Epigallocatechin gallate improves neuronal damage in animal model of ischemic stroke and glutamate-exposed neurons via modulation of hippocalcin expression.

Epigallocatechin gallate (EGCG) is a polyphenolic component of green tea that has anti-oxidative and anti-inflammatory effects in neurons. Ischemic stroke is a major neurological disease that causes irreversible brain disorders. It increases the intracellular calcium concentration and induces apoptosis. The regulation of intracellular calcium concentration is important to maintain the function of the nervous system. Hippocalcin is a neuronal calcium sensor protein that controls intracellular calcium concentration. We investigated whether EGCG treatment regulates the expression of hippocalcin in stroke animal model and glutamate-induced neuronal damage. We performed middle cerebral artery occlusion (MCAO) to induce cerebral ischemia. EGCG (50 mg/kg) or phosphate buffered saline was injected into the abdominal cavity just before MCAO surgery. The neurobehavioral tests were performed 24 h after MCAO surgery and cerebral cortex tissue was collected. MCAO damage induced severe neurobehavioral disorders, increased infarct volume, and decreased the expression of hippocalcin in the cerebral cortex. However, EGCG treatment improved these deficits and alleviated the decrease in hippocalcin expression in cerebral cortex. In addition, EGCG dose-dependently alleviated neuronal cell death and intracellular calcium overload in glutamate-exposed neurons. Glutamate exposure reduced hippocalcin expression, decreased Bcl-2 expression, and increased Bax expression. However, EGCG treatment mitigated these changes caused by glutamate toxicity. EGCG also attenuated the increase in caspase-3 and cleaved caspase-3 expressions caused by glutamate exposure. The effect of EGCG was more pronounced in non-transfected cells than in hippocalcin siRNA-transfected cells. These findings demonstrate that EGCG protects neurons against glutamate toxicity through the regulation of Bcl-2 family proteins and caspase-3. It is known that hippocalcin exerts anti-apoptotic effect through the modulation of apoptotic pathway. Thus, we can suggest evidence that EGCG has a neuroprotective effect by regulating hippocalcin expression in ischemic brain damage and glutamate-exposed cells.

Identification of HPCAL1 as a specific autophagy receptor involved in ferroptosis.

Selective macroautophagy/autophagy maintains cellular homeostasis through the lysosomal degradation of specific cellular proteins or organelles. The pro-survival effect of selective autophagy has been well-characterized, but the mechanism by which it drives cell death is still poorly understood. Here, we use a quantitative proteomic approach to identify HPCAL1 (hippocalcin like 1) as a novel autophagy receptor for the selective degradation of CDH2 (cadherin 2) during ferroptosis. HPCAL1-dependent CDH2 depletion increases susceptibility to ferroptotic death by reducing membrane tension and favoring lipid peroxidation. Site-directed mutagenesis aided by bioinformatic analyses revealed that the autophagic degradation of CDH2 requires PRKCQ (protein kinase C theta)-mediated HPCAL1 phosphorylation on Thr149, as well as a non-classical LC3-interacting region motif located between amino acids 46-51. An unbiased drug screening campaign involving 4208 small molecule compounds led to the identification of a ferroptosis inhibitor that suppressed HPCAL1 expression. The genetic or pharmacological inhibition of HPCAL1 prevented ferroptosis-induced tumor suppression and pancreatitis in suitable mouse models. These findings provide a framework for understanding how selective autophagy promotes ferroptotic cell death.Abbreviations: ANXA7: annexin A7; ARNTL: aryl hydrocarbon receptor nuclear translocator like; CCK8: cell counting kit-8; CDH2: cadherin 2; CETSAs: cellular thermal shift assays; CPT2: carnitine palmitoyltransferase 2; DAMP, danger/damage-associated molecular pattern; DPPH: 2,2-diphenyl-1-picrylhydrazyl; DFO: deferoxamine; EBNA1BP2: EBNA1 binding protein 2; EIF4G1: eukaryotic translation initiation factor 4 gamma 1; FBL: fibrillarin; FKBP1A: FKBP prolyl isomerase 1A; FTH1: ferritin heavy chain 1; GPX4: glutathione peroxidase 4; GSDMs: gasdermins; HBSS: Hanks' buffered salt solution; HMGB1: high mobility group box 1; HNRNPUL1: heterogeneous nuclear ribonucleoprotein U like 1; HPCAL1: hippocalcin like 1; H1-3/HIST1H1D: H1.3 linker histone, cluster member; IKE: imidazole ketone erastin; KD: knockdown; LDH: lactate dehydrogenase; LIR: LC3-interacting region; MAGOH: mago homolog, exon junction complex subunit; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehyde; MLKL: mixed lineage kinase domain like pseudokinase; MPO: myeloperoxidase; MTOR: mechanistic target of rapamycin kinase; OE: overexpressing; OSTM1: osteoclastogenesis associated transmembrane protein 1; PRKC/PKC: protein kinase C; PRKAR1A: protein kinase cAMP-dependent type I regulatory subunit alpha; PRDX3: peroxiredoxin 3; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; SLC40A1: solute carrier family 40 member 1; SPTAN1: spectrin alpha, non-erythrocytic 1; STS: staurosporine; UBE2M: ubiquitin conjugating enzyme E2 M; ZYX: zyxin.

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.

Hippocalcin-like 1 is a key regulator of LDHA activation that promotes the growth of non-small cell lung carcinoma.

BACKGROUND: Hippocalcin-like 1 (HPCAL1), a neuronal calcium sensor protein family member, has been reported to regulate cancer growth. As yet, however, the biological functions of HPCAL1 and its molecular mechanisms have not been investigated in non-small cell lung carcinoma (NSCLC). METHODS: HPCAL1 expression in NSCLC samples was detected using immunohistochemistry, Western blotting and RT-PCR. The anticancer effects of HPCAL1 knockdown were determined by MTT, soft agar, cell cycle, oxygen consumption and reactive oxygen species assays. The effect of HPCAL1 knockdown on in vivo tumor growth was assessed using NSCLC cancer patient-derived xenograft models. Potentially interacting protein partners of HPCAL1 were identified using IP-MS/MS, immunoprecipitation and Western blotting assays. Metabolic alterations resulting from HPCAL1 knockdown were investigated using non-targeted metabolomics and RNA sequencing analyses. RESULTS: We found that HPCAL1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and AJCC clinical staging in lung cancer patients. Knockdown of HPCAL1 strongly increased oxygen consumption rates and the production of reactive oxygen species. HPCAL1 knockdown also inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Mechanistically, we found that HPCAL1 can directly bind to LDHA and enhance SRC-mediated phosphorylation of LDHA at tyrosine 10. The metabolomics and RNA sequencing analyses indicated that HPCAL1 knockdown reduces amino acid levels and induces fatty acid synthesis through regulating the expression of metabolism-related genes. Additionally, rescued cells expressing wild-type or mutant LDHA in HPCAL1 knockdown cells suggest that LDHA may serve as the main substrate of HPCAL1. CONCLUSIONS: Our data indicate that the effect of HPCAL1 knockdown on reducing SRC-mediated LDHA activity attenuates NSCLC growth. Our findings reveal novel biological functions and a mechanism underlying the role of HPCAL1 in NSCLC growth in vitro and in vivo.

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).

Specific Cognitive Changes due to Hippocalcin Alterations? A Novel Familial Homozygous Hippocalcin Variant Associated with Inherited Dystonia and Altered Cognition.

BACKGROUND: Recent research suggested an hippocalcin (HPCA)-related form of DYT2-like autosomal recessive dystonia. Two reports highlight a broad spectrum of the clinical phenotype. Here, we describe a novel HPCA gene variant in a pediatric patient and two affected relatives. METHODS: Whole exome sequencing was applied after a thorough clinical and neurological examination of the index patient and her family members. Results of neuropsychological testing were analyzed. RESULTS: Whole exome sequencing revealed a novel homozygous missense variant in the HPCA gene [c.182C>T p.(Ala61Val)] in our pediatric patient and the two affected family members. Clinically, the cases presented with dystonia, dysarthria, and jerky movements. We observed a particular cognitive profile with executive dysfunctions in our patient, which corresponds to the cognitive deficits that have been observed in the patients previously described. CONCLUSION: We present a novel genetic variant of the HPCA gene associated with autosomal recessive dystonia in a child with childhood-onset dystonia supporting its clinical features. Furthermore, we propose specific HPCA-related cognitive changes in homozygous carriers, underlining the importance of undertaking a systematic assessment of cognition in HPCA-related dystonia.

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.

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.

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.

Ubiquitylome profiling of Parkin-null brain reveals dysregulation of calcium homeostasis factors ATP1A2, Hippocalcin and GNA11, reflected by altered firing of noradrenergic neurons.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.

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.

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.

Loss of Par3 promotes prostatic tumorigenesis by enhancing cell growth and changing cell division modes.

Although cell polarity plays an important role in epithelial tumorigenesis, the consequence of polarity protein loss in prostatic tumorigenesis and the underlying mechanisms remain unclear. Using conditional knockout mouse models, we found in the current study that loss of polarity protein Par3 increases prostatic epithelial cell growth, elevates symmetrical cell divisions in basal cells, and randomizes spindle orientation in luminal cells, causing the development of high-grade prostatic intraepithelial neoplasia (PIN). Mechanistically, loss of Par3 dissociates the Par3/merlin/Lats1 complex, consequently inhibiting phosphorylation of Lats1 to attenuate the Hippo pathway. Furthermore, attenuated Hippo pathway enhances nuclear translocation of Yes-associated protein (YAP), which promotes cell proliferation and symmetrical cell divisions through transcriptional activation of Ki-67 and Sox2. In addition, Lats1 dephosphorylation impairs its interaction with G protein signaling modulator 2 (GPSM2, which is also known as LGN) that causes randomization of spindle orientation in luminal cells. Interestingly, co-deletion of Par3 and Lats1 for complete blockade of the Hippo pathway in mice results in prostate tumor initiation, whereas co-deletion of Par3 and YAP for disrupting YAP nuclear translocation reverses the phenotypes to a relatively normal state. Therefore, our findings highlight combination of Par3 loss and blockade of the Hippo pathway as a novel mechanism for prostatic tumorigenesis.

HPCA confirmed as a genetic cause of DYT2-like dystonia phenotype.

BACKGROUND: HPCA (hippocalcin) is one of the underlying genetic causes of autosomal-recessively inherited forms of dystonia. Here, we describe two consanguineous Turkish DYT-HPCA families carrying the novel HPCA mutations. METHODS: After detailed clinical and neurological examination, whole-exome sequencing was performed. RESULTS: Whole-exome sequencing analysis revealed two homozygous novel truncating mutations (p.W103* and p.P10PfsTer80) in the HPCA gene in two unrelated Turkish dystonia families presenting with complex dystonia. CONCLUSIONS: After identification of HPCA as a genetic cause of DYT-HPCA-like dystonia by Charlesworth et al, this is the second report in the scientific literature that describes dystonia families harboring HPCA mutations. Our findings confirm that HPCA leads to recessively inherited dystonia. © 2018 International Parkinson and Movement Disorder Society.

Measurement of intracellular concentration of fluorescently-labeled targets in living cells.

Estimations of intracellular concentrations of fluorescently-labeled molecules within living cells are very important for guidance of biological experiments and interpretation of their results. Here we propose a simple and universal approach for such estimations. The approach is based upon common knowledge that the dye fluorescence is directly proportional to its quantum yield and the number of its molecules and that a coefficient of proportionality is determined by spectral properties of the dye and optical equipment used to record fluorescent signals. If two fluorescent dyes are present in the same volume, then a ratio of their concentrations is equal to a ratio of their fluorescence multiplied by some dye- and equipment-dependent coefficient. Thus, if the coefficient and concentration of one dye is known then the concentration of another dye can be determined. Here we have demonstrated how to calculate this coefficient (called a ratio factor) and how to use it for concentration measurements of fluorescently tagged molecules. As an example of how this approach can be used, we estimated a concentration of exogenously expressed neuronal Ca2+ sensor protein, hippocalcin, tagged by a fluorescent protein in a dendritic tree of rat hippocampal neurons loaded via a patch pipette with Alexa Fluor dye of known concentration. The new approach should allow performing a fast, inexpensive and reliable quantitative analysis of fluorescently-labeled targets in different parts of living cells.

Diabetes aggravates decreases in hippocalcin and parvalbumin expression in focal cerebral ischemia.

Hyperglycemia is a major risk factor for stroke and increases brain damage during ischemic stroke. Hyperglycemia increases the intracellular calcium concentration after ischemic injury, thereby triggering neuronal cell death. Calcium binding proteins, including hippocalcin and parvalbumin, are critical regulators of intracellular calcium levels. This study aimed to investigate whether hyperglycemic conditions affect hippocalcin and parvalbumin expression during ischemic brain injury. Male adult rats were treated intraperitoneally with streptozotocin (40mg/kg) to induce hyperglycemia. Four weeks later, cerebral ischemic injury was induced via surgical middle cerebral artery occlusion (MCAO). Cerebral cortex samples were collected 24h after MCAO. A proteomic approach showed that the protein levels of hippocalcin and parvalbumin were significantly decreased in streptozotocin-treated animals with MCAO injury compared to streptozotocin-treated animals and animals that underwent MCAO alone. Reverse transcription-PCR and Western blot analyses clearly confirmed the decreased levels of these proteins. These decreases indicate dysregulation of the intracellular calcium balance and induction of cell death. Thus, these results suggest that significantly decreased levels of hippocalcin and parvalbumin exacerbate neuronal cell death in diabetic animals with ischemic brain injury.

Biophysical and functional characterization of hippocalcin mutants responsible for human dystonia.

Dystonia is a neurological movement disorder that forces the body into twisting, repetitive movements or sometimes painful abnormal postures. With the advent of next-generation sequencing technologies, the homozygous mutations T71N and A190T in the neuronal calcium sensor (NCS) hippocalcin were identified as the genetic cause of primary isolated dystonia (DYT2 dystonia). However, the effect of these mutations on the physiological role of hippocalcin has not yet been elucidated. Using a multidisciplinary approach, we demonstrated that hippocalcin oligomerises in a calcium-dependent manner and binds to voltage-gated calcium channels. Mutations T71N and A190T in hippocalcin did not affect stability, calcium-binding affinity or translocation to cellular membranes (Ca2+/myristoyl switch). We obtained the first crystal structure of hippocalcin and alignment with other NCS proteins showed significant variability in the orientation of the C-terminal part of the molecule, the region expected to be important for target binding. We demonstrated that the disease-causing mutations did not affect the structure of the protein, however both mutants showed a defect in oligomerisation. In addition, we observed an increased calcium influx in KCl-depolarised cells expressing mutated hippocalcin, mostly driven by N-type voltage-gated calcium channels. Our data demonstrate that the dystonia-causing mutations strongly affect hippocalcin cellular functions which suggest a central role for perturbed calcium signalling in DYT2 dystonia.

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.

DYT2 screening in early-onset isolated dystonia.

BACKGROUND: Mutations in HPCA, a gene implicated in calcium signaling in the striatum, have been recently described in recessive dystonia cases previously grouped under the term "DYT2 dystonia". Positive patients reported so far show focal onset during childhood with subsequent generalization and a slowly progressive course to adulthood. METHODS: 73 patients with isolated dystonia of various distribution, manifesting within 21 years of age, were enrolled in this Italian study and underwent a mutational screening of HPCA gene by means of Sanger sequencing. RESULTS/CONCLUSIONS: Mean age at onset was 10.2 (±5.1) years and mean age at the time of genetic testing was 33 (±14.2) years. Mean disease duration at the time of enrollment was 22.7 (±12.8) years. None of the patients enrolled was found to carry HPCA mutations, rising suspicion that these probably represent a very rare cause of dystonia in childhood-adolescence. Larger studies will help determining the real mutational frequency of this gene also in different ethnic groups.