 d after 10 wk of experiment. Compared with CON group: 1) Feed conversion ratio (FCR) of LH and HL group decreased significantly (P < 0.05); 2) in serum, total antioxidant capacity and CuZn-superoxide dismutase (SOD) at 0,000 h increased significantly, as well as Ca and Zn concentration of tibia at 0,800 h in LH group (P < 0.05); 3) in duodenum, mRNA expression of calbindin-d28k (CaBP) and NCX1 at 1,600 h in HL group upregulated significantly, as well as Per2 and Per3 at 0,000 h, CLOCK, Cry2, Per2, and Per3 at 1,600 h (P < 0.05). But, Zn5 at 0,800 h in HL group downregulated significantly (P < 0.05). 4) In eggshell gland, the mRNA expression of CaBP at 0,000 h and Zn5 at 1,600 h in HL group downregulated significantly (P < 0.05). However, SOD at 1,600 h in HL group upregulated significantly, as well as Cry1 and Per3 at 0,800 h in HL group upregulated significantly (P < 0.05). In conclusion, circadian Zn feeding diet regime was beneficial to improvement of FCR. The regulation of laying hens' circadian rhythms affected Zn and Ca transporter and interrelationship between Ca and Zn metabolism, also altered antioxidant capacity in present study. Therefore, circadian Zn feeding regime can be considered as a new method to improve laying performance in laying hens.

Effect of light on the calretinin and calbindin expression in skin club cells of adult zebrafish

In the last decade, zebrafish has been used as a model for the study of several human skin diseases. The epidermis of Danio rerio is composed of keratinocytes and two types of secretory cells: mucous cells and club cells. Club cells have multiple biological functions and among them may be important in the protection against ultraviolet damage through the proliferative response or through the increased production of protective substances. Calcium-binding proteins such as calbindin D28K and calretinin are used as markers of nervous and enteric nervous systems, but they are present in numerous other cells. These proteins are involved in a wide variety of cell activities, such as cytoskeletal organization, cell motility and differentiation, cell cycle regulation and neuroprotective function. In this study we demonstrated, for the first time, the presence of calretinin and calbindin D28K in skin club cells of Danio rerio exposed to different wavelengths by immunohistochemistry analysis. Exposure to white-blue light and blue light causes the expression and colocalization of calbindin-D28K and calretinin. These proteins were moderately expressed and no colocalization was observed in the club cells of the control fish. In zebrafish exposed to continuous darkness for 10days, in the club cells the two antibodies did not detect any proteins specifically. These results demonstrate that calbindin and calretinin could be involved in the pathophysiology of skin injury due to exposure to short-wavelength visible light spectrums.

Tacrolimus-induced hypomagnesemia and hypercalciuria requires FKBP12 suggesting a role for calcineurin.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new‐onset diabetes mellitus, and hypercalciuria, which may contribute to post‐transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off‐target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus‐treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX‐1 and Calbindin‐D28k). However, qPCR also showed decreased mRNA expression of NCX‐1 and Calbindin‐D28k, and TRPM6. In contrast, KS‐FKBP12−/− mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved.

Amyloid pathology–produced unexpected modifications of calcium homeostasis in hippocampal subicular dendrites

IntroductionAlzheimer's disease (AD) is linked to neuronal calcium dyshomeostasis, which is associated with network hyperexcitability. Decreased expression of the calcium-binding protein calbindin-D28K (CB) might be a susceptibility factor for AD. The subiculum is affected early in AD, for unknown reasons. MethodsIn AD, CB knock-out and control mice fluorescence Ca2+ imaging combined with patch clamp were used to characterize Ca2+ dynamics, resting Ca2+, and Ca2+-buffering capacity in subicular neurons. CB expression levels in wild-type and AD mice were also analyzed. ResultsThe subiculum and dentate gyrus of wild-type mice showed age-related decline in CB expression not observed in AD mice. Resting Ca2+ and Ca2+-buffering capacity was increased in aged AD mice subicular dendrites. Modeling suggests that AD calcium changes can be explained by alterations of Ca2+ extrusion pumps rather than by buffers. DiscussionOverall, abnormal Ca2+ homeostasis in AD has an age dependency that comprises multiple mechanisms, including compensatory processes.

Role of Calbindin-D28k in Diabetes-Associated Advanced Glycation End-Products-Induced Renal Proximal Tubule Cell Injury

Diabetes-associated advanced glycation end-products (AGEs) can increase extracellular matrix (ECM) expression and induce renal fibrosis. Calbindin-D28k, which plays a role in calcium reabsorption in renal distal convoluted tubules, is increased in a diabetic kidney. The role of calbindin-D28k in diabetic nephropathy still remains unclear. Here, calbindin-D28k protein expression was unexpectedly induced in the renal tubules of db/db diabetic mice. AGEs induced the calbindin-D28k expression in human renal proximal tubule cells (HK2), but not in mesangial cells. AGEs induced the expression of fibrotic molecules, ECM proteins, epithelial-mesenchymal transition (EMT) markers, and endoplasmic reticulum (ER) stress-related molecules in HK2 cells, which could be inhibited by a receptor for AGE (RAGE) neutralizing antibody. Calbindin-D28k knockdown by siRNA transfection reduced the cell viability and obviously enhanced the protein expressions of fibrotic factors, EMT markers, and ER stress-related molecules in AGEs-treated HK2 cells. Chemical chaperone 4-Phenylbutyric acid counteracted the AGEs-induced ER stress and ECM and EMT markers expressions. Calbindin-D28k siRNA in vivo delivery could enhance renal fibrosis in db/db diabetic mice. These findings suggest that inducible calbindin-D28k protects against AGEs/RAGE axis-induced ER stress-activated ECM induction and cell injury in renal proximal tubule cells.

Calbindin-D28k expression in spinal electromotoneurons of the weakly electric fish Apteronotus leptorhynchus during adult development and regeneration.

Additive neurogenesis, the net increase in neuronal numbers by addition of new nerve cells to existing tissue, forms the basis for indeterminate spinal cord growth in brown ghost knifefish (Apteronotus leptorhynchus). Among the cells generated through the activity of adult neural stem cells are electromotoneurons, whose axons constitute the electric organ of this weakly electric fish. Electromotoneuron development is organized along a caudo-rostral gradient, with the youngest and smallest of these cells located near the caudal end of the spinal cord. Electromotoneurons start expressing calbindin-D28k when their somata have reached diameters of approximately 10μm, and they continue expression after they have grown to a final size of about 50μm. Calbindin-D28k expression is significantly increased in young neurons generated in response to injury. Immunohistochemical staining against caspase-3 revealed that electromotoneurons in both intact and regenerating spinal cord are significantly less likely to undergo apoptosis than the average spinal cord cell. We hypothesize that expression of calbindin-D28k protects electromotoneurons from cell death; and that the evolutionary development of such a neuroprotective mechanism has been driven by the indispensability of electromotoneurons in the fish's electric behavior, and by the high size-dependent costs associated with their production or removal upon cell death.

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.

An Improved Method for Differentiating Mouse Embryonic Stem Cells into Cerebellar Purkinje Neurons.

While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure-function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCβ4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.

Admixing MPTP-resistant and MPTP-vulnerable mice enhances striatal field potentials and calbindin-D28K expression to avert motor behaviour deficits

Asian-Indians are less vulnerable to Parkinson’s disease (PD) than the Caucasians. Their admixed populace has even lesser risk. Studying this phenomenon using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6J, MPTP-resistant CD-1 and their resistant crossbred mice revealed differences in the nigrostriatal cyto-molecular features. Here, we investigated the electrophysiological and behavioural correlates for differential MPTP-susceptibility and their outcome upon admixing. We recorded local field potentials (LFPs) from dorsal striatum and assessed motor co-ordination using rotarod and grip strength measures. Nigral calbindin-D28K expression, a regulator of striatal activity through nigrostriatal projections was evaluated using immunohistochemistry.The crossbreds had significantly higher baseline striatal LFPs. MPTP significantly increased the neuronal activity in delta (0.5–4 Hz) and low beta (12–16 Hz) ranges in C57BL/6J; significant increase across frequency bands till high beta (0.5–30 Hz) in CD-1, and caused no alterations in crossbreds. MPTP further depleted the already low nigral calbindin-D28K expression in C57BL/6J. While in crossbreds, it was further up-regulated. MPTP affected the rotarod and grip strength performance of the C57BL/6J, while the injected CD-1 and crossbreds performed well. The increased striatal β-oscillations are comparable to that in PD patients. Higher power in CD-1 may be compensatory in nature, which were also reported in pre-symptomatic monkeys. Concurrent up-regulation of nigral calbindin-D28K may assist maintenance of striatal activity by buffering calcium overload in nigra. Thus, preserved motor behaviour in PD reminiscent conditions in CD-1 and crossbreds complement compensated/unaffected striatal LFPs. Similar electrophysiological correlates and cytomorphological features are envisaged in human phenomenon of differential PD prevalence, which are modulated upon admixing.

Endotoxaemia differentially regulates the expression of renal Ca2+ transport proteins in mice.

Alterations in parathyroid hormone (PTH) and/or vitamin D signalling are frequently reported in patients with sepsis. The consequences on renal and intestinal Ca2+ and Pi regulatory mechanisms are still unclear. We hypothesized that endotoxaemia alters the expression of important renal and intestinal Ca2+ and Pi transport proteins. Male C57BL/6 mice were treated with lipopolysaccharide (LPS; 3mg/kg; i.p.). The mRNA and protein levels of renal and intestinal Ca2+ and Pi transport proteins were measured by RT-qPCR, immunohistochemistry and western blot analysis. Lipopolysaccharide-induced hypocalcaemia and hyperphosphataemia was paralleled by a decrease in glomerular filtration rate and urinary excretion of Ca2+ and Pi . Endotoxaemia augmented plasma levels of PTH and affected the fibroblast growth factor 23 (FGF23)-klotho-vitamin D axis by increasing plasma levels of FGF23 and downregulation of renal klotho expression. Renal expression of CYP27b1 and plasma levels of 1,25-dihydroxyvitamin D3 were increased in response to LPS. Endotoxaemia augmented the renal expression of TRPV5, TRPV6 and PiT1, whereas the renal expression of calbindin-D28K , NCX1, NaPi -2a and NaPi -2c were decreased. Incubation of primary distal tubule cells with LPS increased TRPV6 mRNA levels. Furthermore, LPS decreased the intestinal expression of TRPV6, calbindin-D9K and of NaPi -2b. Our findings indicate that endotoxaemia is associated with hypocalcaemia and hyperphosphataemia and a disturbed FGF23-klotho-vitamin D signaling. Further, LPS-induced acute kidney injury was accompanied by an increased or decreased expression of specific renal and intestinal Ca2+ and Pi transporters respectively. It seems unlikely that LPS-induced hypocalcaemia is due to renal loss of Ca2+ .

Isx9 Regulates Calbindin D28K Expression in Pancreatic β Cells and Promotes β Cell Survival and Function.

Pancreatic β-cell dysfunction and death contribute to the onset of diabetes, and novel strategies of β-cell function and survival under diabetogenic conditions need to be explored. We previously demonstrated that Isx9, a small molecule based on the isoxazole scaffold, drives neuroendocrine phenotypes by increasing the expression of genes required for β-cell function and improves glycemia in a model of β cell regeneration. We further investigated the role of Isx9 in β-cell survival. We find that Isx9 drives the expression of Calbindin-D28K (D28K), a key regulator of calcium homeostasis, and plays a cytoprotective role through its calcium buffering capacity in β cells. Isx9 increased the activity of the calcineurin (CN)/cytoplasmic nuclear factor of the activated T-cells (NFAT) transcription factor, a key regulator of D28K, and improved the recruitment of NFATc1, cAMP response element-binding protein (CREB), and p300 to the D28K promoter. We found that nutrient stimulation increased D28K plasma membrane enrichment and modulated calcium channel activity in order to regulate glucose-induced insulin secretion. Isx9-mediated expression of D28K protected β cells against chronic stress induced by serum withdrawal or chronic inflammation by reducing caspase 3 activity. Consequently, Isx9 improved human islet function after transplantation in NOD-SCID mice in a streptozotocin-induced diabetes model. In summary, Isx9 significantly regulates expression of genes relevant to β cell survival and function, and may be an attractive therapy to treat diabetes and improve islet function post-transplantation.