Łukasz Majewski

Mitochondrial calcium homeostasis involves coordinated uptake via the mitochondrial calcium uniporter (MCU) and efflux through sodium-dependent NCLX (encoded by SLC8B1 ) and/or TMEM65. We investigated TMBIM5, a proposed bidirectional mitochondrial calcium/proton transporter, by generating zebrafish lacking tmbim5 , slc8b1 , plus tmbim5/mcu and tmbim5/slc8b1 double knockouts. Tmbim5-deficient fish exhibited growth impairment, muscle atrophy, and increased brain cell death. tmbim5/mcu double knockouts showed no additive effects, arguing against Tmbim5 functioning as an independent calcium uptake pathway. slc8b1 knockouts had no major phenotype but showed attenuated, although not abolished sodium-dependent mitochondrial calcium efflux. tmbim5/slc8b1 double knockouts showed altered mitochondrial calcium handling with reduced uptake and efflux. Remarkably, brain phenotypes were rescued while muscle dysfunction was exacerbated in double mutants, corresponding to restored mitochondrial membrane potential in brain tissue and decreased calcium levels in muscle. These findings suggest that TMBIM5 functions as an auxiliary calcium efflux pathway cooperating with NCLX in a tissue-specific manner.

Voltage-gated potassium channels (Kv) play a crucial role in maintaining the cell’s resting potential. Mutations in the Kv2.1 voltage-gated potassium channel are associated with developmental epileptic encephalopathy. Previous analysis of the loss-of-function zebrafish mutants kcnb1 ^sq301 and kcng4b ^waw304 , which affect genes that encode Kv2.1 subunits (the α subunit Kcnb1 and the modulatory subunit Kcng4b), has shown that they play an antagonistic role in the development of hollow organs, such as the brain and ear. In this study, we investigated the behavioral effects of these mutations. Under normal light conditions, both mutants exhibited reduced activity at 5 days post-fertilization. However, exposure to a low concentration of 5 mM of the chemoconvulsant pentylenetetrazole increased their locomotor activity and induced seizures. Quantitative RT-PCR (qRT-PCR) analysis of the mutants revealed an increase in the transcript levels of c-fos and gad2, and a decrease in a transcript level of gabra1. This suggests that mutations cause a disruption to inhibitory neurotransmission. Local field potential recordings from the optic tectum of the mutants under baseline conditions showed an increase in spontaneous electrical activity. The kcnb1 mutant is photosensitive and experiences freezing episodes under high-intensity light. Together, these findings suggest that defects in Kv2.1 subunits impact both locomotor behavior and light-evoked responses.

Mitochondrial calcium homeostasis involves coordinated uptake via the mitochondrial calcium uniporter (MCU) and efflux through sodium-dependent NCLX (encoded by SLC8B1 ). Mild Mcu knockout phenotypes suggest additional transport mechanisms. We investigated TMBIM5, a proposed bidirectional mitochondrial calcium/proton transporter, by generating zebrafish lacking tmbim5 , slc8b1 , plus tmbim5/mcu and tmbim5/slc8b1 double knockouts. Tmbim5-deficient fish exhibited growth impairment, muscle atrophy, and increased brain cell death. tmbim5/mcu double knockouts showed no additive effects, arguing against Tmbim5 functioning as an independent calcium uptake pathway. However, tmbim5/slc8b1 double knockouts showed disrupted mitochondrial calcium handling with reduced uptake and efflux. Remarkably, brain phenotypes were rescued while muscle dysfunction was exacerbated in double mutants, corresponding to restored mitochondrial membrane potential in Tmbim5-deficient brain tissue and exacerbated decreased calcium levels in double knockout muscle. We also found broad downregulation of mitochondrial calcium transport proteins and decreased mitochondrial DNA content in double knockout brain tissue, indicating reduced mitochondrial mass and a potential beneficial glycolytic shift in energy metabolism. These findings demonstrate that TMBIM5 functions as a calcium efflux pathway cooperating with NCLX in a tissue-specific manner.

Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca²⁺ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5 days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology.

Store‑operated calcium entry (SOCE) is important for refilling endoplasmic reticulum (ER) Ca2+ stores and Ca2+ signaling. Extracellular Ca2+ is conducted by highly Ca2+‑selective Orai channels that are activated by stromal interaction molecule proteins (STIMs), which are sensitive ER Ca2+ sensors. We found an approximately five‑fold increase in annexin A3a (anxa3a) expression levels in stim2b knockout zebrafish. The present study investigated whether annexin A3 protein is involved in SOCE. We used Ca2+ imaging and electrophysiological recordings to determine the effect of annexin A2, A3, and A6 overexpression on SOCE and Orai-dependent Ca2+ currents (ICRAC) in cultured cells. Annexin A3 increased SOCE amplitude and potentiated Ca2+ ICRAC currents. These results suggest that annexin A3 is a positive modulator of SOCE.

The maintenance of proper cytosolic Ca²⁺ level is crucial for neuronal survival, and dysregulation of Ca²⁺ homeostasis is found in a variety of neurological disorders, including Alzheimer’s disease. According to the “Ca²⁺ hypothesis of aging”, Ca²⁺ disturbances precede the onset of AD symptoms and lead to neurodegeneration. STIM and ORAI proteins are involved in neuronal physiological and pathological processes as essential components of the store-operated Ca²⁺ entry. Our previous data suggested that overexpression of STIM2 and ORAI1 might increase basal neuronal cytosolic Ca²⁺ level. We generated double transgenic mice overexpressing these two genes in neurons, expecting that the increased basal Ca²⁺ concentration will lead to premature neurodegeneration. We observed changes in Ca²⁺ homeostasis and electrophysiological properties in acute brain slices of STIM2/ORAI1 neurons. However, we did not observe any augmentation of neurodegenerative processes, as tested by Fluoro-Jade^® C staining and assessment of amyloidogenesis. The battery of behavioral tests did not show any signs of accelerated aging. We conclude that changes of calcium homeostasis induced by overexpression of STIM2 and ORAI1 had no substantial adverse effects on neurons and did not lead to early neurodegeneration.

Neuronal Store-Operated Ca2+ Entry (nSOCE) plays an essential role in refilling endoplasmic reticulum Ca2+ stores and is critical for Ca2+-dependent neuronal processes. SOCE sensors, STIM1 and STIM2, can activate Orai, TRP channels and AMPA receptors, and inhibit voltage-gated channels in the plasma membrane. However, the link between STIM, SOCE, and NMDA receptors, another key cellular entry point for Ca2+ contributing to synaptic plasticity and excitotoxicity, remains unclear. Using Ca2+ imaging, we demonstrated that thapsigargin-induced nSOCE was inhibited in rat cortical neurons following NMDAR inhibitors. Blocking nSOCE by its inhibitor SKF96365 enhanced NMDA-driven [Ca2+]i. Modulating STIM protein level through overexpression or shRNA inhibited or activated NMDA-evoked [Ca2+]i, respectively. Using proximity ligation assays, immunofluorescence, and co-immunoprecipitation methods, we discovered that thapsigargin-dependent effects required interactions between STIMs and the NMDAR2 subunits. Since STIMs modulate NMDAR-mediated Ca2+ levels, we propose targeting this mechanism as a novel therapeutic strategy against neuropathological conditions that feature NMDA-induced Ca2+ overload as a diagnostic criterion.

Previously, we showed that the overexpression of ORAI1 calcium channel in neurons of murine brain led to spontaneous occurrence of seizure-like events in aged animals of transgenic line FVB/NJ-Tg(ORAI1)Ibd (Nencki Institute of Experimental Biology). We aimed to identify the mechanism that is responsible for this phenomenon. Using a modified Ca²⁺-addback assay in the CA1 region of acute hippocampal slices and FURA-2 acetomethyl ester (AM) Ca²⁺ indicator, we found that overexpression of ORAI1 in neurons led to altered Ca²⁺ response. Next, by RNA sequencing (RNAseq) we identified a set of genes, whose expression was changed in our transgenic animals. These data were validated using customized real-time PCR assays and digital droplet PCR (ddPCR) ddPCR. Using real-time PCR, up-regulation of hairy and enhancer of split-5 (Hes-5) gene and down-regulation of aristaless related homeobox (Arx), doublecortin-like kinase 1 (Dclk1), and cyclin-dependent kinase-like 5 (Cdkl5, also known as serine/threonine kinase 9 (Stk9)) genes were found. Digital droplet PCR (ddPCR) analysis revealed down-regulation of Arx. In humans, ARX, DCLK1, and CDLK5 were shown to be mutated in some rare epilepsy-associated disorders. We conclude that the occurrence of seizure-like events in aged mice overexpressing ORAI1 might be due to the down-regulation of Arx, and possibly of Cdkl5 and Dclk1 genes.

Myosin VI (MVI) is a unique actin-based motor protein moving towards the minus end of actin filaments, in the opposite direction than other known myosins. Besides well described functions of MVI in endocytosis and maintenance of Golgi apparatus, there are few reports showing its involvement in transcription. We previously demonstrated that in neurosecretory PC12 cells MVI was present in the cytoplasm and nucleus, and its depletion caused substantial inhibition of cell migration and proliferation. Here, we show an increase in nuclear localization of MVI upon cell stimulation, and identification of potential nuclear localization (NLS) and nuclear export (NES) signals within MVI heavy chain. These signals seem to be functional as the MVI nuclear presence was affected by the inhibitors of nuclear import (ivermectin) and export (leptomycin B). In nuclei of stimulated cells, MVI colocalized with active RNA polymerase II, BrUTP-containing transcription sites and transcription factor SP1 as well as SC35 and PML proteins, markers of nuclear speckles and PML bodies, respectively. Mass spectrometry analysis of samples of a GST-pull-down assay with the MVI tail domain as a “bait” identified several new potential MVI binding partners. Among them are proteins involved in transcription and post-transcriptional processes. We confirmed interaction of MVI with heterogeneous nuclear ribonucleoprotein U (hnRNPU) and nucleolin, proteins involved in pre-mRNA binding and transport, and nucleolar function, respectively. Our data provide an insight into mechanisms of involvement of MVI in nuclear processes via interaction with nuclear proteins and support a notion for important role(s) for MVI in gene expression.