Lack of Stim2 Affects Vision-Dependent Behavior and Sensitivity to Hypoxia.

Stromal interaction molecules (STIMs) are endoplasmic reticulum-resident proteins that regulate Ca2+ homeostasis and signaling by store-operated calcium entry (SOCE). The different properties and functions of STIM1 and STIM2 have been described mostly based on work in vitro. STIM2 knockout mice do not survive until adulthood. Therefore, we generated and characterized stim2a and stim2b double-knockout zebrafish. The (stim2a;stim2b)-/- zebrafish did not have any apparent morphological phenotype. However, RNA sequencing revealed 1424 differentially expressed genes. One of the most upregulated genes was annexin A3a, which is a marker of activated microglia. This corresponded well to an increase in Neutral Red staining in the in vivo imaging of the (stim2a;stim2b)-/- zebrafish brain. The lack of Stim2 decreased zebrafish survival under low oxygen conditions. Behavioral tests, such as the visual-motor response test and dark-light preference test, indicated that (stim2a;stim2b)-/- larvae might have problems with vision. This was consistent with the downregulation of many genes that are related to light perception. The periodic acid-Schiff staining of retina sections from adult zebrafish revealed alterations of the stratum pigmentosum, suggesting the involvement of a Stim2-dependent process in visual perception. Altogether, these data reveal new functions for Stim2 in the nervous system.

npc2-Deficient Zebrafish Reproduce Neurological and Inflammatory Symptoms of Niemann-Pick Type C Disease.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal storage disease that is caused by a mutation of the NPC1 or NPC2 gene, in which un-esterified cholesterol and sphingolipids accumulate mainly in the liver, spleen, and brain. Abnormal lysosomal storage leads to cell damage, neurological problems, and premature death. The time of onset and severity of symptoms of NPC disease are highly variable. The molecular mechanisms that are responsible for NPC disease pathology are far from being understood. The present study generated and characterized a zebrafish mutant that lacks Npc2 protein that may be useful for studies at the organismal, cellular, and molecular levels and both small-scale and high-throughput screens. Using CRISPR/Cas9 technology, we knocked out the zebrafish homolog of NPC2. Five-day-old npc2 mutants were morphologically indistinguishable from wildtype larvae. We found that live npc2-/- larvae exhibited stronger Nile blue staining. The npc2-/- larvae exhibited low mobility and a high anxiety-related response. These behavioral changes correlated with downregulation of the mcu (mitochondrial calcium uniporter) gene, ppp3ca (calcineurin) gene, and genes that are involved in myelination (mbp and mpz). Histological analysis of adult npc2-/- zebrafish revealed that pathological changes in the nervous system, kidney, liver, and pancreas correlated with inflammatory responses (i.e., the upregulation of il1, nfκß, and mpeg; i.e., hallmarks of NPC disease). These findings suggest that the npc2 mutant zebrafish may be a model of NPC disease.

Knockout of stim2a Increases Calcium Oscillations in Neurons and Induces Hyperactive-Like Phenotype in Zebrafish Larvae.

Stromal interaction molecule (STIM) proteins play a crucial role in store-operated calcium entry (SOCE) as endoplasmic reticulum Ca2+ sensors. In neurons, STIM2 was shown to have distinct functions from STIM1. However, its role in brain activity and behavior was not fully elucidated. The present study analyzed behavior in zebrafish (Danio rerio) that lacked stim2a. The mutant animals had no morphological abnormalities and were fertile. RNA-sequencing revealed alterations of the expression of transcription factor genes and several members of the calcium toolkit. Neuronal Ca2+ activity was measured in vivo in neurons that expressed the GCaMP5G sensor. Optic tectum neurons in stim2a-/- fish had more frequent Ca2+ signal oscillations compared with neurons in wildtype (WT) fish. We detected an increase in activity during the visual-motor response test, an increase in thigmotaxis in the open field test, and the disruption of phototaxis in the dark/light preference test in stim2a-/- mutants compared with WT. Both groups of animals reacted to glutamate and pentylenetetrazol with an increase in activity during the visual-motor response test, with no major differences between groups. Altogether, our results suggest that the hyperactive-like phenotype of stim2a-/- mutant zebrafish is caused by the dysregulation of Ca2+ homeostasis and signaling.

stim2b Knockout Induces Hyperactivity and Susceptibility to Seizures in Zebrafish Larvae.

In neurons, stromal interaction molecule (STIM) proteins regulate store-operated Ca2+ entry (SOCE) and are involved in calcium signaling pathways. However, STIM activity in neurological diseases is unclear and should be clarified by studies that are performed in vivo rather than in cultured cells in vitro. The present study investigated the role of neuronal Stim2b protein in zebrafish. We generated stim2b knockout zebrafish, which were fertile and had a regular lifespan. Using various behavioral tests, we found that stim2b-/- zebrafish larvae were hyperactive compared with wild-type fish. The mutants exhibited increases in mobility and thigmotaxis and disruptions of phototaxis. They were also more sensitive to pentylenetetrazol and glutamate treatments. Using lightsheet microscopy, a higher average oscillation frequency and higher average amplitude of neuronal Ca2+ oscillations were observed in stim2b-/- larvae. RNA sequencing detected upregulation of the annexin 3a and gpr39 genes and downregulation of the rrm2, neuroguidin, and homer2 genes. The latter gene encodes a protein that is involved in several processes that are involved in Ca2+ homeostasis in neurons, including metabotropic glutamate receptors. We propose that Stim2b deficiency in neurons dysregulates SOCE and triggers changes in gene expression, thereby causing abnormal behavior, such as hyperactivity and susceptibility to seizures.

Identification of Zebrafish Calcium Toolkit Genes and their Expression in the Brain.

Zebrafish are well-suited for in vivo calcium imaging because of the transparency of their larvae and the ability to express calcium probes in various cell subtypes. This model organism has been used extensively to study brain development, neuronal function, and network activity. However, only a few studies have investigated calcium homeostasis and signaling in zebrafish neurons, and little is known about the proteins that are involved in these processes. Using bioinformatics analysis and available databases, the present study identified 491 genes of the zebrafish Calcium Toolkit (CaTK). Using RNA-sequencing, we then evaluated the expression of these genes in the adult zebrafish brain and found 380 hits that belonged to the CaTK. Based on quantitative real-time polymerase chain reaction arrays, we estimated the relative mRNA levels in the brain of CaTK genes at two developmental stages. In both 5 dpf larvae and adult zebrafish, the highest relative expression was observed for tmbim4, which encodes a Golgi membrane protein. The present data on CaTK genes will contribute to future applications of zebrafish as a model for in vivo and in vitro studies of Ca2+ signaling.

Behavioral and electrophysiological changes in female mice overexpressing ORAI1 in neurons.

Orai proteins form highly selective Ca2+ release-activated channels (CRACs). They play a critical role in store-operated Ca2+ entry (SOCE; i.e., the influx of external Ca2+ that is induced by the depletion of endoplasmic reticulum Ca2+ stores). Of the three Orai homologs that are present in mammals (Orai1-3), the physiological function of Orai1 is the best described. CRACs are formed by both homomeric assemblies and heteromultimers of Orais. Orai1 and Orai2 can form heteromeric channels that differ in conductivity during SOCE, depending on their Orai1-to-Orai2 ratio. The present study explored the potential consequences of ORAI1 overexpression in neurons where the dominant isoform is Orai2. We established the Tg(ORAI1)Ibd transgenic mouse line that overexpresses ORAI1 in brain neurons. We observed seizure-like symptoms in aged (≥15-month-old) female mice but not in males of the same age. The application of kainic acid and bicuculline to slices that were isolated from 8-month-old (±1 month) female Tg(ORAI1)Ibd mice revealed a significantly lower frequency of interictal bursts compared with samples that were isolated from wildtype mice. No differences were observed in male mice of a similar age. A battery of behavioral tests showed that context recognition decreased only in female transgenic mice. The phenotype that was observed in female mice suggests that ORAI1 overexpression may affect neuronal activity in a sex-dependent manner. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Overexpression of STIM1 in neurons in mouse brain improves contextual learning and impairs long-term depression.

STIM1 is an endoplasmic reticulum calcium sensor that is involved in several processes in neurons, including store-operated calcium entry. STIM1 also inhibits voltage-gated calcium channels, such as Cav1.2 and Cav3.1, and is thus considered a multifunctional protein. The aim of this work was to investigate the ways in which transgenic neuronal overexpression of STIM1 in FVB/NJ mice affects animal behavior and the electrophysiological properties of neurons in acute hippocampal slices. We overexpressed STIM1 from the Thy1.2 promoter and verified neuronal expression by quantitative reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry. Mature primary hippocampal cultures expressed STIM1 but exhibited no changes in calcium homeostasis. Basal synaptic transmission efficiency and short-term plasticity were comparable in slices that were isolated from transgenic mice, similarly as the magnitude of long-term potentiation. However, long-term depression that was induced by the glutamate receptor 1/5 agonist (S)-3,5-dihydroxyphenylglycine was impaired in STIM1 slices. Interestingly, transgenic mice exhibited a decrease in anxiety-like behavior and improvements in contextual learning. In summary, our data indicate that STIM1 overexpression in neurons in the brain perturbs metabotropic glutamate receptor signaling, leading to impairments in long-term depression and alterations in animal behavior. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.