RNA-sequencing showed striking differences in gene expression patterns between the two isolation methods with enzymatically dissociated fibers resembling myopathic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria following enzymatic dissociation. Repeated contractions resulted in a prolonged mitochondrial Ca2+ accumulation in enzymatically dissociated fibers, which was partially prevented by cyclophilin inhibitors. Of importance, muscle fibers of mice with severe mitochondrial myopathy show pathognomonic mitochondrial Ca2+ accumulation during repeated contractions and this accumulation was concealed with enzymatic dissociation, making this an ambiguous method in studies of native intracellular Ca2+ fluxes.Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. By comparing adult mouse skeletal muscle fibers, isolated either by mechanical dissection or by collagenase-induced ECM digestion, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca  •Collagenase treatment disrupts the complex cytoarchitecture of muscle fibers•Muscle fibers rapidly dedifferentiate on collagenase-mediated cell isolation•2+ accumulationDedifferentiation is paralleled by aberrant mitochondrial Ca•2+ accumulationCyclophilin inhibitors limit the aberrant mitochondrial Ca Cellular physiology; Cell biology; Functional aspects of cell biology; Developmental biology. Within the cell, Ca2+ levels are compartmentalized and can differ by orders of magnitude between the cytosol and organelles, such as the sarcoplasmic reticulum (SR). In this context, mitochondrial Ca2+ levels have received increasing attention in recent years for their important roles in energy homeostasis, oxidative stress, and apoptosis.,,2+] ([Ca2+]mit) can stimulate mitochondrial respiration and hence play an integral role in the regulation of cellular metabolism, whereas prolonged and excessive uptake can activate apoptotic and necrotic cell signaling pathways.,,2+]mit seems particularly important in skeletal and cardiac muscle cells where cytosolic free [Ca2+] ([Ca2+]cyt) can reach μM concentrations, and even higher in the vicinity of SR Ca2+ release sites.2+]mit in response to increased [Ca2+]cyt has been observed during in vivo contractions of mouse skeletal muscle, and [Ca2+]mit rapidly returned to the basal level after the end of contraction.The intracellular secondary messenger Ca2+]mit is set by the balance between mitochondrial Ca2+ entry, buffering capacity, and extrusion. The mitochondrial Ca2+ uniporter (MCU) was recently identified as the pore-forming unit of a mitochondrial Ca2+ uptake channel,,2+ extrusion is mediated by the Na+/Ca2+ antiporter in skeletal muscle (Nclx).In general terms, mit is reduced but still measurable, which indicates that additional slower, yet undefined uptake mechanisms exist.,Mitochondrial Ca,,2+ release,2+ handling.2+ exchange between the two organelles, which in turn can entail functional impairments in both organelles.2+]mit after repeated contractions has been observed in skeletal muscle fibers of a transgenic mouse model of desmin myopathy.,2+ overload is regarded as a key pathogenic factor.cis-trans isomerase F , a treatment considered to inhibit the Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP).,,,,,PPID encodes the peptidylprolyl isomerase D, which is a different protein involved in protein folding.Importantly, bidirectional mechanical and biochemical signaling between the extracellular matrix (ECM) and the intracellular cytoskeleton plays a key role in the control of cell structure, including the morphology and function of mitochondria.in vivo and are therefore highly valuable in mechanistic studies of, for instance, the intracellular signaling associated with muscle activation and contraction. Of importance, single muscle fibers are often isolated via collagenase treatment,,,,,,2+ handling. Specifically, by integrating data obtained with second harmonic generation (SHG) and immune-fluorescence imaging, electron microscopy, and RNA-sequencing, we show that enzymatic fiber dissociation, but not mechanical microdissection, causes altered cellular organization and the deterioration of molecular signatures characteristic of mature muscle fibers. Furthermore, repeated tetanic contractions resulted in a marked increase of basal [Ca2+]mit  in enzymatically dissociated but not in mechanically dissected muscle fibers.In mammals, each muscle fiber is under the direct control of a single branch of an α-motoneuron and hence its cellular activation occurs independently of the activity of neighboring muscle fibers. Thus, studies on isolated single muscle fibers will reflect the function of muscle fibers 2+]mit similar to that in enzymatically dissociated fibers has previously been observed in mechanically dissected muscle fibers from a mouse model of severe mitochondrial myopathy, the fast-twitch skeletal muscle fiber-specific mitochondrial transcription factor A knock-out (Tfam KO) mouse, and this increase was implied to have a central role in the disease progress.,2+]mit in mitochondrial myopathy muscle fibers can be detected in enzymatically dissociated fibers, where basal [Ca2+]mit shows a marked increase already in muscle fibers of healthy mice.A pronounced contraction-induced increase in basal mit did not have time to fully return to basal level between contractions when 500 ms tetani were evoked every ∼2s.2+ during repeated contractions resulting in a prolonged elevation of basal [Ca2+]mit (i.e. measured at rest and not during an ongoing contraction), and we here studied this aspect of mitochondrial Ca2+ handling by stimulating isolated FDB muscle fibers at 70 Hz for 350 ms every 2 s. A total of 25 tetanic contractions were evoked and changes in basal [Ca2+]mit were assessed from confocal images of the fluorescence of the [Ca2+]mit indicator rhod-2 obtained before and ∼5 s after the 10th and last contractions. In mechanically dissected fibers, basal [Ca2+]mit did not show any marked increase during the 25 tetanic contractions, which agrees with previous results from FDB fibers of wildtype mice and implies intact mitochondrial Ca2+ control2+]mit in enzymatically dissociated fibers with rhod-2 fluorescence being increased by ∼90% after 10 tetani and by ∼140% after 25 tetani. The increase in basal [Ca2+]mit after 25 tetanic contractions was similar in enzymatically dissociated fibers 4, 24, and 48 h after fiber isolation, hence the increase was not because of an acute stress caused by the isolation procedure as such  or any increase in mitochondrial reactive oxygen species (ROS) levels (measured with the fluorescent indicator MitoSOX Red)  . These f2+]mit in mitochondrial myopathy muscle fibers will escape detection in enzymatically dissociated fibers. First, we used fibers from Tfam KO mice that display important hallmarks of severe mitochondrial myopathy.,2+]mit in mechanically dissected Tfam KO FDB fibers, whereas basal [Ca2+]mit was maintained at a low level in muscle fibers of non-KO littermate controls .Slirp KO mice appear largely healthy with only minor (∼5%) reduction in body weight.2+]cyt-frequency relationship was studied in FDB fibers mechanically dissected from Slirp KO mice and wildtype littermates by producing brief contractions at 1 min intervals. Slirp KO fibers displayed lower [Ca2+]cyt than wildtype fibers at high stimulation frequencies (120–150 Hz) and during tetanic stimulation in the presence of caffeine (5 mM), which facilitates SR Ca2+ release and hence provides an estimate of the total amount of Ca2+ stored in the SR , and we measured basal [Ca2+]mit ∼5 s after contractions. Thus, based on the results of Rudolf et al., only a minor increase in basal [--Ca2+]mit after the current repeated contractions would be expected, which is in accordance with the results obtained in mechanically dissected fibers. On the other hand, the marked increase in basal [Ca2+]mit observed in enzymatically dissociated fibers indicates a defective control of mitochondrial Ca2+ fluxes, i.e. a defect also observed in mechanically dissected muscle fibers of mitochondrial myopathy mouse models  tetanic contraction  or where mechanically dissected fibers were subsequently treated with collagenase and contractions performed under the same conditions as enzymatically dissociated fibers. Nevertheless, we cannot exclude that the observed differences between mechanically dissected and enzymatically dissociated fibers involved other methodological aspects; for instance, mechanically dissected fibers were not exposed to the trituration process.2+ accumulation in enzymatically dissociated fibers occurs partly via a Ppif-dependent pathway. Cyclophilin inhibitors act on Ppif to inhibit opening of mPTP. In situations of severe cellular stress, mPTP enters a high-conductance state. In the present study, however, the acute stress is relatively mild, and the high-conductance state is not entered. We propose that the cyclophilin inhibitors then act by inhibiting opening of a low-conductance state or by increasing the mitochondrial Ca2+ buffering capacity, but further experiments are required to clarify the exact details of their action.Our results imply that contraction-induced mitochondrial Cahakan.westerblad@ki.se).Further information and requests for resources and reagents should be directed to and will be fulfilled by lead contact, Håkan Westerblad  under a 12-h light/dark cycle. Adult mice were used, except when stated otherwise. C57BL/6JRj (Janvier Labs) female mice were used, except when stated otherwise. Fast-twitch skeletal muscle fiber-specific Tfam KO mice were treated with the specific cyclophilin inhibitor, NV556,Some 14 weeks old Flexor digitorum brevis (FDB) muscles were cleaned of tendons, connective tissue, and blood vessels and incubated for ∼2 h at 37°C in 0.3% collagenase type 1 (Merck) in Dulbecco's modified Eagle medium  supplemented with 20% fetal bovine serum. Muscles were then transferred to fresh DMEM and gently triturated to dissociate individual muscle fibers. A volume of 300 μL of the resultant muscle fiber suspension was placed in laminin-coated glass-bottom Petri dishes, and fibers were allowed to attach for 15 min. Thereafter, 3 mL DMEM supplemented with antibiotic antimycotic solution  was added. Experiments were performed ∼4 h after enzymatic dissociation, except when otherwise noted.Single or small bundles (2–5 fibers) of FDB fibers were mechanically dissected as previously described.,,Mechanically dissected and enzymatically dissociated FDB fibers were prepared as described above and fixed in 2.5% glutaraldehyde (TAAB Laboratories). They were then imaged using a multi-photon microscope  equipped with a combination of two water immersion objectives  and a mode-locked ps-pulsed Ti:Sa-laser  tuned to 810 nm to excite the myosin SHG signal.Mechanically dissected or enzymatically dissociated muscle fibers were fixed with 4% methanol-free PFA for 2 h at 4°C. Afterward, fibers were washed 3x with PBS and permeabilized with 0.1% Triton X-100 in PBS at room temperature. After repeated PBS washes, the fibers were incubated with 1:50 rabbit anti-desmin  or 1:50 rabbit anti-plectin  antibodies overnight at 4°C. The primary antibodies were extensively washed out and samples were incubated with AlexFluor488-conjugated secondary goat anti-rabbit antibody for 2h at room temperature. Images were collected on a Zeiss LSM800-Airy confocal microscope.2+ was used during the fixation and contrasting steps with lead aspartate were omitted to reduce overstaining. Samples were trimmed with a glass knife and 70 nm ultrathin sections were picked up on Cu-grids and examined with the TEM Talos L120C  operating at 120 kV. Micrographs were acquired with a Ceta 16M CCD camera using TEM Image & Analysis software ver. 4.17 (Thermo Fisher Scientific).Mechanically dissected and enzymatically dissociated FDB muscle fibers were prepared as described above and fixed in 2.5% glutaraldehyde (TAAB Laboratories), 4% formaldehyde in 0.1 M sodium cacodylate buffer. All samples were processed using Pelco Biowave Pro + microwave tissue processor (Ted Pella) according to,https://imagej.nih.gov/ij/) and the mitochondrial network was reconstructed and analyzed in a final volume of 365 μm3 (7.9 × 4.5 × 10.3 μm). Identified mitochondrial volumes were modeled and measured using the IMOD software package ver. 4.9.13.For focused ion beam scanning electron microscopy (FIB-SEM), a small cube of the sample was cut and glued on an SEM stub with epoxy and silver glue. In the SEM chamber, the specimen was coated with a 5 nm thin layer of platinum to reduce charging. Specimens were imaged using Scios DualBeam SEM and the ‘Auto slice and view 4’ software system (Thermo Fisher Scientific); the electron beam operated at 2 kV and 0.2 nA and was detected with T1 in-lens detector. A 1 μm protective layer of platinum was deposited on the selected area before milling. FIB milling thickness was set to 30 nm and each slice was imaged with pixel sizes 3.55 × 3.55 nm  and 3.74 × 3.74 nm . Images were further processed using the ImageJ plugins ‘Linear Stack Alignment with SIFT’ and ‘Multistackreg’ (https://nf-co.re/rnaseq/usage). Genes with an average number of fragments per kilo base per million mapped reads (FPKM) > 0.5 across all samples were analyzed using Qlucore Omics Explorer . Differential gene expression analysis was conducted using DESeq2 and multiple testing correction was applied using the Benjamini–Hochberg procedure with false discovery rates (FDRs) ≤ 5%. Pathway enrichment analysis was conducted based on the PANTHER gene family classification system using the WebGestalt toolbox.RNA sequencing by poly-A capture was performed using >10 ng RNA input material. Image analysis, base calling and quality checks were performed using the RTA3.4.4 pipeline and Bcl2fastq (v2.20) conversion software (Illumina). Quality control, removal of genomic contaminants and ribosomal RNA, UMI-based read deduplication, transcript assembly and quantification were conducted using the Stringtie nf-core/rnaseq package (2+] ([Ca2+]mit), membrane potential (Δψm) and ROS production (see below), unmounted FDB fibers were loaded with fluorescent indicators for 20minat room temperature. During FDB fiber experiments, cells were superfused at room temperature (∼25°C) with Tyrode solution (in mM): NaCl, 121; KCl, 5.0; CaCl2, 1.8; MgCl2, 0.5; NaH2PO4, 0.4; NaHCO3, 24.0; EDTA, 0.1; glucose, 5.5; 0.2% fetal calf serum. The solution was bubbled with 95% O2–5% CO2. Measurements were performed using a BioRad MRC 1024 confocal unit with a dual Calypso laser  attached to a Nikon Diaphot 200 inverted microscope. Confocal images were obtained before, ∼5 s after 10 and 25 repeated tetani , and at regular intervals after the contractions. Note that confocal imaging was always performed at rest and not during an ongoing contraction due to fiber movement. Confocal images were analyzed using ImageJ and data are expressed as F/F0, i.e., the ratio of the fluorescence intensity after and before the repeated contractions, respectively.For measurements of mitochondrial [Ca2+]mit, fibers were incubated in 5 μM rhod-2 in the membrane permeable AM form (Invitrogen). Rhod-2 was excited with 531 nm light and the emitted light was collected through a 585 nm long-pass filter.For measurements of mit as described above. To investigate the potential sites of Ca2+ entry into mitochondria, enzymatically dissociated fibers were exposed to either Ru360 , CsA or NV556 . Ru360 was first injected into the fibers and they were subsequently superfused with 10 μM Ru360 throughout the experiment. CsA  or NV556 (5 μM) was applied to the fibers for 5 min before and during the 25 repeated tetani.Enzymatically dissociated FDB fibers were loaded with rhod-2 to measure [Ca2+]cyt-frequency relationship was obtained by stimulating fibers for 350 msat 15–150 Hz every 1 min; 150 Hz contractions were also produced in the presence of 5 mM caffeine to assess the SR Ca2+ storage capacity.Intact single FDB fibers were mechanically dissected.2+]cyt. An advantage with high-affinity Ca2+ indicators is that they can measure resting as well as mean tetanic [Ca2+]cyt. However, they cannot readily detect fast transient [Ca2+]cyt increases and hence tetanic [Ca2+]cyt values tend to be lower with high-affinity than with low-affinity Ca2+ indicators. On the other hand, low-affinity Ca2+ indicators cannot accurately measure the low [Ca2+]cyt at rest.2+]cyt using the following equation:d is the apparent dissociation constant of indo 1, β is the ratio of the 495 nm signals at very low and saturating [Ca2+]cyt, Rmin is the ratio at very low [Ca2+]cyt, and Rmax is the ratio at saturating [Ca2+]cyt. Fluorescence was sampled online and stored on a computer for subsequent data analysis. [Ca2+]cyt was measured at rest and as the mean during 70 Hz, 350 ms tetanic stimulation. The steady-state [Ca2+]cyt-frequency relationship was obtained in Slirp KO and control WT fibers by stimulating fibers for 350 msat 15–150 Hz every 1 min; 150 Hz contractions were also produced in the presence of 5 mM caffeine to assess the SR Ca2+ storage capacity.We used the relatively high-affinity fluorescent indicator indo-1 to measure [CaStatistical analyses were performed with SigmaPlot 13 . Student’s paired, unpaired t-tests, one-way ANOVA, or z-test were used as appropriate. Two-way repeated measures ANOVA was used to determine differences between two groups of repeatedly stimulated fibers. The Holm-Sidak post-hoc analysis was used when ANOVA showed a significant difference between groups. Significance was assumed for p< 0.05. Data are presented as mean ± SEM.


The regulation of NCKX transporters enables them to respond to the changing cellular environment thereby helping to shape the extent and kinetics of Ca2+ signals. This review examines the current knowledge of the different ways in which NCKX activity can be modulated. These include (i) cellular and dynamic subcellular location (ii); changes in protein expression mediated at the gene, transcript, or protein level (iii); genetic changes resulting in altered protein structure or expression (iv); regulation via changes in substrate concentration (v); and post-translational modification, partner protein interactions, and allosteric regulation. Detailed mechanistic understanding of NCKX regulation is an emerging area of research with the potential to provide important new insights into transporter function, the control of Ca2+ signals, and possible interventions for dysregulated Ca2+ homeostasis.Potassium-dependent sodium-calcium exchangers (NCKX) have emerged as key determinants of calcium (Ca Consequently, [Ca2+] is tightly regulated by a constellation of different proteins  that is mediated and temporally shaped by NCKX1 activity is a critical factor for the kinetics and termination of the photo-response and for adaptation to changing light levels  is thought to be close to the level found in the cytosol; the membrane potential appears to be low; and the pH is known to be low. The pH gradient is maintained by the V-ATPase and can increase luminal [Na+] via Na+/H+-exchangers. This small Na+ gradient may be sufficient to allow Ca2+ accumulation because of the 4Na+:1Ca2+ transport stoichiometry of NCKX proteins  lead to an increase in NCKX2 expression. Disruption of homeostasis under the extreme conditions of ischemia, however, breaks the loop, resulting in the collapse of NCKX2 expression. The link between Ca2+ signaling and gene expression is well studied, particularly in the context of synaptic plasticity [2+] changes to altered NCKX2 expression remain to be determined.These data suggest a homeostatic loop, whereby physiologically modest sustained increases in cellular  is a limiting substrate for NCKX activity is borne out by detailed kinetic analysis of different Ca2+ transport pathways in neuronal nerve terminals [2+] levels, while NCKX activity predominates over the low µM levels found during signaling events, with mitochondrial accumulation playing an increasingly important role as [Ca2+] rises even further to deleterious and possibly pathological levels.Transport protein activity depends upon the concentration of the substrates and their binding affinities at the transport sites. In the case of the NCKX proteins under normal physiological conditions, where the inward movement of Nay is not ,120,121.erminals . Here, P+ from its extracellular binding site. The affinity for K+ here is in the low mM range , sua region , which i+ ions in one direction to 1 Ca2+ ion and 1 K+ ion in the other, are thermodynamically poised to transport Ca2+ ions out of the cytoplasm under a wide range of ion concentrations and membrane potentials. Nevertheless, under pathological conditions associated with severe ischemia and excitotoxic states, the extent of the alterations in ion concentration can cause the reversal of NCKX transporters, leading to the build-up of toxic levels of Ca2+ [NCKX proteins, because of their transport stoichiometry linking the movement of 4 Na of Ca2+ ,63.2+ binding to two well-characterized domains in the cytosolic loop far away from the transport site activates exchange. Meanwhile, Na+ acts as an allosteric inhibitor, apparently involving the intracellular ion transport sites themselves, although the molecular mechanism is not well understood [+-initiated Ca2+ flux measurements in bovine rod outer segment preparations demonstrate a time-dependent rapid inhibition of activity that prevents NCKX1 from lowering Ca2+ below a threshold value of about 100 nM [+-dependent inhibition has been seen when measuring NCKX2 activity in the HEK293 cell expression system using a reverse-mode assay [+-dependent inhibition of NCX1. It is unclear if the time-dependent inhibition of NCKX2 measured in HEK293 cells is related to the Na+- and time-dependent inhibition observed for NCKX1 in rod outer segments.Substrates can also be allosteric regulators of transporters, as well described for the cardiac NCX1 . In NCX1derstood . No equiderstood . Howevert 100 nM . Second,de assay . AlthougPerhaps the most common form of protein regulation is via allostery, most often in the form of post-translational modifications that influence protein conformation and, hence, activity. In this section of the review, we will discuss the currently known processes by which NCKX proteins are allosterically regulated. 2+ levels in close vicinity to the channel through NCKX1-mediated extrusion [Cross-linking and hydrodynamic studies have identified NCKX1 homodimers within the plasma membrane of bovine rod photoreceptor outer segments . Becausextrusion ,126. It xtrusion . Interesxtrusion . Whether2+ levels and the downstream physiological events in those cells. CNGC is the only Ca2+ entry pathway and NCKX is the only Ca2+ extrusion pathway in photoreceptor outer segments. Hence, by physically and functionally associating Ca2+ entry and exit mechanisms, both the kinetics and spatial extent of Ca2+ signaling can be controlled [2+ signals.This specific arrangement of NCKX homodimers within a protein complex with the CNGC in both rod and cone photoreceptor outer segments may be critical for the control of Cantrolled ,59. Whetntrolled ,87, suggThe NCKX-NCKX and NCKX-CNGC interactions are not isoform-specific. When co-expressed together in heterologous systems, NCKX1 can also be co-immunoprecipitated with NCKX2. Similarly, co-expression of NCKX1 or 2 with CNGA1 or 3 results in complexes between either NCKX and CGNA isoforms . It seem2+ homeostasis in many environments, including muscle contraction and neuronal plasticity [The phosphorylation of proteins represents one of the most common forms of post-translational modification linked to changes in activity. Phosphorylation cascades are well known mediators of regulated Caasticity ,129.2+ signals. Therefore, Ca2+-dependent pathways that regulate this process, such as PKC, are of particular interest. Indeed, the activation of PKC has been shown to stimulate NCKX activity in axon terminals at the calyx of Held [2+-dependent dephosphorylation via calcineurin [2+ flux observed following PKC activation are clear, the molecular mechanisms that allow NCKX to be regulated remain unexplored. It is interesting to speculate how covalent modifications and/or subsequent changes in protein interactions within the NCKX2 cytoplasmic loop can lead to changes in activity. A direct interaction between the cytoplasmic loop and the transport domain in the membrane might mediate these effects, perhaps through the relief of a constraint in the conformational changes associated with transport [NCKX2 has been implicated in the integration of synaptic plasticity ,32, pres of Held . Studiescineurin . While transport . Another2+ signaling [As discussed earlier, NCKX2 has a polarized surface distribution on neurons that depends upon somatodendritic endocytosis and subsequent vesicular trafficking to axon terminals mediated by the kinesin KIF21A . Evidencignaling . Thus, iignaling .2+ signaling dynamics [2+ from the cell. The precise mechanism that leads from the stimulation of PKC and CaMKII to an increase in NCKX4 activity has not yet been identified. As with regulation of NCKX2, allosteric activation of transport activity or recruitment to the plasma membrane from a sub-membrane vesicle pool are possible mechanisms. Intriguingly, the activation of GluR1 that underlies synaptic plasticity also requires a synergistic activation of PKC and CaMKII [Purinergic signaling, mediated by ATP release, is thought to be an important modulatory component of neurotransmitter-dependent Cadynamics . ATP tredynamics . This std CaMKII .2+-dependent regulation processes, binding to helical amphipathic basic motifs [2+ levels, appearing to stabilize the protein but inhibit activity. Following a Ca2+ signal, rapid activation of NCKX4 activity associated with PKC/CaMKII phosphorylation helps to extrude Ca2+ from the cell, hence dampening the signal. Subsequently, a slow Ca2+-dependent reorientation of CaM on NCKX4 causes a reversal of the activation [2+ signaling events. Such a model fits with the role of NCKX4 as an important mechanism for time-dependent adaptation to a signal response in the olfactory and visual systems [Calmodulin (CaM) is a very common mediator of Cac motifs ,136,137.c motifs  reveals c motifs . In thistivation . These c systems ,87. The The reversible thioester bond between a protein cysteine and palmitic acid  is a post-translational modification that regulates many membrane proteins ,141. PalSTRING is a database of protein networks that includes both known and predicted interactions . The ent2+ homeostasis with several NCKXs). In other instances, the two partners are simply both mentioned in the text of a publication . However, none of these predictions are supported by experimental evidence. Nevertheless, some of the connections between NCKXs and proteins linked by physiological or genetic interactions might represent potentially interesting avenues for future investigation.For the unanticipated interactions, further investigation into the underlying data reveals that in most instances the networks represent a connection between proteins whose expression is known to co-localize in the same cells/tissue with the corresponding NCKX protein or is associated with the same physiological process (e.g., visual transduction with NCKX1; pigmentation with NCKX5; general CaD. melanogaster between the NCKX homolog, zydeco, and CaM [In this regard, two examples stand out. First, several NCKX entries in the STRING database highlight a strong genetic interaction observed in  and CaM . This co and CaM , althoug and CaM . Since W and CaM , which i+-dependent Na+/Ca2+-exchangers can be regulated, including changes in cellular location, protein expression, genetics, substrate concentration, and finally, allostery. While these studies have great potential to unlock the relation between NCKX proteins, Ca2+ homeostasis, and physiological function, this area remains under-explored. The topic of allosteric regulation, in particular, has much promise for the future. New post-translational modifications, novel interaction partners, and a deeper mechanistic understanding of how such interactions lead to changes in NCKX transporter function are eagerly anticipated. This new knowledge will deepen our understanding regarding the control of Ca2+ signaling, will have important implications for transporter function in general, and has the potential to provide new therapies to treat pathological states where Ca2+ homeostasis is dysregulated.In this review, we have focused on a variety of modalities by which K


Calcium is a central signal transduction element in biology. Peroxisomes are essential cellular organelles, yet calcium handling in peroxisomes has been contentious. Recent advances show that peroxisomes are part of calcium homeostasis in cardiac myocytes and therefore may contribute to or even shape their calcium-dependent functionality. However, the mechanisms of calcium movement between peroxisomes and other cellular sites and their mediators remain elusive. Here, we review calcium handling in peroxisomes in concert with other organelles and summarize the most recent knowledge on peroxisomal involvement in calcium dynamics with a focus on mammalian cells. ER calcium is released either into the cytosol, or through specialized compartments and membrane contact sites to juxtaposed organelles . Calciume (PMCA) .Peroxisomes are membrane-bound organelles originally identified as sites for production and degradation of hydrogen peroxide, and fatty acid metabolism . Mutatio2+ handling in cardiomyocytes (CMs)  . The defs in CMs .Peroxisomes are highly dynamic organelles capable of fast adaptation to nutritional and environmental changes . The mul2+-sensitive targets involved in peroxisomal metabolism have been described. In Arabidopsis and tobacco, ROS-scavenging efficiency increases with Ca2+-mediated activation of peroxisomal catalase 3  could be detected upon maximal stimulation. The response of D3cpV-px (FRET sensor) and pericam-px (ratiometric sensor) were comparable and were not saturated, suggesting that peroxisomal Ca2+ levels are in the optimal detection range of these sensors  as 2+ levels with a mean value of 600 nM and increase upon stimulation up to 2.4 µM  on the SR, voltage-operated Ca2+ channels in the T-tubules and Na+/Ca2+-exchanger on the plasma membrane. Strict control of cellular Ca2+ levels is of particular importance in cardiomyocytes. Ca2+ overload in CMs results in malfunction of the heart by affecting both the electrical and contractile properties of cardiomyocytes  is the main source of Ca2+ increase in CMs in the excitation-contraction coupling  and human induced pluripotent stem cell-derived CMs (hiPSC-CMs)  . We hypot manner .2+ entry to peroxisomes, if there are tissue or cell type-specific molecular composition and/or abundance of contact sites, and which of these are relevant for CMs.Peroxisomes in hiPSC-CMs occasionally localize in vicinity of ER protein RyR2 and rarely to T-tubular system and LTCC . The str2+ from the SR and localize in the proximity of RyR receptors in CMs, it is plausible that peroxisomes may contribute to or even be essential for normal excitation-contraction coupling. This hypothesis is supported by the fact that patients with mild forms of ZSS occasionally present with cardiac arrhythmias that may become the cause of lethal outcome  also on peroxisomes based on immunoelectron microscopy in rabbit small intestinal tissue. Along with peroxisomal malate dehydrogenase and lactate dehydrogenase  protein subfamily—were found exclusively in mitochondria  beyond its initial maximum  occur. On the other hand, the absence of peroxisomes may contribute to cardiac pathogenesis and promote the development or extent of arrhythmias.As Ca2+ enters peroxisomes of non-excitable and excitable mammalian cells upon near-physiological electrical and chemical stimulation. Peroxisomal Ca2+ handling presents an exciting research area with many open questions: For example, the existence of Ca2+-dependent transporters in peroxisomes as suggested (2+-sensitive targets in the mammalian peroxisome, as known for the plant peroxisome, may yet be identified. Furthermore, it is conceivable that peroxisomal Ca2+ homeostasis may be important in the absence of luminal Ca2+ binding proteins, by buffering local calcium. In excitable cells, peroxisomal Ca2+ dynamics might be of special importance, also with regard to their pathophysiology. The cellular function of peroxisomal Ca2+ and the role of peroxisomal Ca2+ in pathology remain to be studied further.Cauggested  is still


Long noncoding RNAs (lncRNAs) are increasingly emerging as regulators across human development and disease, and many have been described in the context of hematopoiesis and leukemogenesis. These studies have yielded new molecular insights into the contribution of lncRNAs to AML development and revealed connections between lncRNA expression and clinical parameters in AML patients. In this mini review, we illustrate the versatile functions of lncRNAs in AML, with a focus on pediatric AML, and present examples that may serve as future therapeutic targets or predictive factors. Acute myeloid leukemia (AML) accounts for approximately 20% of acute leukemias in children . AlthougLong noncoding RNAs (lncRNAs) — defined as transcripts longer than 200 nucleotides that lack open reading frames — represent the largest group of noncoding RNAs and constitute two-thirds of the human transcriptome . DiffereBased on the mechanistic interaction of lncRNAs with other molecules, four different archetypes of lncRNA functions — namely signal, decoy, guide, and scaffold — have been defined in a seminal work by Wang and Chang in 2011 . As the in cis) as well as on distant genes (function in trans), while cytoplasmic lncRNA mechanisms include competitive miRNA binding and interaction with translational proteins , t, and AML with ) . In the patients .HOTTIP is overexpressed in NPM1-mutated and KMT2A-r AML cases and predicts poor outcome [HOTTIP alters the three-dimensional structure of the nearby HOXA locus and binds to posterior HOXA sites as well as other genes critically involved in hematopoiesis and leukemogenesis, resulting in the activation of an AML-specific transcriptional program. Of note, HOTTIP expression is sufficient to initiate leukemic transformation of HSCs in mice. Knockout of HOTTIP perturbs leukemic proliferation and prolongs the survival in AML mouse models, suggesting a novel therapeutic option for the treatment of pediatric AML [Other studies have focused on the in-depth characterization of individual lncRNAs in pediatric AML. Here, we will summarize lncRNAs that have been implicated in pediatric AML Table  and will outcome . Mechanitric AML .Table 1LCDK6-AS1 as a novel regulator in pediatric AML [CDK6-AS1 was significantly overexpressed and associated with higher minimal residual disease after induction therapy. High CDK6-AS1 levels contributed to an immature phenotype in healthy HSCs and primary AML blasts, whereas silencing of the lncRNA led to increased hematopoietic differentiation of HSCs and to a rescue of the pathogenic undifferentiated state of AML blasts. Mechanistically, the authors could show that CDK6-AS1 regulates expression of its neighboring gene CDK6 by sharing a bidirectional promotor, and that the common CDK6-AS1/CDK6 axis downregulates RUNX1 signaling, which is essential for early hematopoietic differentiation. In addition, CDK6-AS1 activates mitochondrial biogenesis in healthy HSCs as well as in pediatric AML blasts. Interestingly, mitochondrial targeting, using Tigecycline, sensitizes AML blasts with high CDK6-AS1 expression to chemotherapy, supporting the concept of a mitochondrial vulnerability in these blasts. Overall, these findings identified CDK6-AS1 as an important regulator of early hematopoietic differentiation and leukemogenesis of pediatric AML and uncovered therapeutics targeting mitochondrial biogenesis as a novel treatment strategy in pediatric AML [A recent elaborate study has identified lncRNA tric AML . In a petric AML .UCA1 is an additional example of an oncogenic lncRNA in adult and pediatric AML. UCA1 has been shown to be upregulated by CEBPα-p30, the CEBPα isoform that results from CEBPA mutations recurrently found in AML patients [UCA1 is upregulated, and knockdown of the lncRNA impairs leukemic viability, migration, and invasion through binding of various microRNAs, such as miR-126, miR-204, miR96-5p, and miR296-3p [UCA1 contributes to chemoresistance in pediatric AML by tethering miR-125a [patients . In pediiR296-3p –64. FurtmiR-125a .MONC and MIR100HG, which are host genes for the homologous miRNA clusters miR-99a~125b-2 and miR-100~125b-1, respectively. These miRNA clusters are known to promote the progression of AMKL [MONC alters hematopoietic differentiation, independently of the expression of the oncogenic miRNA clusters [Other examples of relevant lncRNAs in childhood AML are  of AMKL , 88. Botclusters .MEG3 is downregulated in adult and pediatric AML, supposedly by epigenetic modifications of its genomic locus. Mechanistically, MEG3 has been shown to activate p53 expression and DNMT3A, thereby inhibiting leukemogenesis [MEG3 promoter is associated with poor prognosis in adult AML patients [MEG3 correlates with better survival [LncRNA ogenesis . Hypermepatients . In pedisurvival .These are only a few examples of lncRNAs that have been characterized in the context of pediatric AML and that show correlation to clinically relevant subgroups and/or prognosis of the patients. In addition, a more general lncRNA scoring system based on the expression of 14 lncRNAs has been proposed to predict overall survival in children with AML .HOXA10-AS was identified as an essential regulator of hematopoiesis and as a novel oncogenic lncRNA in the context of pediatric AML with KMT2A rearrangements (KMT2A-r AML) [HOXA10-AS is located at the posterior end of the HOXA cluster — one of the four highly conserved HOX gene clusters. Tightly controlled spatiotemporal expression of the different HOX genes is crucial for hematopoietic differentiation, and dysregulation of HOX genes such as HOXA9 and HOXA7 by KMT2A fusion proteins is responsible for leukemic transformation in KMT2A-r AML [HOX gene clusters harbor numerous lncRNAs that are expressed in the same specific pattern as their protein-coding neighbors during differentiation, indicating their putative biological importance [HOX lncRNAs are capable of regulating the expression of neighboring or distant protein-coding HOX genes, as well as of independent effects on other signaling pathways [HOX lncRNAs have undergone further characterization, the role of the vast majority of HOX lncRNAs in AML remains unknown.In a recently published study from our group, the antisense lncRNA A-r AML) . Along t2A-r AML . The HOXpathways , 91. AltHOXA10-AS is transcribed from the antisense strand relative to the protein-coding gene HOXA10 and microRNA mir-196b, both of which are involved in hematopoiesis and in the pathogenesis of KMT2A-r AML. In our study, we confirmed that HOXA10-AS is overexpressed in KMT2A-r AML as well  [HOXA10-AS, we found that its effects in hematopoiesis and leukemogenesis were independent of changes in expression of the neighboring oncogenes, arguing against a regulatory role for HOXA10-AS on the HOXA cluster in cis. This was supported by subcellular localization studies, which showed that the HOXA10-AS is mainly located in the cytoplasm. Indeed, microarray-based gene expression analysis uncovered a possible trans mechanism for HOXA10-AS involving the upregulation of NF-κB target genes in HOXA10-AS expressing early monocytic progenitors and KMT2A-r AML  . Regardi2A-r AML .Fig. 1HOHOXA10-AS add important insights on the regulatory roles of lncRNAs in general, as well as crucial knowledge about the specific pathogenesis of pediatric AML, both of which will hopefully contribute to a comprehensive view and new therapies for this disease.LncRNAs are emerging as regulators of hematopoiesis and AML pathogenesis, and knowledge about their individual effects is rapidly increasing. However, extensive functional research is required before we gain a complete understanding of the complex regulatory networks surrounding lncRNAs and their interplay with known oncogenic drivers. While individual examples continue to provide valuable information about the roles of lncRNAs and how they might serve as novel therapeutic targets or prognostic factors in AML, research on lncRNAs in pediatric AML still lags behind adult AML. Thus, investigations of lncRNAs such as


Interaction of CD52-SIGLEC10 between QSCs and monocytes may contribute to immune evading and poor outcomes. Furthermore, we identified that LGALS1 was a promising target for chemoresistant AML, and LGALS1 inhibitor could help eliminate QSCs and enhance the chemotherapy in patient-derived primary AML cells, cell lines, and AML xenograft models. Our results will facilitate a better understanding of the AML chemoresistance mechanism and the development of novel therapeutic strategies for relapsed/refractory AML patients.Chemoresistance and relapse are the leading cause of AML-related deaths. Utilizing single-cell RNA sequencing (scRNA-seq), we dissected the cellular states of bone marrow samples from primary refractory or short-term relapsed AML patients and defined the transcriptional intratumoral heterogeneity. We found that compared to proliferating stem/progenitor-like cells (PSPs), a subpopulation of quiescent stem-like cells (QSCs) were involved in the chemoresistance and poor outcomes of AML. By performing longitudinal scRNA-seq analyses, we demonstrated that PSPs were reprogrammed to obtain a QSC-like expression pattern during chemotherapy in refractory AML patients, characterized by the upregulation of CD52 and LGALS1 expression. Flow cytometric analysis further confirmed that the preexisting CD99 Acute myeloid leukemia (AML) is the most common and lethal adult acute leukemia, characterized by aggressive proliferation, differentiation blockage, and apoptosis disorder of immature blasts in the bone marrow, peripheral blood, and other tissues . ChemothThe high intratumoral heterogeneity (ITH) is one of the most important reasons for drug resistance and relapse in AML , 6. AlthThe emergence of single-cell sequencing technologies makes it possible to quantify the whole genome or transcriptome of every single cell in a tissue mixture and provides an unprecedented opportunity to decipher the complexity of AML cellular heterogeneity , 19–21. Here, we performed an integrated single-cell transcriptomic analysis of bone marrow samples from primary refractory and short-term relapsed AML patients to identify the critical factors in AML chemoresistance. Besides, longitudinal scRNA-seq analysis was performed to trace the dynamic cellular and molecular plasticity induced by chemotherapy in AML patients. By comprehensively dissecting the cellular states of leukemia-like cells in the AML ecosystem, we defined the transcriptional ITH and identified the LSC subpopulation that was involved in the chemoresistance and relapse of AML. Furthermore, the reprogramming of cellular states induced by chemotherapy was described and effective therapeutic targets for chemotherapy sensitization were identified for RR-AML patients.To explore the ITH and identify potential therapeutic targets for RR-AML, we designed a scRNA-seq-based workflow to comprehensively dissect the dynamic cellular and molecular reprogramming during chemotherapy in RR-AML patients Fig. . Firstly+ T cells, and CD8+ T cells , while PSPs enriched in the cell dividing signature , we measured the transcriptional ITH of each patient based on the identified cellular states. Results showed that patients who harbored more AML-related mutations had a higher transcriptional ITH score Fig. , indicatIn order to further evaluate the additional prognostic power of our ITH-index for AML wild-type patients, TCGA samples were divided into four classes for each gene mutation, including mutated/ITH-high, mutated/ITH-low, wild-type/ITH-high, and wild-type/ITH-low. The results showed that a high level of ITH-index could predict the poor OS of TP53, DNMT3A, RUNX1, ASXL1, KRAS, and FLT3 wild-type AML patients  had a low level of QSCs , suggesting the enrichment of QSCs in the chemo-residual cells  and high relapse rate . In contrast, patients with a high level of PSPs had favorable OS  and low relapse rate . These results were also maintained when the APL cases were excluded , and three CR AML patients during the chemotherapy. Figure Some previous studies reported a decrease in LSC signatures in chemo-residual AML cells , 14, 40.LGALS1, CD52, and PDLIM1 were observed in PSPs after chemotherapy. The principal component analysis (PCA) also showed that the gene expressions of PSPs were more similar to QSCs after chemotherapy than the initial diagnosis status in primary refractory patients  cells had significantly higher levels in viable CD45dimSSClow blasts of diagnosis resistant and refractory/early-relapsed patients than diagnostic patients who eventually achieved CR after chemotherapy  cells remarkably increased in chemo-residual resistant cells, compared with the diagnostic samples  cells at diagnosis were chemoresistant, and these cells were further enriched in the persisting residual AML cells.Notably, we found that the currently targetable gene lls Fig. , and theapy Fig. . This obapy Fig. . ResultsCD52 and SIGLEC10 showed specific co-expression in QSCs and mono cells  reduced significantly in the combined treatment group , Ara-C, or combined treatment  and lymphoma , 56, butIn summary, by performing scRNA-seq analysis, this study sheds new light on the cellular and molecular reprogramming induced by chemotherapy in primary refractory and short-term relapsed AML patients and identified the cell subpopulation and molecules involved in the drug resistance and poor outcome of AML. Our results will facilitate a better understanding of the AML chemoresistance mechanism and the development of novel therapeutic strategies for RR-AML patients.Bone marrow aspirates from AML patients were obtained from the First Affiliated Hospital of Nanjing Medical University . All cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, 4 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere of 95% air and 5% COFresh human bone marrow samples of AML patients were processed using density gradient centrifugation at 500 g for 10 min at 4 °C to isolate mononuclear cells. Cell pellets were resuspended in 5 ml RPMI-1640 with 5% FBS and 2-Mercaptoethanol, and filtered using a 40 mm nylon mesh (Thermo Fisher Scientific) with residual cell clumps discarded. We used DAPI (Sigma-Aldrich) for staining to identify live cells, and cell number was determined using a Countess II Automated Cell Counter whenever possible.In total, the cell viability of all samples is above 90%, 10,000 cells per sample were loaded into a Chromium Single-Cell 3′ Chip Kit v2 and processed using a Chromium single cell 3′ Reagent Kits (v2 and v3) (10× Genomics) according to the manufacturer’s instructions. Libraries were constructed using the Single 3′ Library and Gel Bead Kit v2 (PN-120237) and Chromium i7 Multiplex Kit v2 (PN-120236).6/ml) were seeded in 96-well plates and incubated with the indicated concentration of drugs. The absorbance was measured at 450 nm by spectrophotometry.Cell viability assays were performed with CCK-8 reagent . According to the manufacturer’s protocol, cells  with PMSF Protease Inhibitor (Thermo Fisher Scientific) added. Ten micrograms of purified proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% gradient gel (BioRad) and transferred onto NC membranes (EMD Millipore). Membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-Galectin-1 , mouse anti-GAPDH . Anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies  were used as secondary antibodies.2.Cells were mixed with an equal volume of methylcellulose colony assay medium  and then plated in 12-well plates and incubated for 2 weeks. Colonies were calculated with a diameter of more than 0.1 mm of incubation at 37 °C in 5% COFormalin-fixed bone marrow tissues from AML patients were processed through the routine IHC pipeline and stained for a rabbit anti-Galectin-1 . Multiplex IHC staining was performed with Akoya OpalTM seven-color fluorescent platform. Formalin-fixed, paraffin-embedded tissues were cut in 4-μm thick section and stained by Opal Polaris 7 Color Automation IHC Detection Kit  for simultaneous detection and quantitation of CD34 , CD14 , CD52 , SIGLEC10  and DAPI (Sigma-Aldrich). The slides were observed and imaged by Vectra Polaris automated quantitative pathology imaging system. The images were spectrally unmixed by Akoya phenoptics inForm software (inform 2.4.8).Suspended single cell was prepared from BM samples of AML patients. Red cells were lysed with 1× RBC Lysis Buffer  before staining. Cells were incubated with antibodies in Facs buffer (2% FBS in PBS) on ice for 20 min at dark. The following human-specific monoclonal antibodies were used: CD45-Pacific Blue , CD99-FITC , CD49d-PE , CD52-Percp-Cy5.5 . For intracellular staining, surface-marked cells were fixed for 20 min and then permeabilized using an IntraStain Kit  according to the manufacturer’s instructions after washing with 1× PBS  and centrifugation at 400×g for 5 min. Subsequently, the cells were stained with anti-Galectin-1  for 20 min and then washed once. Stained cells were analyzed on FACSAria .6 primary AML cells resuspended in 100 μL phosphate-buffered saline after being sublethally treated with busulfan (30 mg/kg/day) for 24 h. The mice were tail bled weekly to monitor the tumor burden of human cell engraftment by flow cytometry. For drug treatment, all mice were randomly divided into three groups of five mice each after 4 weeks of AML cell transplantation. Mice in the control group were treated with PBS daily for 6 days; mice in the chemotherapy group were intraperitoneally treated with Ara-C using a physiologically relevant dose and schedule (60 mg/kg/day × 6 days); mice in the co-treatment group were injected intraperitoneally with Ara-C (60 mg/kg/day) at the 1, 3, 5 days and with OTX008 (5 mg/kg/day) at the 2, 4, 6 days. Mice were sacrificed on the 9th day, bone marrow from femurs was harvested, crushed with mortar and pestle, and red blood cells lysed using ammonium chloride buffer after filtering. Cells retrieved from bone marrows were stained with mCD45-PeCy7 and hCD45-BV421 and analyzed by flow cytometry to detect the human graft (hCD45+mCD45− population). In addition, percentages of CD99+CD49d+CD52+Galectin-1+ (QSCs) cells in human AML cells (hCD45+) were measured by flow cytometry to evaluate the impact of different treatment groups.Animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. NCG (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) mice were purchased from the Nanjing Biomedical Research Institute of Nanjing University (NBRI). Briefly, NCG mice (6–8-week-old) were injected intravenously via tail vein with 2 × 10CellRanger was used to perform barcode processing and generate gene count profiles. Data integration, unsupervised clustering, visualization, and differential expression analysis were performed using Seurat . Cell-tySupplementary FiguresSupplementary TablesSupplementary Methods


As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration  One suce CaMKII , 14-3-3γe CaMKII , and thee CaMKII  among ote CaMKII . RGS14 ie CaMKII . Unusuale CaMKII , implicaThe hippocampus is the major focus of temporal lobe-induced seizures and is susceptible to cell damage and injury . Of notein vitro. Consistent with this function, we show that mice lacking RGS14 exhibit enhanced oxidative stress, elevated neuronal injury, and altered inflammation following KA-induced seizure in vivo. Together, these finding suggest that RGS14 serves a previously unappreciated protective role in hippocampal seizure pathologyGiven that RGS14 is protective to insult injury in peripheral tissues, and is highly expressed in injury-resistant hippocampal area CA2, we set out to determine whether loss of RGS14 (RGS14 KO) would impact seizure behavior, seizure-induced cell injury and exacerbate hippocampal pathology following KA-SE. Additionally, we sought to determine how RGS14 may be affording this protection. Here, we report that RGS14 protein is upregualated in hippocampus following KA-SE induction, and that loss of RGS14 accelerated the onset of behavioral seizures and mortality during seizure. Using proteomic approaches, we determined changes in the hippocampal proteome in RGS14 KO animals, and identified altered metabolic/mitochondrial protein expression in RGS14 KO mice after seizure activity. Further investigation identified RGS14 localization to mitochondria in area CA2, and we provide evidence that RGS14 suppresses mitochondrial oxygen consumption 2.2.1Male and female C57BL/6J wild-type (WT) and RGS14 null (RGS14 KO) mice ages 3–7 months were used in this study. Animals in all experiments were housed under a 12 h/12 h light/dark cycle with access to food and water ad libitum. All experimental procedures conformed to US NIH guidelines and were approved by the animal care and use committees (IACUC) of Emory University.2.2.Seizure-induction protocols using kainic acid (KA) were followed as described . Wild ty2.3.To collect hippocampal sections for histology, male and female mice were anesthetized with sodium pentobarbital  and euthanized by transcardial perfusion with 4% paraformaldehyde (PFA) in PBS one day after KA or saline injection. After decapitation, mouse brains were dissected and post-fixed in 4% PFA at 4°C for 24 hours. After post-fix, brains were transferred to a 30% sucrose in PBS solution for 72 hours. Mouse brains were embedded in optimal cutting temperature (OCT) medium , and coronal sections of hippocampus were collected at a thickness of 40 μm on a cryostat. Hippocampal sections were stored in 0.02% sodium azide in PBS at 4°C until they were evaluated for neuropathology. For proteomics and Western Immunoblot, mice were anesthetized with isoflurane followed by rapid decapitation and dissection of whole hippocampi. Whole hippocampi were snap frozen in liquid nitrogen and stored at −80°C until homogenization.2.4.2, 1X protease inhibitor  1X Halt phosphatase inhibitor ]. Cells were lysed in modified RIPA buffer for 1 hour at 4°C, end-over-end. Lysates were cleared, and proteins were denatured by boiling in Laemmli buffer for 5 minutes (stored at −20°C until immunoblotting). Samples were resolved on 4–20% Mini-PROTEAN® TGX™ precast protein gels , and gels were UV-activated for 45 seconds for total protein illumination (BioRad Stain-Free Technology)  . Samples2.5.™ 660 Nissl  were diluted in blocking solution, and sections were blocked from light and incubated in secondary antibody solution for 2 hours at room temperature. Sections were washed thrice with PBS and mounted onto SuperFrost Plus microscope slides (Thermo Fisher Scientific). Once dried, sections were coverslipped with DAPI Fluoromount G  and stored away from light at 4°C prior to imaging.To determine protein expression levels in tissue by immunohistochemistry (IHC), free floating hippocampal sections from KA- and saline-treated WT and RGS14 KO mice were subjected to antigen retrieval by boiling sections in 10 mM citrate buffer for 3 minutes and blocked in 5% normal goat serum in 0.1% Triton-PBS (blocking solution) for 1 hour at room temperature. Sections were incubated overnight at 4°C with primary antibody specific for RGS14 , IBA1 , 3-nitrotyrosine , or SOD2  diluted in blocking solution. Sections were washed thrice with PBS prior to incubation with the appropriate fluorescent secondary. Goat anti-mouse Alexa Fluor Plus 488 , goat anti-rabbit Alexa Fluor 546 , or NeuroTrace2.6.Fluorescent micrographs were collected from dorsal hippocampal sections using a Leica DM6000B epifluorescent upright microscope at 5X, 10X, and 20X magnifications using the appropriate wavelength filters. Images magnified at 20X were processed, analyzed, and quantified using ImageJ software. For RGS14 and SOD2, standardized regions of interests were drawn around each layer  of each CA field  and the dentate gyrus (DG). For 3-nitrotyrosine, a standardized ROI was drawn around each CA field incorporating the three layers . Quantification for 3-nitrotyrosine was performed this way because 3-nitrotyrosine accumulation was consistent throughout all layers, while immunofluorescence of RGS14 and SOD2 expression was noticeably different depending on the layer of analysis. For quantification of raw immunofluorescence, background correction and intensity thresholding (Otsu method) were performed consistently across all images for each ROI prior to quantifying raw immunofluorescence.2.7.2, circularity 0.05–1) were applied consistently across all images for each ROI using ImageJ software. Cell counts and area of IBA1 positive cells were quantified for each image. Microglia density is expressed as the number of cells divided by the area of ROI, and average size is expressed at the mean area of IBA1 positive cells.For the microglia marker ionized calcium binding adaptor molecule 1 (IBA1), a standardized region of interest was drawn for each CA subfield incorporating the stratum radiatum (SR), stratum pyramidale (SP), and stratum oriens (SO) and the DG incorporating the hilus, granule cell layer, and molecular layer. Background subtraction, intensity thresholding (Otsu method), and size and shape criteria for microglia  HALT(-EDTA) protease and phosphatase inhibitor cocktail (Pierce)) in a 1.5 mL Rino tube (Next Advance) harboring stainless steel beads (0.9–2 mm in diameter). Samples were homogenized twice for 5-minute intervals in the cold room (4 °C). Protein homogenates were transferred to 1.5 mL Eppendorf tubes on ice and were sonicated  thrice for 5 sec each with 5 sec intervals of rest at 30% amplitude to disrupt nucleic acids and were subsequently centrifuged at 4° C. Protein concentration was determined by the bicinchoninic acid (BCA) method, and samples were frozen in aliquots at −80 °C. Protein homogenates (100 μg) were treated with 1 mM dithiothreitol (DTT) at room temperature for 30 min, followed by 5 mM iodoacetimide at room temperature for 30 min in the dark. Protein samples were digested with 1:25 (w/w) lysyl endopeptidase (Wako) at room temperature overnight. Next day, samples were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and were further digested overnight with 1:25 (w/w) trypsin (Promega) at room temperature. The resulting peptides were desalted with HLB column (Waters) and were dried under vacuum.2.9.The data acquisition by LC-MS/MS was adapted from a published procedure . Derived2.10.Label-free quantification analysis was adapted from a published procedure . Spectra2.11.https://github.com/edammer/GOparallel). Briefly, this script performs one tailed Fisher’s exact tests (FET) enabled by functions of the R piano package for ontology enrichment analysis on gene sets downloaded from http://baderlab.org/GeneSets, which is maintained and updated monthly to pull in current gene sets from more than 10 different database sources including those mentioned above , Wikipathway, Reactome and molecular signatures database (MSigDB) term enrichment in our gene sets of mouse network module members and significantly differentially changed protein subsets was determined by gene set enrichment analysis (GSEA) using an in-house developed R script  and Seahorse CF Cell Mito Stress Test Kit  according to manufacturer’s instructions. Briefly, cells were seeded into a Seahorse XFe96 microplate  at 15,000 cells/well and incubated for 24 hr at 37°C, 5% CO2. FLAG-RGS14 or pcDNA3.1 (negative control) plasmids  and equilibrated in this medium for 30 minutes. Oxygen consumption rate (OCR) was measured prior to and after sequential treatment with 1 μM oligomycin , 0.5 μM carbonilcyanide p-trifluoromethoxyphenylhydrazone (FCCP) (ATP synthase inhibitor and proton uncoupler), and 0.5 μM rotenone/antimycin A (Complex I/III inhibitor). Basal respiration, mitochondrial ATP-linked respiration, maximal respiration, proton leak, spare respiratory capacity, and non-mitochondrial linked respiration were determined using the XF Wave 2.1 software. Cell lysates were collected in lysis buffer, and Pierce BCA assay was used to determine protein concentration. OCR values were normalized to HEK293T protein concentration in the same sample and were expressed as mean ± SEM. Western immunoblotting to confirm protein expression was performed as stated above using anti-FLAG HRP-conjugated antibody  to verify transfection and expression of FLAG-RGS14.The measurement of mitochondrial respiration was performed as previously described with modifications . Brieflyplasmids  were tra2.14.2O for 2 minutes each. Sections were incubated with 0.06% KMnO4 (dissolved in ddH2O) for 10 minutes and rinsed twice with ddH2O for 2 minutes each to remove all traces of KMnO4. Sections were incubated in a 0.0004% solution of Fluoro-Jade B for 30 minutes covered from light. Sections were then rinsed thrice with ddH2O for 1 minute each and air dried for 24 hours while covered from light. Sections were cleared with xylenes for 5 minutes and coverslipped with DPX  mounting medium. FJB labeled neurons were visualized and imaged using a Leica DM6000B epifluorescent upright microscope with filters for fluorescein or FITC at 10X and 20X magnifications. Representative images were collected from dorsal hippocampal CA fields. A standardized region of interest (ROI) was drawn around CA3 cell bodies in the stratum pyramidale (SP) from 20X magnified images. Fluoro-Jade B positive cells were quantified using DotDotGoose . Cell counts are represented as the total number of Fluoro-Jade B positive cells in each representative section for each animal.To assess for neurodegeneration one day after KA-SE, FluoroJade B (FJB)  was used to label and quantify degenerating neurons  accordin2.15.All statistical analysis was performed in GraphPad Prism (version 9.3.1). Data were subjected to Grubb’s outlier analysis prior to statistical comparisons. For behavioral seizure experiments, Gehan-Breslow-Wilcoxon test was used to determine differences between survival curves, and Fisher’s exact test was used to determine contingency of genotype on binned mortality and overall mortality. Multiple t-tests were used to compare mean behavioral seizure score at each time point during the 90 minute behavioral seizure period, and unpaired Student’s t-test was used to compare mean latency to Stage 3 seizure activity between genotypes. Two-way ANOVA with Sidak post-hoc comparison was used to assess effects of genotype and sex on latency to mortality and Stage 3 seizure activity. One-way ANOVA with Dunnett’s post-hoc comparisons was used to compare mean RGS14 expression (band density from immunoblot) in hippocampal lysates, and unpaired Student’s t-tests were used to compare mean RGS14 immunofluorescence between SAL and 1d KA treatments. Unpaired Students t-tests were used to compare OCR means between control and RGS14 overexpression groups, SOD2 mean immunofluorescence in CA1, and mean number of FJB+ neurons in CA3 between WT and RGS14 KO mice after KA-SE. For comparison 3-NT and SOD2 mean immunofluorescence and IBA1+ microglia cell density and mean area, two-way ANOVA with Tukey’s or Sidak’s post-hoc analysis was used.3.3.1.Recent evidence has supported a role for hippocampal area CA2 in modulating hippocampal network activity and seizure generation . BecauseAfter KA administration, we observed that all mice regardless of genotype either entered SE  or died at higher stage seizures (data not shown). Comparison of survival curves between WT and RGS14 KO mice demonstrate that RGS14 KO mice die significantly quicker than WT mice , and theInitial analysis of behavioral seizure scores demonstrated that WT and RGS14 KO mice progress similarly through behavioral seizure staging following KA . When la3.2.Several studies have demonstrated that RGS14 expression is significantly altered in animal and cellular models of cardiac hypertrophy and hepatic ischemic-reperfusion injury , and thastratum pyramidale, SP) and dendrites  of area CA2 PCs  was performed on dorsal hippocampal sections collected from WT mice one day after KA-SE or saline (SAL) –H. In saC was per CA2 PCs . Additio CA2 PCs . One day CA2 PCs , we dete CA2 PCs , where R CA2 PCs . Like CA CA2 PCs , particuThis demonstrates that RGS14 expression is robustly induced by KA-SE within hippocampal areas CA2 and CA1 , F, whic3.3.To better understand how RGS14 may be influencing seizure activity in the hippocampus and the effects of losing RGS14 induction following seizure activity, we performed a proteomics analysis on hippocampal tissue taken from WT and RGS14 KO mice 1 day after saline or KA-SE treatment (n=5 per genotype and treatment) . Whole hGene ontology (GO) analysis was performed on up- and downregulated DEPs in SAL-treated mice identify common cellular pathways and processes associated with the DEPs that may influence sensitivity to KA . GO termTo determine alterations in cellular processes caused by loss of RGS14 induction following seizure activity, we performed GO analysis 1 day after KA-SE. After KA treatment, significantly downregulated proteins in RGS14 KO hippocampi are associated with GO terms “Diol Biosynthetic Process”, “Diol Metabolic Process”, “Aldehyde Catabolic Processes”, “Oxidoreductase Activity on Ch-Ch Group”, and “Fatty Acid Metabolism” , left, sComplimentary to this analysis, we also determined DEPs altered after KA-SE (between treatment) in either WT or RGS14 KO hippocampi (within genotype) , B. We f3.4.Among the protein pathways most affected by loss of RGS14, those associated with mitochondrial function and oxidative stress were most surprising and of interest. To explore a possible role for RGS14 in mitochondrial function, we first tested whether RGS14 localizes to the mitochondria in area CA2 PCs. We used immunogold-labeled electron microscopy (EM) to visualize RGS14 in subcellular structures within area CA2 PCs of untreated WT mice . Immunog3.5.Superoxide and other reactive metabolites are generated by seizure activity, and this seizure-induced oxidative stress plays an important role in mediating seizure pathology . SOD2 isBecause RGS14 localizes to mitochondria in area CA2  and loss2−) and nitric oxide (NO). 3-NT is commonly used as a marker for oxidative stress and can greatly impact protein function and signaling  using IHC one day after saline or KA-SE. Tyrosine oxidizes to 3-NT when exposed to peroxynitrite, a highly reactive species that forms from superoxide  and potential subregional differences in microglial activation between WT and RGS14 KO hippocampi, we quantified the density of IBA1+ microglia  and area of IBA1+ microglia for each hippocampal subregion. In CA1 , we founIn contrast to area CA1 and CA2, we found that IBA1+ microglia were similarly activated between WT and RGS14 KO in area CA3 . In CA3,4.in vitro. Loss of RGS14 promotes the marked accumulation of 3-nitrotyrosine  and prevents seizure induction of SOD2  in CA2 PCs, suggesting exacerbated oxidative stress in the region. Mice lacking RGS14 exhibit increased neuronal injury in area CA3 PCs (but not CA2) while lacking microgliosis in areas CA1 and CA2 following KA-SE. Together, these results provide compelling evidence for the idea that RGS14 is a protective modulator of behavioral and pathological seizure response to KA in the hippocampus, likely by influencing the cellular excitability and metabolic profile of CA2 PCs.Our group and others have shown that RGS14 is integral for area CA2 pyramidal cell (PC) signaling and function . Based o4.1.Faster onset of SE and mortality in RGS14 KO suggests enhanced hippocampal excitability and/or propagation of seizure activity from the hippocampus . CA2 PCsAgainst this interpretation, chronic inhibition of CA2 results in CA3 hyperactivity, epileptiform discharges in CA3, and increased sensitivity to KA-induced behavioral seizures . In thisImportantly, the capacity of RGS14 to modulate CA2 activity may be clinically relevant as CA2 has been hypothesized to promote epileptogenesis and generate seizure activity in the absence of CA1 and CA3 , which a4.2.Proteomic analysis of RGS14 KO hippocampus , 4, S1–24.3.in vitro, which corroborates a previous finding that RGS14 KO increases mitochondrial respiration in brown adipose tissue   and NADPH oxidase (NOX), respectively, at the membrane  and utilize pharmacological inhibition to elucidate their source(s).Tyrosine nitrosylation (3-NT) (elevated in RGS14 KO CA2 ) is caus2−) , and sug2−) . This fi2−) , 4. By r2−) , RGS14 me stress . Additioe stress . Notably neurons , while Nmembrane . By limi2− detoxifying enzyme) in CA2 after KA-SE, which could explain, at least in part, the elevation of 3-NT caused by seizure activity. 3-NT impacts the structure and activity of proteins susceptible to tyrosine nitrosylation, especially those in the mitochondria which can have deleterious effects on its function  and their synthesis by COX-2 are mediators of microgliosis after seizure (Although lasting neuroinflammation is typically associated with worse seizure pathology and outcomes, several studies suggest early microgliosis following seizure is protective in the hippocampus . Loss of seizure , dysfunc seizure , and RGS seizure . Lack ofex vivo activation of microglia and astrocytes isolated from WT and RGS14 KO hippocampi.Although some microglial/macrophage populations express RGS14 mRNA , we have4.6.Overall, our findings suggest that RGS14 is neuroprotective in the hippocampus by dampening behavioral seizures and regulating CA2 PC function by engaging mitochondria oxidative stress pathways as illustrated in our proposed working model . ConsistSupplement 1


Thus, Ca2+ signaling also provides a target for virus invasion, replication, proliferation and release. After hijacking the host cell, viruses exploit Ca2+ signaling to regulate apoptosis and resist host immunity to establish persistent infection. In this review, we discuss cellular Ca2+ signaling and channels, interaction of calcium-associated proteins with viruses, and host cell fate, as well as the role of Ca2+ in cell death and antiviral response during viral infection.Calcium, as a second intracellular messenger, participate in various physiological and biochemical processes, including cell growth and proliferation, energy metabolism, information transfer, cell death, and immune response. Ca The mitochondria are Ca2+-buffering organelles that maintain Ca2+ homeostasis by absorbing and releasing Ca2+ to establish contacts with the cytoplasm and other organelles in response to various intracellular signals  viral proteins directly or indirectly disrupt intracellular Ca2+ homeostasis by modulating calcium channels and pumps or host membrane permeability; (2) Ca2+-regulated proteins or Ca2+-dependent pathways are involved in regulation of the virus life cycle; and (3) viral proteins bind directly to Ca2+ to hijack the host and destroy the integrity of cellular structure and function  signals resists exogenous pathogenic challenges and endogenous danger signals , as the largest intracellular Ca2+ storage organelle, is several hundred micromolar , receptor-operated calcium channels (ROCs), and store-operated calcium channels (SOCs)] mediate movement of Ca2+ across the plasma membrane and entry of Ca2+ from the extracellular medium, while the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) extrude Ca2+ from the cell ] involved in regulating cellular Ca2+ uptake to increase intracellular Ca2+, thereby facilitating viral entry and replication. For example, human immunodeficiency virus type 1 (HIV-1) gp120 and Tat protein, and porcine rotavirus (RV), activate VGCCs to increase the levels of intracellular Ca2+ , Human betaherpesvirus 5 (CMV), and flaviviruses  ] . Intracencoating . For porproteins . The strectivity .2+-mediated functional coupling also exists at the lysosome-ER interface , Betapolyomavirus macacae (SV40), Middle East respiratory syndrome coronavirus (MERS-CoV) and Zaire Ebola virus  to support viral RNA transcription and establish persistent infection  and viral RNA metabolism  nonstructural phosphoprotein NS2 is a dedicated Ca2+-binding protein that significantly enhances NS2 phosphorylation, triggering viral inclusion body formation and facilitating viral replication and assembly (2+ facilitates hepatitis B virus (HBV) core assembly, which is regulated by the viral multifunctional regulatory protein HBx  on the plasma membrane and stromal interaction molecule (STIM1) on the ER, which are stimulated by the depletion of internal Ca2+ stores. The budding process of enveloped viruses depends on the host Ca2+ signal mediated by SOCs (STIM1/ORAI1). Some viruses activate SOCs or depend on STIM1 and ORAI1 interaction to enhance cellular Ca2+ uptake for the budding of mature virus particles. Such viruses include DENV, EBOV, Marburgvirus (MARV) and Argentinian mammarenavirus  C, as an activator of the caspase family, is required for caspase-dependent apoptosis. It is released into the cytoplasm and binds to apoptotic protease activating factor (Apaf)-1 to form apoptosomes, triggering the caspase cascade via caspase-9 activation (2+ uptake in mitochondria and lead to reactive oxygen species (ROS) production and apoptosis , which mediates the regulation of caspase-independent apoptosis . AIF is 2+ homeostasis imbalance, mitochondrial damage, and release of various apoptotic macromolecules are often associated with virus-induced apoptosis. From the perspective of virus, it exploits host resources by destroying intracellular Ca2+ homeostasis and regulates apoptosis to achieve the purpose of promoting its own replication. Similarly, apoptosis is also regarded by the host as an antiviral method, and the virus has achieved the establishment of persistent infection by regulating the activity of Ca2+ channels to prevent apoptosis.In conclusion, Ca2+ uptake is thought to serve as a safety buffer, maintaining cellular Ca2+ homeostasis in the event of a temporary intracellular Ca2+ overload, and promoting mitochondrial oxidative phosphorylation and ATP synthesis  , and collaborative molecules such as cytosolic chaperone Grp75 are involved in regulation  , and thegulation . The molgulation . Apoptoschondria . For exand VDACs . Mitochotiporter   is activated and allows rapid reuptake of cytosolic Ca2+ by the ER , and mitochondrial matrix protein cyclophilin D (Cyp D). Cyp D is also considered to be the key to MPTP opening, and is blocked by cyclosporine A  Figure . In rece2+ channels in the ER and mitochondria. At the initiation stage of oxidative stress apoptosis, the transient reversible opening of MPTP increases and ROS accumulation causes damage to cells , including Toll-like receptors (TLRs), retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs), cytosolic DNA sensors, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), which cause the production of interferons (IFNs) and stimulate inflammatory cytokines after activation stimulated by microbial or cellular damage  3/7, initiating IFN-associated innate immune responses, and enhancing NF-κB-related inflammatory responses  have been demonstrated to participate in cytokine production, activation of immunocytes, inflammation, and antiviral innate responses by regulating Ca2+ signaling  channels to induce Ca2+ entry in endothelial cells, secondary to TLR4-induced diacylglycerol generation  through calcination, thereby promoting HIV replication -1, and interferon (IFN) regulatory factor (IRF) family members, to cause a further inflammatory reaction and IFN-dependent antiviral immune response . TLRs anignaling . In partneration . TLR3, Tlication . By continocytes . Extraceesponses . In gene2+ is a key regulator of the RLR pathway by regulating expression of many molecules involved in RLR signaling, or via regulation of Ca2+ channels. Studies have found that high levels of Ca2+ induce dsRNA sensors like MDA5 and RIG-I  increases expression of RIG-I and MDA5 , three of which are encoded in the human genome , and enhance IFN production and play an important role in RNA virus infections . RIG-I and RIG-I . In addiand MDA5 . Many re+ levels . Murine  channel . However channel . Pestivioduction . Notably2+ and related signaling proteins regulate cGAS–STING  to produce type I interferons (IFNs), suggesting that cytosolic DNA sensing is the important mechanism by which the innate immune system detects pathogens . SeveralAS–STING . cGAS deAS–STING . RecogniAS–STING . ActivatAS–STING .2+-binding site; when STING forms a homodimer, two ions are shared between the two monomers of the protein, so Ca2+ directly participates in the regulation of STING activation -mediated Ca2+ depletion and ionomycin-mediated Ca2+ elevation suppress STING-mediated IFN-β production. The function of STING is also restricted when virus-infected cells are treated with the IP3R inhibitor 2-APB or SERCA inhibitors 3 is one of the best-identified DNA sensors associated with inflammasomes in the NLR family, and is activated by invading pathogens or endogenous danger signals, leading to the formation of NLRP3 inflammasomes. The NLRP3 inflammasome is a multiprotein platform that includes NLRP3, apoptosis-associated speck-like protein (ASC) and pro-caspase-1, which leads to caspase-1-dependent secretion of proinflammatory cytokines IL-1β and IL-18 . The rolammasome . For exaammasome ; The relammasome ; Emanateammasome . Until ntivation . Similartivation . What’s tion , the National Natural Science Foundation of China (grant no. 32030108), and the Foundation of Key Laboratory of Veterinary Biotechnology (no. shklab202001).The authors declare this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.