Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta.

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells-especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.

MicroRNA-24-3p regulates neuronal differentiation by controlling hippocalcin expression.

Hippocalcin (HPCA) is a neuron-specific calcium-binding protein predominantly expressed in the nervous system. In the present study, we demonstrate that HPCA regulates neuronal differentiation in SH-SY5Y cells. We observed that the expression level of HPCA was increased during neuronal differentiation. Depletion of HPCA inhibited both neurite outgrowth and synaptophysin (SYP) expression, whereas overexpression of HPCA enhanced neuronal differentiation. Interestingly, we also found that the expression of HPCA mRNA was modulated by miR-24-3p. Using a dual-luciferase assay, we showed that co-transfection of a plasmid containing the miR-24-3p binding site from the 3'-untranslated region (3'UTR) of the HPCA gene and an miR-24-3p mimic effectively reduced luminescence activity. This effect was abolished when miR-24-3p seed sequences in the 3'UTR of the HPCA gene were mutated. miR-24-3p expression was decreased during differentiation, suggesting that the decreased expression level of miR-24-3p might have upregulated mRNA expression of HPCA. As expected, upregulation of miR-24-3p by an miRNA mimic led to reduced HPCA expression, accompanied by diminished neuronal differentiation. In contrast, downregulation of miR-24-3p by an antisense inhibitor promoted neurite outgrowth as well as levels of SYP expression. Taken together, these results suggest that miR-24-3p is an important miRNA that regulates neuronal differentiation by controlling HPCA expression.

HPCAL1 promotes glioblastoma proliferation via activation of Wnt/ß-catenin signalling pathway.

Glioblastoma (GBM) is the most prevalent primary malignancy of the central nervous system with obvious aggressiveness, and is associated with poor clinical outcome. Studies have indicated that calcium ion (Ca2+ ) can positively regulate the initiation of malignancy with regard to GBM by modulating quiescence, proliferation, migration and maintenance. Hippocalcin like-1 protein (HPCAL1) serves as a sensor of Ca2+ . However, the understanding of HPCAL1 activity in GBM is limited. The present study revealed that the gene HPCAL1 was up-regulated by Ca2+ in the tissues and cells of GBM. Ectopic expression of HPCAL1 promoted proliferation of cells. Exhaustion of HPCAL1 inhibited cell growth not only in vivo, but also in vitro. In addition, HPCAL1 enhanced the Wnt pathway by stimulating ß-catenin accumulation and nuclear translocation in GBM cells, while ß-catenin silencing significantly inhibited the proliferation and growth of the GBM cells. Our results showed that Ser9 phosphorylation of GSK3ß was significantly decreased after HPCAL1 knockdown in GBM cells, and knockdown of the gene GSK3ß in GBM cells enhanced cell proliferation and promoted transcription of the genes CCND1 and c-Myc. Furthermore, the phosphorylation of ERK was decreased in the cells with HPCAL1 knockdown, while it was promoted via overexpression of HPCAL1. The suppression or depletion of the gene ERK decreased proliferation triggered by overexpression of HPCAL1 and impaired transcription of the genes c-Myc and CCND1. These studies elucidate the tumour-promoting activity of HPCAL1. They also offer an innovative therapeutic strategy focusing on the HPCAL1-Wnt/ß-catenin axis to regulate proliferation and development of GBM.

Proteomic identification of hippocalcin and its protective role in heatstroke-induced hypothalamic injury in mice.

Heatstroke is a devastating condition that is characterized by severe hyperthermia and central nervous system dysfunction. However, the mechanism of thermoregulatory center dysfunction of the hypothalamus in heatstroke is unclear. In this study, we established a heatstroke mouse model and a heat-stressed neuronal cellular model on the pheochromocytoma-12 (PC12) cell line. These models revealed that HS promoted obvious neuronal injury in the hypothalamus, with high pathological scores. In addition, PC12 cell apoptosis was evident by decreased cell viability, increased caspase-3 activity, and high apoptosis rates. Furthermore, 14 differentially expressed proteins in the hypothalamus were analyzed by fluorescence two-dimensional difference gel electrophoresis and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Expression changes in hippocalcin (HPAC), a downregulated neuron-specific calcium-binding protein, were confirmed in the hypothalamus of the heatstroke mice and heat-stressed PC12 cells by immunochemistry and western blot. Moreover, HPAC overexpression and HPAC-targeted small interfering RNA experiments revealed that HPAC functioned as an antiapoptotic protein in heat-stressed PC12 cells and hypothalamic injury. Lastly, ulinastatin (UTI), a cell-protective drug that is clinically used to treat patients with heatstroke, was used in vitro and in vivo to confirm the role of HPAC; UTI inhibited heat stress (HS)-induced downregulation of HPAC expression, protected hypothalamic neurons and PC12 cells from HS-induced apoptosis and increased heat tolerance in the heatstroke animals. In summary, our study has uncovered and demonstrated the protective role of HPAC in heatstroke-induced hypothalamic injury in mice.

Perturbed Ca2+-dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

Single-column purification of the tag-free, recombinant form of the neuronal calcium sensor protein, hippocalcin expressed in Escherichia coli.

Hippocalcin is a 193 aa protein that is a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. Mice that lack the function of this protein are compromised in the long term potentiation aspect of memory generation. Recently, mutations in the gene have been linked with dystonia in human. The protein has no intrinsic enzyme activity but is known to bind to variety of target proteins. Very little information is available on how the protein executes its critical role in signaling pathways, except that it is regulated by binding of calcium. Further delineation of its function requires large amounts of pure protein. In this report, we present a single-step purification procedure that yields high quantities of the bacterially expressed, recombinant protein. The procedure may be adapted to purify the protein from inclusion bodies or cytosol in its myristoylated or non-myristoylated forms. MALDI-MS (in source decay) analyses demonstrates that the myristoylation occurs at the glycine residue. The protein is also biologically active as measured through tryptophan fluorescence, mobility shift and guanylate cyclase activity assays. Thus, further analyses of hippocalcin, both structural and functional, need no longer be limited by protein availability.

Childhood-Onset Choreo-Dystonia Due to a Recurrent Novel Homozygous Nonsense HPCA Variant: Case Series and Literature Review.

Background: Biallelic variants in HPCA were linked to isolated dystonia (formerly DYT2) in 2015. Since then, the clinical spectrum of HPCA-related disorder has expanded up to including a complex syndrome encompassing neurodevelopmental delay, generalized dystonia with bulbar involvement, and infantile seizures. Cases: We report four individuals with a new phenotype of childhood-onset choreo-dystonia belonging to two unrelated Iranian pedigrees and harboring a novel homozygous nonsense pathogenic variant NM_002143.3:c.49C>T p.(Arg17*) in HPCA. Although the families are both Iranian, haplotype analysis of the exome data did not reveal a founder effect of the variant. Literature Review: A systematic review of articles on HPCA and dystonia published since the disease gene discovery (PubMed; search on July 09, 2022; search strategy "HPCA AND dystonia", "HPCA AND movement disorder", "hippocalcin AND dystonia", and "hippocalcin AND movement disorder"; no language restriction) resulted in 18 references reporting 10 cases from six families. HPCA-related dystonia was isolated or in various combinations with neurodevelopmental delay, intellectual disability, seizures, cognitive decline, and psychiatric comorbidity. Onset of dystonia ranged from infancy to early adulthood. Dystonia started in the limbs or neck and became generalized in most cases. Brain MRI was unremarkable in nearly all cases where performed. There was poor or no response to common antidystonic medications in most cases. Conclusions: Our case series expands the pheno-genotypic spectrum of HPCA-related disorder by describing childhood-onset choreo-dystonia as a new phenotype, reporting on a recurrent novel pathogenic nonsense variant in HPCA, and suggesting that exon 2 of HPCA might be a mutational hotspot.

Hippocalcin-Like 1 blunts liver lipid metabolism to suppress tumorigenesis via directly targeting RUVBL1-mTOR signaling.

Rationale: Hepatocellular carcinoma (HCC) is one of the most severe cancers worldwide, with few effective targeted therapies for HCC. Lipid metabolic reprogramming is emerged as a hallmark of cancer metabolism that guides response to antitumoral therapies. Such lipid metabolic alteration in cancers is critically regulated by the mammalian target of rapamycin mTOR, which is considered as a promising therapeutic target. Despite efforts, mTOR inhibitors (mTORi) have produced limited response clinically, partly due to incomplete knowledge of mTORC1 addiction in cancers. Methods: CRISPR-Cas9 system was used to establish Hpcal1 null mice. The liver cancer model in mice was generated using Hpcal1-deficient mice with diethylnitrosamine (DEN) /CCL4 or MYC/Trp53-/- via hydrodynamic tail-vein injection. RNA-sequencing (RNA-seq) was used to identify potential signaling pathways. The expression of HPCAL1 and mTOR signaling were determined using quantitative polymerase chain reaction (qPCR), western blot and immunohistochemistry. The role of Hpcal1 in liver tumorigenesis and its response to mTORi was assessed by CCK-8 measurements, colony formation assay and in mouse model. Results: In this study, we identified hippocalcin-like protein 1 (HPCAL1) as an important negative regulator of de novo lipid biosynthesis and mTOR signaling activation, limiting liver tumorigenesis and establishing a metabolic vulnerability of HCC in mice. Genetic loss of HPCAL1 rendered HCC mTORC1-addicted and sensitive to mTORi AZD-8055 in vitro and in vivo. Importantly, HPCAL1 expression was inversely correlated with the levels of mTOR phosphorylation and several critical lipid biosynthesis enzymes in human specimens. Mechanistically, HPCAL1 directly bound to RuvB Like AAA ATPase 1 (RUVBL1), inhibiting the assembly of TEL2-TTI1-TTI2 (TTT)-RUVBL complex and subsequent leading the mTOR signaling suppression. Conclusion: We uncover a metabolic vulnerability and mTOR addiction in HCC with HPCAL1 loss that provides a selective therapeutic window for HCC with mTORC1 hyperactivation using mTORi.

Novel Biomarker Panels Discriminating between Pancreatic Ductal Adenocarcinoma and Extrahepatic Bile Duct Carcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) and extrahepatic bile duct carcinoma (EBDC) are distinct entities with different clinicopathological implications. Therefore, research to differentiate between the two diseases is compulsory. In this study, four biomarkers were selected (Hippocalcin-like 1 (HPCAL1); annexin A10 (ANXA10); MUC5AC; sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1)) and focus was placed on clarifying the diagnostic performance of each biomarker and pioneering novel-combined biomarker panels to discriminate between PDAC and EBDC. PROCEDURES: An immunohistochemical microarray analysis of HPCAL1, ANXA10, MUC5AC, and ATP1B1 was conducted for surgically resected 55 PDACs and 77 EBDCs. The diagnostic performance discriminating between PDAC and EBDC was evaluated using four biomarkers and the combined biomarker panels. RESULTS: PDACs exhibited more positive expressions for HPCAL1, ANXA10, and MUC5AC, whereas EBDCs exhibited more ATP1B1-positive expressions. The PDAC panel with the best diagnostic performance was the profile of (+ in ≥ 2 among HPCAL1, ANXA10, MUC5AC)/ATP1B1-. The immunophenotype pattern of (- in ≥ 1 among HPCAL1, ANXA10, MUC5AC)/ATP1B1+ is the EBDC panel with the most excellent discriminating power. CONCLUSION: The suggested combined biomarker panels demonstrate the distinguishing diagnostic ability between PDAC and EBDC is better than previous studies. Therefore, for differentiation between PDAC and EBDC, these panels are expected to help unravel the clinicopathological enigma as promising biomarker panels in the future.

Autoantibodies Against Ubiquitous and Confined Antigens in Patients With Ocular, Neuro-Ophthalmic and Congenital Cerebral Toxoplasmosis.

Toxoplasma gondii infection can trigger autoreactivity by different mechanisms. In the case of ocular toxoplasmosis, disruption of the blood-retinal barrier may cause exposure of confined retinal antigens such as recoverin. Besides, cross-reactivity can be induced by molecular mimicry of parasite antigens like HSP70, which shares 76% identity with the human ortholog. Autoreactivity can be a determining factor of clinical manifestations in the eye and in the central nervous system. We performed a prospective observational study to determine the presence of autoantibodies against recoverin and HSP70 by indirect ELISA in the serum of 65 patients with ocular, neuro-ophthalmic and congenital cerebral toxoplasmosis. We found systemic autoantibodies against recoverin and HSP70 in 33.8% and 15.6% of individuals, respectively. The presence of autoantibodies in cases of OT may be related to the severity of clinical manifestations, while in cases with CNS involvement they may have a protective role. Unexpectedly, anti-recoverin antibodies were found in patients with cerebral involvement, without ocular toxoplasmosis; therefore, we analyzed and proved cross-reactivity between recoverin and a brain antigen, hippocalcin, so the immunological phenomenon occurring in one immune-privileged organ (e.g. the central nervous system) could affect the environment of another (egg. the eye).

Quercetin Reduces Ischemic Brain Injury by Preventing Ischemia-induced Decreases in the Neuronal Calcium Sensor Protein Hippocalcin.

Calcium acts as a second messenger that mediates physiologic functions, such as metabolism, cell proliferation, and apoptosis. Hippocalcin is a neuronal calcium sensor protein that regulates intracellular calcium concentration. Moreover, it prevents neuronal cell death from oxidative stress. Quercetin has excellent antioxidant properties and preventative effects. We studied modulation of hippocalcin expression by quercetin treatment in cerebral ischemic injury and glutamate-induced neuronal cell damage. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO). Male Sprague-Dawley rats were injected with vehicle or quercetin (10 mg/kg) 1 h prior to pMCAO, and cerebral cortical tissues were isolated 24 h after pMCAO. Quercetin improved pMCAO-induced neuronal movement deficit and infarction. pMCAO induced a decrease in hippocalcin expression in the cerebral cortex. However, quercetin treatment attenuated this pMCAO-induced decrease. In cultured hippocampal cells, glutamate excitotoxicity dramatically increased the intracellular calcium concentration, whereas quercetin alleviated intracellular calcium overload. Moreover, Western blot and immunocytochemical studies showed reduction of hippocalcin expression in glutamate-exposed cells. Quercetin prevented this glutamate-induced decrease. Furthermore, caspase-3 expression in hippocalcin siRNA transfection conditions is higher than caspase-3 expression in un-transfection conditions. Quercetin treatment attenuated the increase of caspase-3. Taken together, these results suggest that quercetin exerts a preventative effect through attenuation of intracellular calcium overload and restoration of down-regulated hippocalcin expression during ischemic injury.

Moderate-Intensity Exercise Induces Neurogenesis and Improves Cognition in Old Mice by Upregulating Hippocampal Hippocalcin, Otub1, and Spectrin-α.

Exercise increases the levels of neurogenic factors and enhances neurogenesis, memory, and learning. However, the molecular link between exercise and neurogenesis is not clear. The purpose of this study was to examine the effects of exercise intensity on cognitive function and protein expression in the hippocampus of old mice. To compare the effects of aerobic exercise intensity on cognition in old mice, we exposed 18-month-old mice to low- and moderate-intensity treadmill exercise for 4 weeks. Moderate-intensity exercise improved cognitive function in the old mice, while low-intensity exercise did not. To investigate the underlying mechanisms, two-dimensional electrophoresis was used to examine protein expression. Using peptide fingerprinting mass spectrometry, we identified 19 proteins that were upregulated in the hippocampus following exercise training, and seven of these proteins were normalized by the control value. Among them, the levels of 14-3-3 zeta and heat shock protein 70, which have been shown to be induced by exercise training and related to neurogenesis, were dramatically increased by moderate exercise. Hippocalcin, α-spectrin, ovarian tumor domain-containing ubiquitin aldehyde-binding protein 1 (otub1), mu-crystallin, serine racemase, and rho GDP dissociation inhibitor 1, which are related to neurogenesis, neuroprotection, and synaptic strength, were upregulated in the hippocampus by moderate exercise. In addition, we confirmed that neurogenic markers, including doublecortin and the neuronal nuclei antigen, and hippocalcin, otub1, and spectrin-α are potential molecular links between hippocampal neurogenesis and exercise in the elderly. Thus, these results showed that steady moderate-intensity exercise delayed the declines in cognitive function in the elderly through the activation of multiple factors.

Inhibition of DREAM-ATF6 interaction delays onset of cognition deficit in a mouse model of Huntington's disease.

The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a multifunctional neuronal calcium sensor (NCS) that controls Ca2+ and protein homeostasis through gene regulation and protein-protein interactions. Downregulation of DREAM is part of an endogenous neuroprotective mechanism that improves ATF6 (activating transcription factor 6) processing, neuronal survival in the striatum, and motor coordination in R6/2 mice, a model of Huntington's disease (HD). Whether modulation of DREAM activity can also ameliorate cognition deficits in HD mice has not been studied. Moreover, it is not known whether DREAM downregulation in HD is unique, or also occurs for other NCS family members. Using the novel object recognition test, we show that chronic administration of the DREAM-binding molecule repaglinide, or induced DREAM haplodeficiency delays onset of cognitive impairment in R6/1 mice, another HD model. The mechanism involves a notable rise in the levels of transcriptionally active ATF6 protein in the hippocampus after repaglinide administration. In addition, we show that reduction in DREAM protein in the hippocampus of HD patients was not accompanied by downregulation of other NCS family members. Our results indicate that DREAM inhibition markedly improves ATF6 processing in the hippocampus and that it might contribute to a delay in memory decline in HD mice. The mechanism of neuroprotection through DREAM silencing in HD does not apply to other NCS family members.

Hippocalcin Promotes Neuronal Differentiation and Inhibits Astrocytic Differentiation in Neural Stem Cells.

Hippocalcin (HPCA) is a calcium-binding protein that is restricted to nervous tissue and contributes to neuronal activity. Here we report that, in addition to inducing neurogenesis, HPCA inhibits astrocytic differentiation of neural stem cells. It promotes neurogenesis by regulating protein kinase Cα (PKCα) activation by translocating to the membrane and binding to phosphoinositide-dependent protein kinase 1 (PDK1), which induces PKCα phosphorylation. We also found that phospholipase D1 (PLD1) is implicated in the HPCA-mediated neurogenesis pathway; this enzyme promotes dephosphorylation of signal transducer and activator of transcription 3 (STAT3[Y705]), which is necessary for astrocytic differentiation. Moreover, we found that the SH2-domain-containing tyrosine phosphatase 1 (SHP-1) acts upstream of STAT3. Importantly, this SHP-1-dependent STAT3-inhibitory mechanism is closely involved in neurogenesis and suppression of gliogenesis by HPCA. Taken together, these observations suggest that HPCA promotes neuronal differentiation through activation of the PKCα/PLD1 cascade followed by activation of SHP-1, which dephosphorylates STAT3(Y705), leading to inhibition of astrocytic differentiation.

Time-course changes of hippocalcin expression in the mouse hippocampus following pilocarpine-induced status epilepticus.

Hippocalcin participates in the maintenance of neuronal calcium homeostasis. In the present study, we examined the time-course changes of neuronal degeneration and hippocalcin protein level in the mouse hippocampus following pilocarpine-induced status epilepticus (SE). Marked neuronal degeneration was observed in the hippocampus after SE in a time-dependent manner, although neuronal degeneration differed according to the hippocampal subregions. Almost no hippocalcin immunoreactivity was detected in the pyramidal neurons of the cornu ammonis 1 (CA1) region from 6 h after SE. However, many pyramidal neurons in the CA2 region showed hippocalcin immunoreactivity until 24 h after SE. In the CA3 region, only a few hippocalcin immunoreactive cells were observed at 12 h after SE, and almost no hippocalcin immunoreactivity was observed in the pyramidal neurons from 24 h after SE. Hippocalcin immunoreactivity in the polymorphic cells of the dentate gyrus was markedly decreased from 6 h after SE. In addition, hippocalcin protein level in the hippocampus began to decrease from 6 h after SE, and was significantly decreased at 24 h and 48 h after pilocarpine-induced SE. These results indicate that marked reduction of hippocalcin level may be closely related to neuronal degeneration in the hippocampus following pilocarpine-induced SE.

Data on the identity and myristoylation state of recombinant, purified hippocalcin.

In this data article we report on the purity and post translation modification of bacterially expressed and purified recombinant hippocalcin (HPCA): a member of the neuronal calcium sensor protein family, whose functions are regulated by calcium. MALDI-TOF in source decay (ISD) analysis was used to identify both the myristoylated or non-myristoylated forms of the protein. MALDI-TOF ISD data on the identity of the protein, amino acid sequence and myristoylation efficiency are provided. This data relates to the article "Single-Column Purification of the Tag-free, Recombinant Form of the Neuronal Calcium Sensor Protein, Hippocalcin Expressed in Eschericia coli" [1].

Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells.

Hippocalcin (Hpca) is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs). When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), and brain-derived neurotrophic factor (BDNF), together with the proneural basic helix loop helix (bHLH) transcription factors NeuroD and neurogenin 1 (Ngn1), increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP), an astrocyte marker, and in branch outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, NeuroD, and Ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727), and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201), suggesting that STAT3 (Ser727) activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and branch outgrowth in HNPCs.

Hydrogen-rich water protects against ischemic brain injury in rats by regulating calcium buffering proteins.

Hydrogen-rich water (HRW) has anti-oxidant activities, and it exerts neuroprotective effects during ischemia-reperfusion brain injury. Parvalbumin and hippocalcin are two calcium buffering proteins, which are involved in neuronal differentiation, maturation and apoptosis. The aim of this study was to investigate whether HRW could moderate parvalbumin and hippocalcin expression during ischemic brain injury and glutamate toxicity-induced neuronal cell death. Focal brain ischemia was induced in male Sprague-Dawley rats by middle cerebral artery occlusion (MCAO). Rats were treated with H2O or HRW (6 ml/kg per rat) before and after MCAO, and cerebral cortical tissues were collected 1, 7 and 14 days after MCAO. Based on our results, HRW treatment was able to reduce brain infarct volume and improve neurological function following ischemic brain injury. In addition, HRW prevented the ischemia-induced reduction of parvalbumin and hippocalcin levels in vivo and also reduced the glutamate toxicity-induced death of neurons, including the dose-dependent reduction of glutamate toxicity-associated proteins in vitro. Moreover, HRW attenuated the glutamate toxicity-induced elevate in intracellular Ca(2+) levels. All these results suggest that HRW could protect against ischemic brain injury and that the maintenance of parvalbumin and hippocalcin levels by HRW during ischemic brain injury might contribute to the neuroprotective effects against neuron damage.

The EF-hand Ca(2+)-binding protein super-family: a genome-wide analysis of gene expression patterns in the adult mouse brain.

In mice, 249 putative members of the superfamily of EF-hand domain Ca(2+)-binding proteins, manifesting great diversity in structure, cellular localization and functions have been identified. Three members in particular, namely, calbindin-D28K, calretinin and parvalbumin, are widely used as markers for specific neuronal subpopulations in different regions of the brain. The aim of the present study was to compile a comprehensive atlas of the gene-expression profiles of the entire EF-hand gene superfamily in the murine brain. This was achieved by a meticulous examination of the in-situ hybridization images in the Allen Brain Atlas database. Topographically, our analysis focused on the olfactory bulb, cerebral cortex (barrel cortex in the primary somatosensory area), basal ganglia, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, midbrain, pons and medulla, and on clearly identifiable sub-structures within each of these areas. The expression profiles of four family-members, namely hippocalcin-like 4, neurocalcin-δ, plastin 3 and tescalcin, that have not been hitherto reported, at either the mRNA (in-situ-hybridization) or the protein (immunohistochemical) levels, are now presented for the first time. The fruit of our analysis is a document in which the gene-expression profiles of all members of the EF-hand family genes are compared, and in which future possible neuronal markers for specific cells/brain areas are identified. The assembled information could afford functional clues to investigators, conducive to further experimental pursuit.

Estradiol alleviates the ischemic brain injury-induced decrease of neuronal calcium sensor protein hippocalcin.

Estradiol has protective and reparative effects in neurodegenerative diseases. Hippocalcin is a neuronal calcium-sensor protein that acts as a calcium buffer to regulate the intracellular concentration of Ca(2+). This study was investigated to elucidate whether estradiol regulates hippocalcin expression in a focal cerebral ischemia model and glutamate-treated neuronal cells. An ovariectomy was performed in adult female rats, and vehicle or estradiol was administered before middle cerebral artery occlusion (MCAO). Cerebral cortex tissues were collected at 24h after MCAO. A proteomic approach revealed that hippocalcin expression decreased in vehicle-treated animals with combined MCAO, while estradiol treatment attenuated this decrease. Reverse transcription-PCR and Western blot analyses also showed that estradiol administration prevented the MCAO injury-induced decrease in hippocalcin expression. In cultured hippocampal cells, glutamate exposure increased the intracellular Ca(2+) concentration, which was rescued by the presence of estradiol. Moreover, glutamate toxicity decreased hippocalcin expression, whereas estradiol attenuated this decrease. Together, these findings suggest that estradiol has a neuroprotective function by regulating hippocalcin expression and intracellular Ca(2+) levels in ischemic brain injury.