Alternaria alternata is one of the important diseases of pear fruit during storage. Isothiocyanates are known as being strong antifungal compounds in vitro against different fungi. The aim of this study was to assess the antifungal effects of the volatile compound 2-phenylethyl isothiocyanate (2-PEITC) against A. alternata in vitro and in pear fruit, and to explore the underlying inhibitory mechanisms. The in vitro results showed that 2-PEITC significantly inhibited spore germination and mycelial growth of A. alternata—the inhibitory effects showed a dose-dependent pattern and the minimum inhibitory concentration (MIC) was 1.22 mM. The development of black spot rot on the pear fruit inoculated with A. alternata was also significantly decreased by 2-PEITC fumigation. At 1.22 mM concentration, the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation. Further results of the leakage of electrolyte, increase of intracellular OD260, and propidium iodide (PI) staining proved that 2-PEITC broke cell membrane permeability of A. alternata. Moreover, 2-PEITC treatment significantly decreased alternariol (AOH), alternariolmonomethyl ether (AME), altenuene (ALT), and tentoxin (TEN) contents of A. alternata. Taken together, these data suggest that the mechanisms underlying the antifungal effect of 2-PEITC against A. alternata might be via reduction in toxin content and breakdown of cell membrane integrity.Black spot caused by  Alternaria alternata is a filamentous fungus [Alternaria brown spot of citrus [Hylocereus undatus [Alternaria rot of netted melon [A. alternata, the causal agent of black spot in pear, initially infects the fruit via styles or peel during the growing season and remains latent until fruit maturity [Alternaria species produce mycotoxins during growth that pose potential threat to animal and human health [A. alternata [s fungus  that caus fungus , moldy cs fungus , Alternaf citrus , black r undatus , and Alted melon . A. altematurity , which rmaturity . Most Aln health . These mn health ,11. Therlternata ; howeverlternata ,14. TherBrassica such as cauliflower, broccoli, cabbage, canola, kale, turnip, radish, and various mustards produce a good deal of potentially antimicrobial compounds called glucosinolates (GSLs) [Monilinia laxa of nectarine and peach [Botrytis cinerea of strawberry [Aspergillus parasiticus of pizza crust [Aspergillus ochraceus of peanuts, green coffee, grapes, and soybeans [Phymatotrichopsis omnivora of cotton [A. alternata of tomato [Penicillium expansum of pears [Brassica species. Studies have shown that the antimicrobial activity of 2-PEITC against soil microorganisms [A. alternata both in vitro and in pear fruit, as well as to further ascertain the antifungal mechanisms of 2-PEITC by analyzing membrane integrity and toxin production.Vegetables of the genus s (GSLs) ,16. Isots (GSLs) . They mas (GSLs) . Studiess (GSLs) ,20 and cs (GSLs) . Furthers (GSLs) , nematods (GSLs) ,24, bacts (GSLs) ,26, inses (GSLs) , and wees (GSLs) . Isothios (GSLs) . Studiesnd peach , Botrytirawberry , and Aspza crust ; BITC agsoybeans , Phymatof cotton , and A. f tomato ; and 2-pof pears . Howeverrganisms , plant prganisms , and cliA. alternata was significantly inhibited after 2-PEITC fumigation, and a positive correlation  was observed between the concentration of 2-PEITC and the inhibitory effect  of 2-PEITC was 1.22 mM.2-PEITC effectively inhibited the mycelial growth of t manner B,C; 2-PEA. alternata in pear fruit. The disease severity, indicated by the lesion diameter of black spot, was effectively reduced by 2-PEITC in a dose-dependent manner. The lesion diameter was reduced after 2-PEITC application at a concentration of 0.61 mM. At 1.22 mM (MIC), the lesion diameter was only 39% of that in the control fruit at 7 days after inoculation , which has the ability to permeate broken cell membrane and emit red fluorescence, was used to test plasma membrane integrity in A. alternata. After 120 min of incubation, electric conductivity of 0.5 × MIC, MIC, and 2 × MIC 2-PEITC-treated A. alternata cells was increased by 10%, 33%, and 37% compared with the control, respectively , AOH, AME, and ALT were extracted and detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-TOF-ESI-MS) in the mycelium of Brassica, are promising alternatives to synthetic chemical fungicides. Evidence has shown that different isothiocyanates possess strong antifungal activity against B. cinerea, Rhizopus stolonifer, M. laxa, Mucor piriformis, and Penicillium expansum [−1 inhibited M. laxa, whereas 0.93 g L−1 of the same compound was needed to inhibit P. expansum; p-hydroxybenzyl-isothiocyanate inhibited M. laxa and P. expansum at 0.09 g L−1 concentration; however, 1.8 g L−1 was needed to inhibit R. stolonifer. In addition, 2-PEITC inhibited mycelial growth of Sclerotinia sclerotiorum in a dose-dependent manner with 100% inhibition at a concentration of 0.67 mM [A. alternata 2 days after treatment . Membrane permeability of Penicillium cyclopium increased with increasing concentrations of E-2-hexena, evidenced by cell constituent release and leakage of potassium ions [A. alternata cells and fluorescence value in the fungal suspensions visibly increased with 2-PEITC treatment  in fruit and vegetables [Aspergillus parasiticus growth and aflatoxin (AF) production. However, some studies found that volatile compounds only reduced the amount of mycotoxins. The effects of BITC on Aspergillus ochraceus morphology were related to a decrease in ochratoxin (OT) production, both ochratoxin A (OTA) and ochratoxin B (OTB) [A. alternata were degraded after cinnamaldehyde treatment (0.200 mL L−1) and incubation for 120 min. However, the underlying mechanism 2-PEITC employed to modulate toxin synthesis in A. alternata needs to be further explored.Mycotoxins are common virulence factors in the host infected by plant pathogens . Alternagetables ,51. Somegetables  reported B (OTB) . In the  B (OTB) , who repS. sclerotiorum [B. cinerea [P. expansumon [Although results from the present study and several previous studies showed that isothiocyanates including 2-PEITC derived from plants are biofumigant fungicides against postharvest pathogens such as rotiorum , B. cine cinerea , and P. pansumon  through pansumon  and isotpansumon ,54,55 haA. alternata in pear fruit, and the MIC was 1.22 mmol/L after in vitro experiment. Moreover, 2-PEITC disrupted the permeability and integrity of the fungal membrane that led to leakage of cytoplasmic contents and finally cell death. In addition, 2-PEITC treatment significantly decreased the content of mycotoxins, including TEN, AOH, AME, and ALT, in A. alternata. These results suggest that 2-PEITC could be an alternative fungicide for the control of black spot in pear fruit caused by A. alternata.In conclusion, this work elucidated 2-PEITC as an effective antifungal compound to control postharvest black spot disease caused by Pyrus bretschneideri Rehd) were commercially picked from the Tiaoshan Farm in Jingtai County, Gansu Province, China. Pears of uniform size, with no mechanical damage, no pests, and diseases were selected, and the fruit were individually packaged in a plastic mesh bag, transported the laboratory, and stored at 4 °C. A total of 28 pears were required for this experiment.“Zaosu” pear  and stored at −20 °C for HPLC analysis. 2-PEITC was purchased from Shanghai Macklin Biochemical Co., Ltd. .A. alternata was measured according to a previously reported method [5 CFU mL−1) was dripped onto the center of each Petri dish mounted with potato dextrose agar (PDA) medium. The amount of 2-PEITC was calculated according to the space of the Petri dish (0.0635 mm3), and a sterilized filter paper disc (20 mm diameter) was attached on the center of the lid inner surface with different concentrations of 2-PEITC  added onto the paper, and then the plate was quickly covered. Filter paper without 2-PEITC was used as the control. After the plates were incubated at 28 ± 1°C for 2, 4, and 6 days, the diameter (cm) of the colony area was determined. Each treatment comprised three replicates. The lowest concentration in which no visual hyphal growth was observed after 2 days of incubation was the minimum inhibitory concentration (MIC).Mycelial growth of d method . In shorA. alternata, the spore suspension (1 × 105 CFU mL−1) was dripped onto the slide and placed in a large Petri dish with wet filter paper (humidity = 95%). The amount of 2-PEITC was calculated according to the space of the Petri dish (0.1905 mm3), and a sterilized filter paper disc (20 mm diameter) was attached on the center of the lid inner surface with different concentrations of 2-PEITC  added onto the paper, and then the plate was quickly covered. Treatment without adding 2-PEITC was the control. Spore germination rate was observed and calculated after 2, 4, 6, and 8 h of incubation. Each treatment comprised three replicates.To evaluate the effects of 2-PEITC fumigation on spore germination of 6 CFU mL−1. After 2 h incubation, the inoculated fruit were fumigated in plastic boxes (lid was drilled with air holes). The amount of 2-PEITC was calculated according to the space of the plastic boxes (200 × 130 × 50 mm), a sterilized filter paper disc (30 mm diameter) was attached on the center of the lid inner surface with different amount of 2-PEITC  added onto the paper, and then the plate was quickly covered. Treatment without adding 2-PEITC was the control. Each treatment contained nine fruit. Fruit were fumigated in plastic boxes with humidity (55%) at room temperature (23 ± 1 °C) for 1 day and later taken out and stored in a paper carton (humidity = 55%) at room temperature. Disease severity was determined 3, 5, and 7 days after the treatment by measuring the lesion diameter.The in vivo assay was carried out according to a previously described method with minor modifications . Zaosu pA. alternata cells exposed to 0.5 × MIC, MIC, and 2 × MIC 2-PEITC was a slightly modified version of a previously described method [6 CFU mL−1), incubated in a moist chamber at 28 ± 1 °C for 30 min, centrifuged at 8000 rpm for 5 min, washed three times with phosphate-buffered saline , and stained with propidium iodide (PI) for 5 min at 30 °C. After centrifugation, fungal suspension was washed three times with PBS solution  to remove the residual dye, and was observed under a fluorescence microscope  at an excitation wavelength of 620 nm.Membrane integrity of d method . InitialA. alternata cells was measured according to a previously described method [6 CFU mL−1) and incubated for 0, 30, 60, and 120 min. Subsequently, the electric conductivity was evaluated in a conductivity meter .Electrolyte leakage of d method  with minA. alternata culture grown for 4 days in 0.5 g PDA was collected and resuspended in 15 mL PBS (pH 7.0). The suspension was treated with 2-PEITC at different concentrations  and incubated at 28 ± 1 °C for 0, 30, 60, and 120 min. Subsequently, the suspension was centrifuged at 12,000 rpm for 2 min and the supernatant was collected. Absorbance of the supernatant (1 mL) was evaluated at 260 nm with UV-2450 UV/VIS Spectrophotometer (Shimadzu). Each treatment comprised three replicates. The control group was calibrated with PBS (pH = 7.0).Nucleic acid content of the supernatant was measured following the method of Paul et al.  with somA. alternata mycelium liquid nitrogen grinding, subsequently the hyphae were transferred to 10mL sterile centrifuge tubes, Subsequently, 2.5 mL of acetonitrile and water extract (4:1 v/v) containing 0.3% formic acid was added. The solution was vortexed, and was then mixed with 2.5 r s−1 extraction at room temperature for 30 min. Then, 0.25 g anhydrous MgSO4 and 0.04 g NaCl were added, and the mixture was shaken vigorously for 1 min, followed by centrifugation at 180 r s−1 for 10 min at 4 °C. Finally, the supernatant was taken through the 0.22 micron organic filter, quasi-determined to 1.2 mL, and the sample was prepared for HPLC analysis [For mycotoxin extraction, the fungi were cultured in PDA at 28 °C for 4 days and fumigated with 2-PEITC , followed by accurately weighed 0.5 g of analysis .−1; total running time, 7.1 min.Separation and qualitative analysis of TEN, AOH, AME, and ALT were performed using a mass spectrometer  equipped with the electrospray ionization (ESI) source. HPLC conditions were as a follows: column, C18 ; column temperature, 35 °C; injection volume, 5 μL; mobile phase A was deionized water, mobile phase B was methanol; gradient elution conditions were A after 70% retention for 1 min, after falling to 50% within 2 min, continued to drop to 10% within 1 min, kept for 2 min, rose to 90% within 0.1 min, kept for 2 min; tassel 0.005 mL s+); mass spectrometry scanning mode, multiple reaction monitoring (MRM); sheath gas temperature, 350 °C; nebulizer pressure, 35 psi; sheath gas flow, 0.183 L s−1; capillary voltage, 4000 V; other parameters were adjusted to optimum by the instrument. The mass spectrometric parameters such as monitoring ion, cone voltage, and collision voltage of four Alternaria species are shown in Following the mass spectrometry conditions: ion source mode, positive ion mode .


It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.The glucosinolate–myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca 2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels or light-induced stomatal opening.Allyl isothiocyanate (AITC) induces Ca The temperature and relative humidity in the growth chamber were 22±2 °C and 60±10%, respectively. Twice or three times a week, 0.1% Hyponex solution  as a fertilizer was provided to the plants. All the plants used for experiments were from 4 to 6 weeks old.Arabidopsis wild-type plants (Columbia) were grown in pots containing a mixture of 70% (v/v) vermiculite  and 30% (v/v) Kureha soil  in a growth chamber  with their adaxial surface upward in the dark for 2 h to close the stomata. After adding AITC, the leaves were kept in the light (80 μmol m−2 s−1) for 2 h before measurement. For assays of stomatal closure, leaves were floated on the assay solution in the light for 2 h to open the stomata. Then AITC was added, and the leaves were kept in the light for 2 h before measurement. Inhibitors were added 30 min before AITC treatment. For measurement of stomatal apertures, the leaves were shredded for 30 s, and epidermal tissues were collected using nylon mesh. Thirty stomatal apertures were measured for each sample.Fully expanded young leaves from 4- to 5-week-old plants were excised for stomatal aperture measurements as described previously  in the light for 2 h at 22 °C to promote stomatal opening. Turgid guard cells were used to measure [Ca2+]cyt. The observation chamber was perfused with the solution using a peristaltic pump. Guard cells were incubated in the absence of AITC for 5 min and then in the presence of AITC. Inhibitors were added at the time point indicated. For dual-emission ratio imaging of YC3.6, we used an FF-02-438/24-25 (Semrock) excitation filter, a 445DRLP (Omega) dichroic mirror, an XF3075 480AF30 (Omega) emission filter for cyan fluorescent protein (CFP), and an XF3011 535DF25 (Omega) emission filter for yellow fluorescent protein (YFP). The CFP and YFP fluorescence intensity of guard cells were imaged and analysed using the W-View system and AQUA COSMOS software (Hamamatsu Photonics). CFP and YFP fluorescence were simultaneously monitored.Four- to six-week-old wild-type plants expressing YC3.6 were used for the measurement of cyt was assessed by χ 2 test. Differences were considered significant for P<0.05.The significance of differences between datasets was assessed by Student’s To investigate AITC-induced stomatal closure, intact leaves were initially incubated in the light to open the stomata followed by AITC treatment in the light. For investigation of AITC inhibition of light-induced stomatal opening, leaves were initially incubated in the dark to close the stomata followed by AITC treatment in the light. AITC induced stomatal closure in a dose-dependent manner , which i2+]cyt elevation. However, it remains unknown whether the [Ca2+]cyt elevation is essential for AITC-induced stomatal closure. Application of a Ca2+ channel blocker, La3+, and a Ca2+ chelator, EGTA, inhibited AITC-induced stomatal closure in a dose-dependent manner  and ruthenium red (RR) , was investigated. Treatment of 50 μM AITC significantly inhibited FC-induced stomatal opening in the dark (+-ATPases by increasing the phosphorylation level of the penultimate Thr (penThr) of H+-ATPases , yeast elicitor, and amino acids  family members, glutamate receptor homologs, and cyclic nucleotide-gated channels are ICa channels activated by different stimuli (Activation of Ird cells . On the on cells . Studies2+ influx but also Ca2+ release from intracellular stores is essential for [Ca2+]cyt elevations induced by ABA, methyl jasmonate and flg22 (2+ release pathway involving cADPR, which is sensitive to NA (2+]cyt elevations, suggesting that the NA-sensitive Ca2+ release mechanism is conserved among different signaling pathways.Previous studies have shown not only Cand flg22 . ABA and+-ATPase. Ca2+ positively regulates S-type anion channels but negatively regulates H+-ATPase through Ca2+ sensor-dependent pathways (2+ is essential for stomatal closure induced by many stimuli such as ABA, MeJA, and yeast elicitor (2+]cyt elevation but not inhibition of light-induced stomatal opening (2+]cyt by three inhibitors impaired AITC-induced stomatal closure but not inhibition of light-induced stomatal opening (2+]cyt elevation is essential for AITC-induced stomatal closure but not inhibition of light-induced opening. Taken together, these results suggest that signaling leading to stomatal closure does not overlap with signaling leading to inhibition of light-induced stomatal opening in different stress responses.The driving force for stomatal closure is membrane depolarization induced by activation of S-type anion channels, while the driving force for stomatal opening is membrane hyperpolarization induced by activation of H opening , 3, indi+in channels suppresses stomatal opening driven by activation of H+-ATPase (+in channels in the absence of [Ca2+]cyt elevation (+in channels contributes to AITC inhibition of light-induced stomatal opening.Inhibition of Klevation . Furtherlevation , 8. Thes+in channels in guard cells and target KAT1, a main K+in channel in guard cells, expressed in a heterologous system using Xenopus oocytes (+in channels through a microtubule-dependent pathway.As an electrophile, AITC is prone to forming covalent adducts with amino acid residues, such as Cys, Lys, and His, to modify protein function. It is known that the covalent modification of Cys is reversible under physiological conditions ( oocytes . However oocytes , suggest2+ influx and Ca2+ release to induce [Ca2+]cyt elevation, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels or light-induced stomatal opening.The presented results suggest that AITC triggers CaJXB online.Supplementary data are available at 3+, VERA, and RR on AITC-induced elevation of [Ca2+]cyt in guard cells.Fig. S1. Effects of Gderaa073_suppl_Supplementary_Figure_S1Click here for additional data file.


Trypanosoma cruzi. In the search for new drugs to treat Chagas disease, potential therapeutic targets have been identified by exploiting the differences between the mechanisms involved in intracellular Ca2+ homeostasis, both in humans and in trypanosomatids. In the trypanosomatid, intracellular Ca2+ regulation requires the concerted action of three intracellular organelles, the endoplasmic reticulum, the single unique mitochondrion, and the acidocalcisomes. The single unique mitochondrion and the acidocalcisomes also play central roles in parasite bioenergetics. At the parasite plasma membrane, a Ca2+-−ATPase (PMCA) with significant differences from its human counterpart is responsible for Ca2+ extrusion; a distinctive sphingosine-activated Ca2+ channel controls Ca2+ entrance to the parasite interior. Several potential anti-trypansosomatid drugs have been demonstrated to modulate one or more of these mechanisms for Ca2+ regulation. The antiarrhythmic agent amiodarone and its derivatives have been shown to exert trypanocidal effects through the disruption of parasite Ca2+ homeostasis. Similarly, the amiodarone-derivative dronedarone disrupts Ca2+ homeostasis in T. cruzi epimastigotes, collapsing the mitochondrial membrane potential (ΔΨm), and inducing a large increase in the intracellular Ca2+ concentration ([Ca2+]i) from this organelle and from the acidocalcisomes in the parasite cytoplasm. The same general mechanism has been demonstrated for SQ109, a new anti-tuberculosis drug with potent trypanocidal effect. Miltefosine similarly induces a large increase in the [Ca2+]i acting on the sphingosine-activated Ca2+ channel, the mitochondrion and acidocalcisomes. These examples, in conjunction with other evidence we review herein, strongly support targeting Ca2+ homeostasis as a strategy against Chagas disease.There is no effective cure for Chagas disease, which is caused by infection with the arthropod-borne parasite,  Trypanosoma cruzi. The strategic rationale for current investigative efforts has been directed by consideration of the biological differences between the parasite and the host  cells. The basic premise of this approach recognizes that Ca2+ is an essential signaling messenger in all eukaryotic cells studied so far, including trypanosomatids, and that fluctuation of the intracellular Ca2+ concentration ([Ca2+]i) is finely regulated, by diverse mechanisms at the plasma membrane level and by intracellular organelles. It has been extensively demonstrated that the various mechanisms responsible for regulation of [Ca2+]i in trypanosomatids differ in many important features, from those in the host counterpart. Disruption of intracellular Ca2+ homeostasis by any means is lethal for all mammalian cells, since this is a driver to apoptotic processes or to necrosis , an important alternative energy coin in T. cruzi, as we will discuss below. For this reason, early in evolution cells were forced to develop sophisticated mechanisms to maintain a very low concentration of Ca2+ in the cytoplasm . Accordingly, Ca2+ has largely been compartmentalized in intracellular organelles, in which its concentration is very similar to the millimolar range encountered outside the cell, i.e., 4 orders of magnitude higher than in the cytoplasm. The extreme difference between the concentration of Ca2+ in the cytoplasm and in the exterior milieu, is even more remarkable when considered in the context of ionic distributions predicted for other cations by the Nernst equation. The difference between the intracellular and the extracellular concentrations calculated is far larger for Ca2+ than for any other ion normally present inside the cells . Maintaining the difference is associated with a high energy cost. However, throughout evolution, cells have taken advantage of this large Ca2+ electrochemical gradient to use it as an essential signaling messenger. The role of Ca2+ in cell signaling has been widely recognized in all eukaryotic cells so far studied including T. cruzi  has been shown to modulate proliferation in T. cruzi  to the host cell cultures, reducing the infective capacity of T. cruzi to 72 %  halting the ability of the parasites to enter the cells  by reducing the fusogenic properties of the membrane  in its acidocalcisomes involved in the Ca2+ signaling pathways. It has been shown that the presence of this receptor is necessary for the growth and establishment of infection of these organisms  p41 to the cytoskeleton, as well as for successful assembly of dimers and microtubular assembly. Even though p41 is normally found bound to cytoskeleton and preassembled microtubules of trypanosome tubulins, it remains tightly bound when calcium ions are present  release in T. brucei  in humans  that utilizes the electrochemical H+ gradient across the internal mitochondrial membrane , have been studied by the use of the CRISPR/Cas9 system, demonstrating that albeit their overexpression does not significantly affect mitochondrial Ca2+ uptake, their ablation has a large effect on the cation uptake by the mitochondria supporting their role in the stabilization of the MCU complex . By the use of the CRISPR/Cas9 system it has been shown that overexpression of these genes drives an increase of mitochondrial Ca2+ uptake. Conversely, knockout of any of these genes leads to a loss of Ca2+ uptake, although the mitochondrial membrane potential is maintained  that is sensitive to physiological Ca2+ concentration and is able to stimulate the activity of a mitochondrial pyruvate dehydrogenase, stimulating energy metabolism through the Krebs cycle activation, has provided further insight into the role of Ca2+ in T. cruzi bioenergetics plasmic reticulum Ca2+-ATPase (SERCA) that accumulates large amounts of Ca2+ by transporting the cation at the expense of ATP hydrolysis, and has high Ca2+ affinity  (The endoplasmic reticulum possesses a Cand 2 mM) .T. cruzi, a Ca2+-ATPase (TcSCA) that localizes at the endoplasmic reticulum (ER), has been partially characterized  and the Ryanodine receptor , that allow the Ca2+ release to the cytoplasm when they are opened by a particular signal. The activity of IP3R is modulated by IP3, the product of the hydrolysis of PIP2 by several isoforms of PLC . These organelles possess several different transporters, pumps and exchangers, for the accumulation of Ca2+ and other cations, such as a Vacuolar type H+ -ATPase, a Ca2+-ATPase similar to that found at the plasma membrane (PMCA), as well as other distinct Ca2+ transport mechanisms  that allows the accumulation of H+, and hence the acidification of this organelles. The name of the organelle is derived from this capability, together with its capacity for Ca2+ accumulation , polyphosphates and particularly pyrophosphate (PPi), which is particularly important for T. cruzi, and other trypanosomatids and apicomplexan parasites .The activity of the SERCA in mammalian cells is inhibited by tapsigargin and ciclopiazonic acid, and also by the sphingolipid sphingosine  has been cloned, expressed, and associated with proliferation, differentiation virulence, and infectivity , which has been identified in all eukaryotic cells so far described, including T. cruzi , the also ubiquitous Ca2+-binding protein, present in all eukaryotic cells  has not been characterized fully, recent studies on Trypanosoma equiperdum (a T. brucei-related hemoflagellate parasite that causes infection in cattle), have demonstrated that the CaM-binding domain of the PMCA of T. equiperdum possesses a non-canonical sequence  CaM binding motif , can still form an α-helix predicted to be able to bind CaM .The endoplasmic reticulum, the mitochondrion and acidocalcisomes act in concert whenever an elevation of the i, and is, at the same time, paradoxically enhanced by absence of external calcium  amazonensis in humans  a systemic antifungal once thought to exert its anti-parasitic effect through disruption of CaT. cruzi, this drug inhibits the biosynthesis of phosphatidylcholine 10–20 times more potently when compared to mammalian cells , and to induce a rapid alkalinization of the parasites acidocalcisomes through direct action  have also shown promising therapeutic applications in trypanosomatid infections  have proved effective for inhibiting 2+ homeostasis of these parasites is depicted in An abbreviate list of the most important functional targets of the main drugs affecting intracellular Ca2+ homeostasis and intracellular Ca2+ storage compartments as strategic targets for treatment of trypanosomatids infections in humans.The identification of new intracellular calcium-linked anti-parasitic targets is rapidly expanding the set of potential therapeutic options against trypanosmatid infections. Because target-based toxicity and side effects may arise due to cross reactivity with human homologues caution is advised when looking at structural differences between species while preferably choosing exquisitely selective anti-parasitic inhibitors. Many of these compounds are already a relevant part of the current clinical arsenal to treat Chagas disease and are likely to remain so for the foreseeable future; examples include the antiarrhythmic agent amiodarone and emerging benzofuran derivatives, as well as the calcium channel blockers. Others, such as the azole derivatives  and the new antituberculosis drug SQ109, are gaining relevance as they are repurposed as anti-parasitic drugs. An increasing body of experimental evidence supports the disruption of parasite CaGB conceive and wrote the review. AP-M, ES, and NM-S participate directly in the writing of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, we identified novel FLT3 inhibitors and investigated 5-(4-fluorophenyl)-N-phenyloxazol-2-amine  as candidates for the treatment of AML. The results showed that 7c inhibited the activities of FLT3 and mutated FLT3 in a cell-free kinase assay and Molm-13 and MV4-11 cells, as well as the proliferation of FLT3-ITD+ AML cells, increasing apoptosis. The anti-leukemic activity of 7c was confirmed by in vivo tumor growth inhibition in MV4-11 xenograft mice. Besides, 7c suppressed the expression of DNA damage repair genes. Combination treatment with 7c and olaparib (a poly (ADP-ribose) polymerase [PARP] inhibitor) synergistically inhibited cell proliferation in Molm-13 and MV4-11 cells. Our findings demonstrated that 7c is a therapeutic candidate targeting FLT3 for AML treatment and suggested that combination treatment with 7c and a PARP inhibitor may be an effective therapy regimen for FLT3-mutated AML.Internal tandem duplication (ITD) of FMS-like tyrosine kinase 3 (FLT3) is the most common mutation in patients with acute myeloid leukemia (AML). FLT3-ITD Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by rapid clonal proliferation or poor differentiation of myeloid lineage cells in the hematopoietic system. The incidence of AML increases with age and occurs mainly in adults, accounting for nearly 80% of AML cases . The staFLT3), a well-characterized oncogenic mutation; it occurs in approximately 30% of patients. Approximately 25% of patients with AML have an internal tandem duplication (ITD), whereas 5–10% of patients with AML have point mutations in the tyrosine kinase domain (TKD), typically at codon D835 [Genomic studies on AML have identified several recurrently mutated genes that enable its classification and diagnosis using predictive biomarkers to help develop anti-leukemic agents. One of the most frequently mutated genes in AML is the FMS-like tyrosine kinase 3 receptor gene  approved FLT3-targeting therapeutic agents, allowing targeted therapy for FLT3-mutated AML. First-generation FLT3 inhibitors, such as sunitinib, midostaurin, lestaurtinib, sorafenib, ponatinib, and tandutinib, are multi-kinase inhibitors approved by the FDA in 2017 for the treatment of FLT3-mutated AML. Inhibition of multiple RTKs induces AML cell death via the inhibition of FLT3; however, these inhibitors are cytotoxic to healthy cells owing to unclear target specificity. Second-generation FLT3 inhibitors, such as KW-2449, crenolanib, gilteritinib, and quizartinib, which mainly target FLT3 mutants, have relatively low toxicity . Of thes+ AML cells generate high levels of reactive oxygen species (ROS) that induce DNA damage, mutations, double-strand DNA breaks (DSB), and chromosomal instability, but they can survive owing to enhanced DNA repair activities [Previous studies suggest that FLT3-ITDtivities ,10,11,12tivities ,14. Ther+ AML cells. Our study will provide additional data for the use of FLT3 inhibitors in AML treatment protocols.In this study, to identify tyrosine kinase inhibitors targeting FLT3, we designed and synthesized 11 oxazol-2-amine-based small-molecule compounds. We aimed to investigate the anti-leukemic effect of the novel compounds and the combination therapeutic effect of the novel FLT3 compound with a PARP1 inhibitor in FLT3-ITD10c, 11c, 5c, 2c, 1c, 6c, and 4c showed the highest FLT3 inhibitory activity at 100 nM. Of the eight compounds that showed the inhibitory kinase activity against FLT3-ITD and FLT3-D835Y in vitro, five compounds  inhibited the growth of Molm-13 and MV4-11 cells at 100 nM, but not that of AML HL-60 (FLT3-null) cells at 10,000 nM (7c (5-(4-fluorophenyl)-N-phenyloxazol-2-amine) was the most effective in inhibiting the growth of Molm-13 and MV4-11 cells and was further examined as a novel putative FLT3 inhibitor  [7c showed a pi–alkyl interaction with Val675, Lys644, Cys828, Leu818, Val624, Ala642, and Leu616, as well as a pi–pi T-shaped interaction with Phe830  . The aroh Phe830 b. The reectively . We furtll lines b. In viv control c. Besidel groups d. Overal7c inhibited FLT3 signaling in FLT3-ITD+ Molm-13 and MV4-11 cells in vitro. Consistent with the results of the in vitro kinase assay, phosphorylated FLT3 was inhibited by 7c treatment in a concentration-dependent manner in both cell lines. Besides, we also observed that 7c inhibited FLT3-downstream effector STAT5 phosphorylation and total STAT5 protein in Molm-13 and MV4-11 cells  and NHEJ  genes in both cell lines  in combination therapy. Olaparib is the first PARP inhibitor targeting mainly PARP 1 and 2 approved by the FDA to treat advanced ovarian cancer patients with germline BRCA mutations and is widely studied in combination with DNA damage-induced drugs or radiotherapy. Our results showed that 7c and olaparib exerted synergistic growth inhibition in Molm-13 and MV4-11 cells  and used without any correction. Proton nuclear magnetic resonance (NMR) spectra were recorded using the Avance-500 spectrometer  at 500 MHz. Chemical shifts are reported in ppm, and Me4Si was used as the reference standard. Mass spectra were recorded using JMS-600W VG Trio-2 GC-MS . Reaction products were purified by flash column chromatography using silica gel 60 (230–400 mesh) and monitored by thin-layer chromatography with precoated silica gel 60 F254 . Spots were visualized under UV light (254 nm) after staining with PMA or Hanessian solution.5-(4-Fluorophenyl)-N-phenyloxazol-2-amine (7c). Pale yellow solid, mp = 160–162 °C, 1H NMR  δ 10.31 , 7.57–7.68 , 7.26–7.35 , 6.89–7.03 ; MS (FAB) m/z 254 (M+).2CO3, and extracted with DCM several times. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to generate the product (86% yield). Isooxazole was produced as previously described [3 (1.0 eq) was added to a solution of azide (1.0 eq) and isothiocyanate (1.0 eq) in dioxane (0.2 M). The resulting mixture was heated to 90 °C and stirred for 4 h. The solvent was cooled at room temperature and evaporated under reduced pressure. The residue was purified by flash column chromatography over silica gel with EtOAc/hexane (1:10) to generate the product (32% yield).Azide was produced as previously described . Brieflyescribed . Briefly2, and 95% relative humidity.Human AML cell lines, Molm-13, MV4-11, and HL-60 were maintained in Roswell Park Memorial Institute (RPMI)- 1640  and Iscove’s Modified Dulbecco’s Medium (IMDM)  media supplemented with 10% fetal bovine serum (Welgene) and 1% penicillin–streptomycin (Gibco). Culture flasks were incubated at 37 °C, 5% CO3, compound 7  was administered intraperitoneally (i.p.) once every 2 day for three times. Tumor size was measured twice a week using vernier calipers and tumor volume calculated as follows:MV4-11 cells were resuspended in 10% Matrigel for tumor solidification and subcutaneously (s.c.) implanted into the right hind leg thigh of 6-week-old BALB/c nu/nu mice. When the tumor volume reached approximately 80 mm2/2 mm3.Tumor volume = (longest tumor axis × shortest tumor axis)All animal experiments were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care Use Committee .2, 2 × 103, and 1.2 × 103 cells per well, respectively. Experimental groups were treated with 7c and incubated at 37 °C and 95% relative humidity for 5 d, whereas the control groups were treated with dimethyl sulfoxide (DMSO) and incubated under the same conditions. CCK-8 solution was added to each well with cells and the absorbance of the sample was measured at 450 nm using a 96-well plate reader .Cell viability was determined using the Cell Counting Kit-8 (CCK-8) . Molm-13, MV4-11, and HL-60 were seeded in 96-well plates at a density of 8 × 10Proteins were extracted from cell lysates using TNN (40 mM Tris-HCl (pH 8.0), 0.2% NP-40, 120 mM NaCl) buffer or RIPA lysis buffer  supplemented with protease inhibitor . Proteins samples, loaded at equal amounts, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 6–15% gels, and then transferred on to nitrocellulose membranes . The membranes were washed and blocked with 5% skim milk in TBS-T , and then incubated overnight with primary antibodies at 4 °C. Secondary antibodies conjugated to peroxidase and visualized immunoreactive proteins enhanced by ECL reagent were detected using ImageQuan LAS 4000 mini .®  to synthesize cDNA using CycleScript reverse transcriptase . cDNA was mixed with target sequence primers and Maxima SYBR Green/Fluorescein qPCR Master Mix . The sequence of the designed primers targeting DNA repair genes is as follows: BRCA1, forward 5′-CTGAAGACTGCTCAGGGCTATC-3′, reverse 5′-AGGGTAGCTGTTAGAAGGCTGG-3′; BRCA2, forward 5′-GGCTTCAAAAAGCACTCCAGATG-3′, reverse 5′-GGATTCTGTATCTCTTGACGTTCC-3′; RAD51, forward 5′-CTCAGCCTCCCGAGTAGTTG-3′, reverse 5′-CATCACTGCCAGAGAGACCA-3′; BARD1, forward 5′-GCCAAAGCTGTTTGATGGAT-3′, reverse 5′-CGAACCCTCTCTGGGTGATA-3′; XRCC4, forward 5′- CATTGTTGTCAGGAGCAGGA-3′, reverse 5′-TCTGCAGGTGCTCATTTTTG-3′; XRCC5, forward 5′-CGACAGGTGTTTGCTGAGAA-3′, reverse 5′-TCACATCCATGCTCACGATT-3′; XRCC6, forward 5′-AAAAGACTGGGCTCCTTGGT-3′, reverse 5′-TGTGGGTCTTCAGCTCCTCT-3′. 18S ribosomal RNA was used as a housekeeping gene to normalize samples. The housekeeping gene sequence is as follows: forward 5′-CACGCCAGTACAAGATCCCA-3′, reverse 5′-TTCACGGAGCTTGTCCA-3′. The conditions of qRT-PCR were as follows: initial duration at 95 °C for 10 min, followed by 50 cycles at 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. The PCR products were detected and analyzed using the LightCycler 96 system . The mRNA level was indicated as the fold change relative to the control. Experiments were carried out in triplicate.The total RNA was extracted from the cell lysate using TRIzolIn vitro kinase profiling of the 11 synthesized compounds was performed by Reaction Biology Corporation .http://www.rcsb.org). Before docking, the original crystal ligand quizartinib (AC220) and water molecules were removed from the protein–ligand complexes. Hydrogen atoms were added using the CHARMm force field and the Momany–Rone partial charge as default settings in Discovery Studio 2019. The ligand-binding site was extracted from RCSB-PDB and designated as active site 1. Docking analysis of compounds with the FLT3 protein in the presence of quizartinib was performed using the CDOCKER module. The number of generated poses was set at 100 for each ligand, whereas default settings were selected for other parameters.Ligand preparation and optimization were conducted using the Prepare Ligands module of Discovery Studio 2019 . The FLT3 protein structure, as described previously , was dowt-test was carried out to identify significant differences at p < 0.05.Data are presented as mean ± standard deviation or standard error. Analysis of variance in conjunction with Student’s


Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (Aβ) causes cytosolic Ca2+ overload, but the effects of Aβ on mitochondrial Ca2+ levels in Alzheimer’s disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral β-amyloidosis. Naturally secreted soluble Aβ applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD.Mitochondria contribute to shape intraneuronal Ca Calvo-Rodriguez et al. show elevated calcium levels in neuronal mitochondria in a mouse model of cerebral β-amyloidosis after plaque deposition, which precede rare neuron death events in this model. The mechanism involves toxic extracellular Aβ oligomers and the mitochondrial calcium uniporter. Aβ neurotoxicity has also been associated with intraneuronal Ca2+ dyshomeostasis3. The calcium hypothesis of AD4 posits that activation of the amyloidogenic pathway remodels neuronal Ca2+ signaling, altering normal Ca2+ homeostasis and the mechanisms responsible for learning and memory. Previous work from our group and others has shown that Aβ aggregates increase cytosolic Ca2+ levels in vivo both in a mouse model of AD6, and after application of soluble Aβo to the brain of naive wild-type (Wt) mice8. However, these investigations assessed Ca2+ levels in the cytosol, and whether Aβ affects Ca2+ levels in mitochondria in vivo remains elusive.Alzheimer’s disease (AD), the most common age-related neurodegenerative disorder, is characterized by deposition of extracellular senile plaques, neurofibrillary tangles and neurodegeneration, which lead to cognitive impairment and dementia. Despite the extensive efforts to understand its pathophysiology, the exact mechanisms underlying AD remain unknown. The most influential hypothesis for AD postulates that abnormal accumulation of the amyloid β (Aβ) peptide is the precipitating event that triggers a pathogenic cascade of adverse events, and that Aβ oligomers (Aβo), rather than amyloid plaques themselves, are the neurotoxic species of Aβ9. Studies on brains from AD patients and AD mouse models have shown impaired mitochondrial function, manifested as decreased bioenergetics and ATP synthesis10, morphological abnormalities11, imbalance of mitochondrial dynamics12 and redistribution of mitochondria13. Mitochondria participate in intracellular Ca2+ signaling as modulators, buffers and sensors14. After a cytosolic Ca2+ increase, mitochondria rapidly take up Ca2+ to prevent Ca2+ overload into the cytosol. However, excessive Ca2+ taken up into mitochondria, i.e., mitochondrial Ca2+ overload, results in increased reactive oxidative species (ROS) production, ATP synthesis inhibition, mitochondrial permeability transition pore (mPTP) opening, release of cytochrome c, activation of caspases and apoptosis15. In vitro studies have reported that Aβo induce mitochondrial Ca2+ uptake17, and Ca2+ transfer from ER to mitochondria18 in cultured rat primary neurons, but whether these findings apply in vivo remains unknown. In the present study, we assess whether Aβ aggregates alter mitochondrial Ca2+ levels in neurons in the mouse brain in vivo. We find elevated Ca2+ levels in neuronal mitochondria in transgenic mice, but only after plaque deposition, and preceding neural death. This mitochondrial Ca2+ overload in vivo involves toxic extracellular Aβ oligomers and requires the mitochondrial Ca2+ uniporter. We propose reducing mitochondrial Ca2+ overload by blocking the mitochondrial Ca2+ uniporter as a novel potential therapeutic target for AD.Mitochondrial malfunction has been proposed as an early event in AD and other aging-related neurodegenerative disorders2+ overload5 and mitochondrial membrane potential decrease11 in the APPswe/PSEN1∆E9 (APP/PS1) Tg mouse after amyloid plaque deposition. Here we hypothesized that Aβ aggregates could also increase neuronal mitochondrial Ca2+ levels in vivo. To study free Ca2+ concentration in mitochondria ([Ca2+]mit) in neurons, we targeted the expression of the ratiometric Ca2+ indicator Yellow Cameleon 3.6 (YC3.6)19 to neuronal mitochondria  imaging between CFP and YFP, quantitatively measuring absolute Ca2+ concentrations. YC3.6 is one of the brightest reporters among the YC versions, and exhibits a large dynamic range and excellent signal-to-noise ratio19. Detailed characterization of hSyn.2mtYC3.6 expression was performed in vitro using neuroblastoma Neuro2a cells (N2a) and mouse primary cortical neurons, and ex vivo after immunofluorescence of sections from a Wt adult mouse brain injected with AAV.hSyn.2mtYC3.6. The punctate and perinuclear pattern expression of 2mtYC3.6 . An increase in the YFP/CFP fluorescence ratio of YC3.6 corresponds to an increase in [Ca2+]. Ratios of YFP/CFP fluorescence intensities were plotted against [Ca2+] and fitted with a sigmoidal curve , with an apparent Kd (Kd′) of 4.21 µM and a Hill coefficient of 1.5720. Either the mitochondrial environment or the fusion of two mitochondrial sequences shifted the affinity of YC3.6 for Ca2+ to higher values, making it more suitable for Ca2+ measurements in mitochondria. This calibration was used to obtain [Ca2+]mit in the subsequent experiments. To verify that AAV.hSyn.2mtYC3.6 was functional in vivo, we applied KCl topically onto the brain of C57BL/6J mice injected with AAV.hSyn.2mtYC3.6 to induce neuronal depolarization, and imaged the neuronal mitochondria with multiphoton microscopy. Figure 2+]mit (pseudocolored images and Ca2+ recording), immediately after KCl application. Therefore, AAV.hSyn.2mtYC3.6 specifically targets mitochondria in neurons, can be expressed in vivo and provides a quantitative readout of neuronal [Ca2+]mit.We calibrated hsyn.2mtYC3.6 in N2a cells. Cells were transfected with hSyn.2mtYC3.6, and 24 h later, cells were permeabilized and exposed to solutions containing increasing known mit. However, mitochondria exhibit different kinetics, which justified the need to address the mitochondrial Ca2+ impairment independent of cytosolic Ca2+ in the disease model.We compared the kinetics of cytosolic Ca2+ contributing to a general Ca2+ dyshomeostasis in AD, we examined [Ca2+]mit in the APP/PS1 Tg mouse model of cerebral β-amyloidosis in vivo using multiphoton microscopy. Age-matched Wt littermates were used as controls. 8-months-old (mo-old) APP/PS1 Tg mice were intracortically injected with AAV.hSyn.2mtYC3.6 targeting layers II/III, and 5 mm diameter cranial windows were implanted over the somatosensory cortex. Imaging was conducted 3 weeks later to allow for protein expression. Methoxy X0-4 was injected intraperitoneally 24 h before to label amyloid plaques21  was observed for APP/PS1 Tg mice. Ca2+ overload was defined as YFP/CFP ratio greater than two standard deviations (SD) above the mean YFP/CFP ratio obtained for all the mitochondria in all Wt mice22 . Twice as many mitochondria from APP/PS1 Tg mice showed Ca2+ overload compared to Wt mice , in either somas or neurites  , which mainly contains low molecular weight oligomers (dimers to tetramers)28. CM from Wt-littermate neurons (WtCM) and Aβ-immunodepleted TgCM were used as controls. The three media were subjected to an Aβ40 sandwich ELISA. Aβ40 concentration in the media was 0.3, 5 and 0.7 nM for WtCM, TgCM and Aβ-immunodepleted TgCM, respectively. Aβo concentration in TgCM represents around 10% of the total amount of Aβ407.We have previously shown that acute exposure of the brain of naive (Wt) living mice to soluble Aβo leads to disruption of cytosolic Ca2+]mit in the healthy brain, a cranial window was implanted over the somatosensory cortex of 4- to 5-mo-old C57BL/6J mice injected with AAV.hSyn.2mtYC3.6, the mouse was imaged at baseline, the window was reopened to apply either WtCM, TgCM or Aβ-depleted TgCM onto the brain for 1 h, and finally, the mouse was reimaged in the same volumes imaged at baseline ). Application of TgCM significantly increased [Ca2+]mit, causing a shift towards higher YFP/CFP ratios (higher [Ca2+]mit) in the [Ca2+]mit histogram . Per ELISA measurements, immunodepletion of Aβ effectively removed about 85% of Aβ present in TgCM, from 5 to 0.7 nM . Application of Aβ-immunodepleted TgCM did not increase [Ca2+]mit  acquired before and after application of CM , whereas only a few z-stacks showed a ≥5% increase for WtCM and Aβ-immunodepleted TgCM. Using these ratios, we calculated the relative change in YFP/CFP ratio (∆R/R0) for each z-stack. Application of TgCM induced a relative change in the YFP/CFP ratio significantly higher than WtCM or Aβ-immunodepleted TgCM  complex, which includes the MCU pore, located in the inner mitochondrial membrane30, and the combination of two regulatory subunits MiCU1 and MiCU2 , which regulate the MCU response to extra-mitochondrial Ca2+ 32. Other proteins involved in the complex include the essential MCU regulator (EMRE)33, MCUb34, or MiCU335. Mitochondrial Ca2+ efflux depends mainly on the electrogenic Na+-dependent Ca2+ release exchanger (NCLX)36. Given the increased number of mitochondria with Ca2+ overload in APP/PS1 Tg mice after amyloid plaque deposition, we performed Western blots for MCU, MiCU1, MiCU2, and NCLX in mitochondrial-enriched fractions of the brain of 10-mo-old Wt and APP/PS1 Tg mice to test whether Aβ affects the expression levels of these proteins. No significant differences were detected between genotypes in any of these mitochondrial proteins 37. As in Fig. 2+]mit was assessed in basal conditions. The window was then reopened and the underlying brain was pre-treated with Ru360 (100 µM) for 15 min. TgCM mixed with Ru360 was next applied onto the brain for 1 h, and the mouse was reimaged in the same areas. TgCM increased neuronal [Ca2+]mit (arrow heads) as expected, but application of TgCM mixed with Ru360 did not ), was not significantly different , whereas for the majority the increase was ≤5% (light lines)  for each volume   Fig. . Averageume Fig.  showed t2+ could have a detrimental effect in Ca2+ overloaded mitochondria and, eventually, also in the neurons containing these mitochondria. We examined the time course of Aβo-driven neuronal mitochondrial Ca2+ overload in the C57BL/6 J Wt mouse brain, and specifically asked whether this was permanent or reversible and, if the latter, how long after Aβo application. At 8 h post-application of TgCM, neuronal [Ca2+]mit had already returned to basal levels  compared to basal conditions  mitochondrial Ca2+ overload using longitudinal multiphoton microscopy serial imaging over a 24–48 h period in 9-mo-old APP/PS1 Tg mice. First, we evaluated whether the static mitochondrial Ca2+ levels were stable longitudinally. We observed that over 3 imaging sessions (48 h), the absolute mitochondrial Ca2+ levels were constant in the same cell, for both low and intermediate Ca2+ levels ) were lost within 24 h , and the soma lost its of YFP/CFP fluorescence  41, as verified by calcein/cobalt fluorescence, prevented the decrease of ∆Ψm, but did not inhibit the increase of [Ca2+]mit elicited by TgCM. These results suggest that the reduction of ∆Ψm is driven by the increase in mitochondrial Ca2+ after TgCM application. Blocking mPTP with CsA showed a trend towards decreasing the number of neurons with caspase 3/7 activation  Knowledge Portal42. We evaluated 25 publicly available microarray and RNA-Sequencing datasets from the Mount Sinai Brain Bank (MSBB)43 and the Religious Orders Study and Memory and Aging Project (ROSMAP)44 cohorts, and compared the transcript levels of mitochondrial Ca2+-related genes between subjects with AD (Braak stages V–VI) and control individuals (Braak stages 0–I–II), specifically MCU, MCUB, MCUR1, MICU1, MICU2, MICU3 and SMDT1 for mitochondrial Ca2+ influx, and SLC8B1 (encoding NCLX) for mitochondrial Ca2+ efflux. Surprisingly, the mitochondrial Ca2+ uniporter (Mcu) and its regulatory subunits  are significantly downregulated (p < 0.05 and FDR < 25%) in AD compared to control subjects after adjusting for neuronal loss with the pan-neuronal marker Map2 45 produced similar results (Supplementary Table 2+ influx were downregulated (FDR < 20%), whereas Slc8b1 was upregulated. Mcur1 was also upregulated in this case. These results suggest that the expression of most of human genes involved in mitochondrial Ca2+ transport is altered in AD patients, denoting a counteracting effect to avoid mitochondrial Ca2+ overload.Finally, we sought to test whether mitochondrial Ca2+ overload in AD. Aβ-induced mitochondrial Ca2+ uptake requires MCU, as blocking MCU with the specific inhibitor Ru360 prevents the Ca2+ overload. In addition, excessive Ca2+ taken up by mitochondria leads to opening of the mitochondrial permeability transition pore (mPTP), caspase activation and, eventually, neuronal cell death  lead to mitochondrial Caath Fig. .Fig. 8Sc2+ homeostasis and mitochondrial dysfunction, which contribute to synaptic and neuronal damage46. An extensive array of in vitro and in vivo studies has shown that Aβ aggregates alter cytosolic Ca2+ homeostasis. However, prior studies linking Aβ with mitochondrial Ca2+ overload17 have used cultured cells and high concentrations of synthetic Aβo, and whether there is mitochondrial Ca2+ overload elicited by Aβ aggregates in vivo remained an open question to date. In the present study, we provide evidence of in vivo mitochondrial Ca2+ overload in a Tg mouse model of cerebral β-amyloidosis, and after topical application of natural soluble Aβo at concentrations similar to those found in the AD brain.Proposed mechanisms of neurotoxicity in AD include the disruption of Ca2+ overload in the APP/PS1 Tg mouse, a model that recapitulates an accelerated deposition of amyloid plaques. This overload was observed in the Tg mice once the Aβ plaques were present. The mitochondrial Ca2+ increase was of a higher magnitude than the cytosolic Ca2+ levels observed in the same mouse model5. Whereas Ca2+ levels in the neuronal cytosol upon exposure to Aβo were in the nM range7, mitochondrial Ca2+ levels were in the µM range. This was not surprising as mitochondria take up large amounts of Ca2+ during physiological Ca2+ elevations. Indeed, effective mitochondria Ca2+ buffering capacity was reported ~0.2–2 µM in isolated brain mitochondria47 and in neuronal mitochondria in situ48. In vivo, we found that mitochondrial Ca2+ overload only occurs at levels higher than ~1.6–1.7 µM, comparable to the reported buffering capacity in vitro.We found mitochondrial Ca9. However, several studies have reported no change in resting cytosolic Ca2+ levels or neuronal dysfunction before amyloid plaque deposition in AD mouse models25. In agreement with these studies, there was no appreciable mitochondrial Ca2+ overload in young mice before plaque deposition (2.5–3-mo-old), suggesting that Aβ deposition is necessary to trigger mitochondrial Ca2+ overload in neurons in AD. Further longitudinal studies will be necessary to clarify whether mitochondrial Ca2+ elevation occurs concomitantly with plaque formation. Surprisingly, no association was found between mitochondrial Ca2+ overload and distance to amyloid plaques, in contrast to cytosolic Ca2+ overload, wherein there was a higher likelihood of detecting Ca2+ overload in the immediate vicinity of individual plaques5. This suggests that diffusible soluble Aβ oligomeric species rather than plaques are likely responsible for the increase of [Ca2+]mit in the neurons of the APP/PS1 mouse, or that mitochondria are less vulnerable than cytosol to the pathophysiological insult mediated by Aβo. The intrinsic properties of mitochondria with regards to Ca2+ kinetics, and as dynamic entities that undergo axonal transport, fusion/fission, and mitophagy when damaged, could also explain this difference.Mitochondrial dysfunction has been suggested as an early event in AD, before plaque deposition2+ overload is triggered by soluble Aβo, we directly applied naturally secreted Aβo (TgCM) to the naive living mouse brain. Application of TgCM to the cortical surface of the mouse brain increased [Ca2+]mit in neurons within 1 h, whereas WtCM or Aβ-immunodepleted TgCM did not. This result supports our previous findings in the APP/PS1 model, and confirms a direct effect of Aβ aggregates altering Ca2+ homeostasis in neurons. The reversibility of TgCM-induced mitochondrial Ca2+ overload supports a normal extrusion via mitochondrial Ca2+ exchangers, and/or clearance of the Aβo from the brain parenchyma.To confirm that mitochondrial Ca49. However, the mechanism(s) by which Aβ evokes mitochondrial Ca2+ uptake in vivo has not been elucidated. Proposed mechanisms include formation of Ca2+-permeable pores in the plasma membrane and also in the membranes of intracellular organelles50, or formation of a non-specific ion channel in the plasma and mitochondrial membranes51. On the other hand, Ca2+ uptake into the mitochondrial matrix is mediated by the highly conserved MCU complex30, which is selectively blocked by Ru360. Our results show that pre-treatment with Ru360 abolishes TgCM-induced mitochondrial Ca2+ uptake, providing evidence that the MCU complex is required for the Aβ-driven mitochondrial Ca2+ increase in neurons in the brain in vivo, and arguing against the formation of Ca2+-permeable pores or non-specific ion channel in the mitochondrial membrane. However, assessment of protein expression of the mitochondrial-enriched fraction extracted from Wt and APP/PS1 Tg mouse brains did not show any significant differences between groups. Western blot might not be sensitive enough to detect changes in protein levels that affect the small subset of Ca2+-overloaded mitochondria seen in vivo. Alternatively, Aβ may alter the levels of a component of the pore or the NCLX exchanger not examined in this model due to lack of adequate antibodies, or may impact their activity rather than expression. Along these lines, recent work by Jadiya et al.52 has observed a decrease in the expression of NCLX in mitochondria in human AD brains and in mouse models of AD, linked to impaired Ca2+ efflux from mitochondria, which could be responsible for the disease progression.Mitochondria have been reported as a direct site of Aβ accumulation in neurons in both human AD brains and AD Tg mouse models2+ dyshomeostasis in the human AD brain, we took advantage of publicly available microarray and RNA-Seq datasets, and compared the expression levels of Ca2+-related mitochondrial genes in AD patients (Braak stages V–VI) and control subjects (Braak stages 0–I–II). Most of the genes involved in Ca2+ influx into mitochondria were significantly downregulated in AD, whereas, the only Ca2+ efflux gene was significantly upregulated. These results most certainly point towards a compensatory effect in AD to handle the mitochondrial Ca2+ overload, and confirm the existence of a mitochondrial Ca2+ dyshomeostasis in the human AD brain.To examine the relevance of mitochondrial Ca2+, and in turn, Ca2+ modulates mitochondrial activity. Excessive Ca2+ accumulation into mitochondria leads to mitochondrial function impairment53, which could lead to decreased ∆Ψm11 and increased ROS production40. Eventually, cytochrome c release and cell death via apoptosis will take place39. Remarkably, when APP/PS1 Tg mice were imaged longitudinally, the small fraction of neurons containing mitochondria with elevated [Ca2+] in their cell bodies were no longer visible 24 h later, and their nucleus was condensed, whereas neurons with physiological [Ca2+]mit and a healthy spherical nucleus remained. Surprisingly, whereas the cells with high [Ca2+]mit in the soma were likely to die, sporadic increases of mitochondrial Ca2+ within the neurites were not as lethal. This feature suggests that within the soma, high [Ca2+]mit represents an “all or none”-phenomena, and not an isolated event in individual mitochondria. The results obtained here in vivo are supported by previous studies in vitro showing neuronal cell death related to mitochondrial Ca2+ overload17, where a subtle or partial depolarization of mitochondria to avoid Aβ-related mitochondrial Ca2+ overload in culture cells was protective for neuronal death. Furthermore, our in vitro data show that soluble Aβ increases [Ca2+]mit, leading to activation of mPTP, ∆Ψm decrease and activation of caspases. Previous studies have already linked Aβ, mPTP and cell death in AD in neurons56 and astrocytes58, but here we show that activation of mPTP is downstream mitochondrial Ca2+ overload, as preventing mPTP activation did not forestall mitochondrial Aβo-driven Ca2+ overload, but avoided mitochondrial depolarization and caspase activation. Moreover, we have previously shown that there is a rapid cell death once Aβ-triggered oxidative stress invades neuronal cell bodies in vivo in the same mouse model40. Oxidative stress started in neurites, but in a small fraction of neurons tracked towards the cell bodies leading to rapid initiation of apoptosis. Mitochondrial Ca2+ overload has an important role in the oxidative stress-induced neuronal cell death59. Our findings of occasional rapid neuronal loss after mitochondrial Ca2+ overload support our previous findings and, more importantly, suggest a close link between high [Ca2+]mit, oxidative stress and neuronal apoptosis via mitochondria in AD.Optimal mitochondrial activity is crucial to maintain neuronal function and health. Mitochondria buffer Ca2+ elevation, but also involved changes in mitochondrial size and shape. Indeed, the APP/PS1 Tg mouse model exhibits structural abnormalities in mitochondria, including mitochondria swelling and fragmentation11, and impaired balance of mitochondrial fission and fusion proteins has been shown in brains from AD patients60. Aβ-induced mitochondrial fragmentation has been reported to precede neuronal cell death61. Further experiments are necessary to monitor the morphological changes and altered dynamics of mitochondria induced by Aβo in vivo.Of note, the neurotoxic effect of Aβo on mitochondria were not restricted to Ca2+ overload, pointing to a critical role of MCU in the mitochondrial Ca2+ overload mediated by Aβo. Multiple lines of evidence support MCU as a candidate therapeutic target against AD: MCU overexpression in mitochondria increased [Ca2+]mit following excitotoxicity through activation of NMDA receptors in vitro, leading to mitochondrial membrane depolarization and cell death62. Besides, inhibition of MCU by Ru360, or silencing by siRNA or miRNA, conferred resistance to oxidative stress to the cells in vitro63. Also, reducing mitochondrial Ca2+ uptake by a null mutation in the gene encoding MCU, improved mitochondrial function and suppressed neurodegeneration in a C. elegans model of AD; and preventing mitochondrial Ca2+ uptake by pre-treatment with Ru360 decreased ROS levels in familial AD fibroblasts64. Our in vitro work shows that preventing activation of the mPTP downstream mitochondrial Ca2+ overload could also serve as a therapeutic approach against AD, to protect mitochondria from depolarization (loss of ∆Ψm) and subsequent activation of apoptosis.Here we show that blocking the MCU complex in vivo prevents the Aβ-related mitochondrial Ca2+ overload in vivo via the MCU complex, leading to neuronal death. We have unveiled a pathophysiological link between Ca2+ dyshomeostasis and mitochondrial dysfunction hypotheses of AD pathogenesis and propose the inhibition of the MCU complex and blocking of the mPTP activation as novel therapeutic approaches against AD.In summary, our findings demonstrate that Aβ accumulation provokes mitochondrial CaAPP gene carrying the Swedish mutation and exon 9 deletion mutation in the PS1 gene along with age-matched Wt-littermate controls; and (2) C57BL/6J males  for the application of the conditioned media and Ru360. For preparation of the conditioned media, Tg2576 males 2576Kha, IMSR Cat# TAC:1349, RRID:IMSR_TAC:1349), which heterozygously overexpress human APPswe under the PrP promoter, were mated with Wt females for preparation of primary cortical neurons. Mice were socially housed at 3–4 animals per cage with ad libitum access to food and water on a 12/12 h light/dark cycle with controlled conditions of temperature and humidity.Mouse experiments were performed with the approval of the Massachusetts General Hospital Animal Care and under the guidelines of Institutional Animal Care and Use Committees  for the use of experimental animals. Mice used included: (1) APPswe/PSEN1∆E9 double Tg mice 85Dbo/Mmjax, MMRRC Cat# 034832-JAX, RRID:MMRRC_034832-JAX)  of either sex, expressing both human 2+ indicator Yellow Cameleon 3.6 (YC3.6)19 was targeted to mitochondria by using the mitochondrial targeting sequence cytochrome oxidase subunit 8 A (COX8A) at the N terminus, duplicated in tandem to enhance the specificity of its localization65. 2mtYC3.6 was inserted on the 5′-end and in frame with the cDNA of YC3.6, which we had previously cloned in an AAV.CBA.2mtYC3.6-WPRE  backbone. We then cut out the 2mtYC3.6 cDNA (NheI/XhoI-fill-in) and subcloned it into an AAV-hSyn backbone obtained from the UPENN vector core, obtaining the final AAV.hSyn.2mtYC3.6-WPRE plasmid. The integrity of the inverted terminal repeats (ITRs) was verified by sequencing and SmaI digestion. Flanked by the ITRs, the expression cassette included the following components, (1) a 1.7-kb sequence containing human synapsin 1 gene promoter, (2) 2mt , (3) YC3.6, (4) WPRE and (5) Simian virus 40 (SV40). See construct in Fig. 12 viral genomes copies per mL.The expression of the Ca2+ concentration we used the construct pAAV.CBA.YC3.6-WPRE5. The plasmid contained AAV terminal repeats (ITRs) and the expression cassette, which included the following components: the hybrid cytomegalovirus (CMV) immediate-early enhancer/chicken β-actin promoter/exon1/intron; yellow cameleon 3.6 cDNA and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). High titers of AAV serotype 8 were produced using triple transfection in HEK293 cells by the Schepens Eye Research Institute Gene Transfer Vector Core. The virus titer was 1.1 × 1012 viral genomes copies per mL.To measure cytosolic Ca2 without further media exchange5.Primary neuronal cultures were obtained from cerebral cortex of CD1 mouse embryos (Charles Rives Laboratories) at gestation day 14–16. Neurons were dissociated using Papain dissociation system  and were maintained for 10–14 days in vitro (DIV) in Neurobasal medium containing 2% B27 supplement (Gibco), 1% penicillin/streptomycin (Gibco) and 1% glutamax (Gibco) in a humidified 37 °C incubator with 5% CO2.N2a cells were maintained in OptiMEM (Gibco) supplemented with 5% Fetal Bovine Serum (FBS)  and 1% penicillin and 1% streptomycin (Gibco) in a humidified 37 °C incubator with 5% COCells were transiently transfected at 70–80% confluence with 4 µg of hSyn.2mtYC3.6 and mRuby-Mito-7  plasmids using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions.2, 2 mM KH2PO4, 20 mM HEPES, 0.5 mM EGTA, 5 mM succinate, 1 mM ATP, pH 7), followed by three washes with intracellular medium devoid of digitonin. After permeabilization, cells were exposed to solutions containing known Ca2+ concentration (ranging from 0 µM to 1 mM) and 10 µM FCCP, to depolarize mitochondria and equilibrate the [Ca2+] in the medium and in the organelle66. Calibration solutions with free Ca2+ concentration <20 µM were prepared by mixing intracellular media with the chelators EGTA or HEDTA and 1 M CaCl2 according to MaxChelator Ca-EGTA Calculator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/CaEGTA-NIST.htm) and to Park and Palmer67. Calibration solutions with free Ca2+ concentration >20 µM were made by adding various volumes of 1 M CaCl2 to the intracellular media solution. 2 photon excitation was used at 860 nm, and emitted light was collected in three channels in the range of 380–480, 500–540 and 560–650 nm. Images were acquired on an Olympus FluoView FV1000MPE multiphoton laser-scanning system mounted on an Olympus Bx61WI microscope, and photomultiplier settings were selected and maintained for the intravital imaging. YFP/CFP fluorescence ratios were plotted against free Ca2+ concentration and fitted with a sigmoid equation. The apparent Kd (Kd′) and Hill coefficient (n) were determined as described previously20. Custom-written ImageJ software (https://imagej.nih.gov/ij/) was used for analysis and calculation of YFP/CFP ratios. Note that ratios increased with increasing [Ca2+]. Ratios were then converted to free Ca2+ concentration with standard ratiometric equations20:N2a cells were plated in 35 mm glass-bottom dishes (MatTek Corporation) and transfected with lipofectamine 2000 (Life Technologies) and 4 μg of the hSyn.2mtYC3.6 construct. 24 h after transfection, cells were permeabilized for 1 min with 100 µM digitonin in intracellular medium  containing 2% B27 supplement (Gibco), 1% penicillin/streptomycin (Gibco) and 1% glutamax (Gibco) for 14 DIV. Conditioned media from either Tg cultures (TgCM) or their Wt littermates (WtCM) was collected, and the concentration of Aβ40 was measured by mouse/human ELISA kit (Wako #294-64701 Human/Rat Aβ1–40).Primary neuronal cultures were prepared as described above1-40) according to the manufacturer’s instructions.Immunodepletion of human Aβ from the TgCM was performed overnight by using the mouse antibody 6E10  and protein G sepharose beads (Sigma-Aldrich). Briefly, protein G beads were washed with cold Neurobasal medium in order to avoid non-specific binding. Then, 1 mL of TgCM was incubated with 40 µL of the pre-washed protein G beads and 6 µg of 6E10 antibody overnight at 4 °C under rotation. Finally, the supernatant was collected and the Aβ concentration was quantified by mouse/human ELISA kit  and 860 nm (for 2mtYC3.6), and emitted light was collected in the range of 380–480, 500–540 and 560–650 nm. Either images were acquired in basal conditions and after treatment with conditioned media, or time-lapse recordings were acquired following stimulation with KCl (50 mM) or TgCM. Background fluorescence corresponding to regions devoid of cells was subtracted. Images were analyzed using custom-written MATLAB scripts .Primary cortical neurons obtained from CD1 embryos were cultured for 12–15 days in vitro (DIV) and infected with AAV.hSyn.2mtYC3.6 to target mitochondria. Experiments were performed using sister cultures obtained on the same day to control for any culture-to-culture variation. Three days after infection, cells were incubated with fresh conditioned media and subjected to multiphoton microscopy. 5–6 fields of view were imaged randomly. For some experiments cortical neurons infected with the mitochondrial reporter hSyn.2mtYC3.6 were loaded with the calcium indicator calbryte 590 (0.5 µM) for 30 min at room temperature to measure Ca68. Briefly, primary cortical neurons cultured for 12–15 DIV were co-loaded with calcein-AM 1 µM, CoCl2 1 mM and Hoechst 33342 1 µM for 15 min at room temperature and imaged with confocal microscopy. The effect of TgCM was evaluated longitudinally. Images were acquired in the green channel (488 nm) every 10 min. Cells were maintained at 37 °C and 5% CO2 during the recording. Fluorescence traces from mitochondria in individual cells were normalized relative to their value before addition of any solutions (t = 0 min). Background fluorescence corresponding to regions devoid of cells was subtracted. In some experiments, cells were pre-incubated with cyclosporine A 1 µM for 15 min before and co-applied during the recording.mPTP opening was directly assessed by the calcein/cobalt method2 during the recording. Excitation of the neurons was performed at 361 nm for Hoechst and 549 nm for TMRE, and images were taken every 10 min. Fluorescence traces from individual cells were normalized relative to their value before addition of any solutions (t = 0 min). Background fluorescence corresponding to regions devoid of cells was subtracted. In some experiments, cells were pre-incubated with cyclosporine A 1 µM for 15 min before and co-applied during the recording.The effects of TgCM on ∆Ψm were evaluated by confocal microscopy in cells loaded with the ∆Ψm sensitive probe TMRE. Primary cortical neurons cultured for 12–15 DIV were loaded with TMRE (10 nM) and Hoechst 33342 1 µM for 15 min at room temperature and imaged with confocal microscopy. The effect of TgCM was evaluated longitudinally. Cells were maintained at 37 °C and 5% CO2 during the recording. Fluorescence traces from individual cells were normalized relative to their value before addition of any solutions (t = 0 min). Background fluorescence corresponding to regions devoid of cells was subtracted. In some experiments, cells were pre-incubated with cyclosporine A 1 µM for 15 min before and co-applied during the recording.Caspase activation was detected with CellEvent Caspase 3/7 Green Detection Reagent in primary cortical neurons at 12–15 DIV. Neurons were labeled with CellEvent Caspase 3/7 5 µM and Hoechst 33342 1 µM for 15 min at room temperature. Then media was replaced by the conditioned media and green fluorescence was detected under confocal microscopy. Excitation of the neurons was performed at 361 nm for Hoechst and 488 nm for CellEvent, and images were recorded every 10 min. Cells were maintained at 37 °C and 5% CO7. Briefly, mice were anesthetized with 1.5% (vol/vol) isoflurane and positioned on a stereotactic frame (Kopf Instruments). Body temperature was maintained throughout the surgery with a heating pad. 3 µL of viral suspension were injected in the somatosensory cortex using a 33-gauge sharp needle  attached to a 10 µL Hamilton microsyringe , at a rate of 0.15 µL/min. Stereotactic coordinates of the injection sites were calculated from bregma .Four- to five-mo-old C57BL/6 J Wt mice were injected intracortically with AAV.hSyn.2mtYC3.6 (serotype 2/8) or pAAV.CBA.YC3.6-WPRE (serotype 2/8). Stereotactic intracortical injections were performed as previously describedEight- to nine-mo-old APP/PS1 Tg mice were injected intracortically with AAV.hSyn.2mtYC3.6 at the moment of the cranial window implantation with 3 µL of viral suspension in the somatosensory cortex (left hemisphere) at 0.15 µL/min, followed by cranial window implantation. In some cases, 1 µl of Hoechst 33342 1uM was co-injected with AAV.hSyn.2mtYC3.6 to label nuclei in the area of injection. Mice were given buprenorphine (0.1 mg/kg) for 3 days following surgery.7. Dura matter was removed before window installation and the window was sealed containing PBS. Texas Red dextran  was retro-orbitally injected to provide a fluorescent angiogram. An imaging session was first performed to determine the basal resting [Ca2+]mit. Then, the window was removed and sealed again with WtCM, TgCM, Aβ-immunodepleted TgCM, Ru360  or Ru360 + TgCM . In the last case, Ru360 was pre-incubated for 15 min previous application of TgCM. After 1 h, each mouse was reimaged at the same fields of view to determine the relative changes in [Ca2+] (ΔR/R0).In the acute conditioned media experiments, a 6 mm cranial window was implanted in the C57BL/6J Wt mice three to four weeks after AAV.hSyn.2mtYC3.6 or pAAV.CBA.YC3.6-WPRE injection. Mice were anesthetized with 1.5% (vol/vol) isoflurane and placed in a stereotactic apparatus. A piece of skull over somatosensory cortex was removed and replaced with an 8 mm diameter glass coverslip7. Mice were given buprenorphine (0.1 mg/kg) for 3 days following surgery. Expression of the virus was allowed for 3–4 weeks before imaging. Methoxy-XO4 (4 mg/kg) was intraperitoneally injected 1 day before the imaging session to label amyloid plaques21.For the APP/PS1 Tg mice, cranial windows were implanted to the animals following the intracortical virus injection. Mice were anesthetized with 1.5% (vol/vol) isoflurane and placed in a stereotactic apparatus. A piece of skull over left somatosensory cortex was removed, replaced with a 5 mm diameter glass coverslip and fixed with Krazy Glue and dental cement2) for induction, and a reduced concentration of isoflurane (~1.5%) was used during the imaging. The animal’s body temperature was maintained at ~37.5 °C during the imaging session with a heating pad; ophthalmic ointment was applied to protect the eyes. Mice were visually monitored throughout the duration of the imaging session (which lasted on average 2 h). Images of AAV.hSyn.2mtYC3.6 or pAAV.CBA.YC3.6-WPRE expressing neurons, amyloid pathology and Texas Dextran Red angiograms were obtained by using an Olympus FlouView FV1000MPE multiphoton laser-scanning system mounted on an Olympus Bx61WI microscope and an Olympus 25× dipping objective (NA = 1.05). A Deep-See Mai Tai Ti:Sapphire mode-locked laser  generated multiphoton excitation at 860 nm, and three photomultiplier tubes (PMTs) (Hamamatsu) collected the emitted light in the range of 380–480, 500–540 and 560–650 nm69. Images were taken at 5× zoom. Five to fifteen cortical volumes  were acquired per mouse, at a resolution of 512 × 512 pixels. CFP and YFP PMTs settings remained unchanged throughout the different imaging sessions. Laser power was adjusted as needed and kept below 50 mW to avoid phototoxicity70.Mice were anesthetized with isoflurane  and brains were removed. The hemisphere injected with AAV.hSyn.2mtYC3.6 was fixed with 4% paraformaldehyde and 15% glycerol for 24 h, frozen in mounting media on ice cold isopropanol and sliced into 20 µm sections on a cryostat (Leica). The other hemisphere was flash frozen with liquid nitrogen and stored at −80 °C until use.At the end of the last imaging session, APP/PS1 Tg mice were euthanized under COz-stacks. To obtain Yellow Cameleon ratio values, the data were preprocessed by subtracting the background from the CFP and YFP channels  and applying a mean filter with radius 2 to each channel. The ratio for each mitochondrion was calculated by dividing the sum of the intensity values corresponding to the mitochondrion in the YFP channel by the sum of intensity values in the CFP channel. The YFP/CFP ratios were mapped to mitochondrial Ca2+ concentrations using the in situ calibration curves . Calcium pseudocolored images were created using MATLAB by first assigning the [Ca2+] values to a RGB  colormap with the color range determined by the empirical values for Rmin and Rmax. The RGB image was then converted to an HSV  image with the ‘value’ field set to the mean of the YFP and CFP intensity images. Distance to plaque was measured based on the centroid of the mitochondrion to the edge of the nearest plaque. To assess changes in mitochondrial morphology, a unique slice per z-stack was selected, matching the region across the three different time points . Slices were selected within 30-60 μm depth. ROIs were drawn around individual mitochondria with the free hand selection tool in ImageJ based on the pseudocolor images. About 100–125 random individual mitochondria were taken. Matlab was used to measure the parameters of interest  for each ROI. Area and perimeter were determined as the total number of pixels within the ROI and the number of pixels outlining the ROI, respectively, and then converted to physical units of μm2 and μm. Circularity was calculated using the formula, Perimeter2/4πArea. Images presented in the figures are either a single slice from the z-stack or 2D maximum intensity projections of the z-stack.Multiphoton microscopy imaging data, acquired in vivo, were analyzed by using a custom-written MATLAB (MathWorks) script. To perform automatic segmentation of mitochondria, an adaptive thresholding procedure was applied to generate binary images and individual mitochondria were selected using a constraint on object size. Amyloid plaques were manually extracted from the g for 30 min at 20 °C. The supernatant was collected, aliquoted and stored at −80 °C until use. Mitochondrial-enriched fraction was obtained using a mitochondrial isolation kit  according to the manufacturer’s instructions. A bicinchonic acid assay  was used to determine protein sample concentration. Supernatants were subjected to SDS-PAGE on NuPAGE Bis-tris gels (Life Technologies). Briefly, 20 µg of total protein per well were loaded on 4–12% Bis-Tris SDS-PAGE gels and run in MES buffer (Invitrogen). Proteins were then transferred to PVDF membranes (Millipore), which were incubated overnight with the primary antibodies: rabbit anti-MCU , rabbit anti-MiCU1 , rabbit anti-EFHA1 (MiCU2) , rabbit anti-SLC24A6 (NCLX) , mouse anti-VDAC1/porin , mouse anti-β-tubulin I . The next day, blots were incubated with infrared secondary goat anti-mouse IgG IRDye680 or goat anti-rabbit IgG IRDye800  for 1 h at room temperature, and imaged on an Odyssey Infrared Imaging System (LICOR). Blots were converted to gray scale and densitometry analysis was performed using Image Studio Lite Ver5.2.Frozen hemibrains devoid of cerebellum from APP/PS1 mice were lysed in Tris-buffered saline (TBS) with 2% sodium dodecyl sulfate (SDS) and protease inhibitor (Roche Diagnostics) at 10% (w/vol) in a 5 mL glass dounce homogenizer. Tissue was dounce homogenized with 25 up/down strokes by hand and incubated for 30 min at 37 °C. The homogenate was then centrifuged at 100,000 × 20 µm coronal sections were subjected to antigen retrieval, permeabilized with 0.5% triton X-100 and incubated with antibodies against GFP , HSP60 , NeuN  and GS  overnight at 4 °C. Appropriate secondary antibodies  were applied and incubated for 1 h at room temperature. Slices were mounted with DAPI Vectashield (Vector Laboratories) and subjected to fluorescence imaging.43 and the Religious Orders Study and Memory and Aging Project (ROSMAP)44 cohorts, spanning 19 brain regions, were analyzed as described in Bihlmeyer et al.71 Four of these brain regions had both microarray and RNA-Seq data. The samples were grouped by the Braak score, which assesses the distribution of tau neurofibrillary tangles in the subject’s brain72. The expression of 8 mitochondrial genes involved in mitochondrial Ca2+ homeostasis  was compared in AD Braak stages (V–VI) versus control subjects (Braak 0–I–II) and by CERAD analysis (frequent versus absent neuritic amyloid plaques). Data were adjusted for neuronal loss with the pan-neuronal marker MAP2. Multiple comparisons corrections were performed using the Benjamini–Hochberg method73. For the purpose of selection for visual display, a FDR threshold of 0.25 was used. All results for the differential expression analysis are listed in Supplementary Tables 25 microarray and RNA-Sequencing (RNA-seq) datasets from the Mount Sinai Brain Bank (MSBB)2+ overload and ∆R/R0), a linear mixed effects model was fitted with treatment group as fixed effect and mouse as random effect. The least square mean and standard error estimates and the p-values resulting from these models are presented in the figure legends. Similarly, estimates for basal, 1 h post CM application and 8 h post CM application mean and SE measurements were generated from a linear mixed effects model with a fixed effect of an intercept only and a random effect of mouse. Kruskal–Wallis test was used for the analysis of the relationship between percent of Ca2+ overload and distance to the edge of the plaque. p < 0.05 was considered significant.Statistical analyses were conducted in SAS and GraphPad 5.0. Data are reported as mean ± SEM. To assess differences between groups , global and pair-wise, for all measurements of interest (YFP/CFP Ratio, CaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Movie 1


The casein kinase 1 enzymes (CK1) form a family of serine/threonine kinases with seven CK1 isoforms identified in humans. The most important substrates of CK1 kinases are proteins that act in the regulatory nodes essential for tumorigenesis of hematological malignancies. Among those, the most important are the functions of CK1s in the regulation of Wnt pathways, cell proliferation, apoptosis and autophagy. In this review we summarize the recent developments in the understanding of biology and therapeutic potential of the inhibition of CK1 isoforms in the pathogenesis of chronic lymphocytic leukemia (CLL), other non-Hodgkin lymphomas (NHL), myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and multiple myeloma (MM). CK1δ/ε inhibitors block CLL development in preclinical models via inhibition of WNT-5A/ROR1-driven non-canonical Wnt pathway. While no selective CK1 inhibitors have reached clinical stage to date, one dual PI3Kδ and CK1ε inhibitor, umbralisib, is currently in clinical trials for CLL and NHL patients. In MDS, AML and MM, inhibition of CK1α, acting via activation of p53 pathway, showed promising preclinical activities and the first CK1α inhibitor has now entered the clinical trials. Phosphorylation, mediated by protein kinases, is a common posttranslational modification with 500,000 potential phosphorylation sites in the human proteome and 25,000 phosphorylation events described for 7000 human proteins . Since pWhereas some protein kinases have been long recognized as attractive targets, others have come to the forefront of biomedical research only recently. These include the family of casein kinase 1 (CK1). CK1 proteins are important regulators of signal transduction, involved in diverse signaling pathways and acting in most cell types. In human, the CK1 protein family includes seven isoforms, α, γ1, γ2, γ3, δ, ε and less well described α-like, with CK1α, δ and ε being the most studied to date . All CK1CK1 family members are involved in various cellular processes such as mitotic checkpoint signaling, DNA repair, apoptosis and p53 pathway, protein translation, circadian rhythm, endocytosis and autophagy, immune response and inflammation, centrosome-associated processes, and play a key regulatory role in several developmental pathways such as Wnt, Hedgehog, NF-κB or Yap/Taz signaling  binds to a specific Frizzled (FZD) receptor (there are 10 FZD genes [2+ pathway or the Wnt/planar cell polarity (PCP) pathway, in what is called the non-canonical pathway. The same Frizzled can bind either a ligand that preferentially activates the canonical  or the non-canonical  branch of the Wnt signaling [CK1ε and redundantly functioning CK1δ are well-established positive regulators of Wnt signaling pathways ,18,19,20ZD genes ), a co-rignaling .2+ release but also, for example, phosphorylation of NFAT [Dishevelled (DVL) is a phosphoprotein and a crucial CK1δ/ε substrate that is required for both canonical Wnt/β-catenin and non-canonical Wnt signaling. Wnt activation stimulates CK1δ/ε kinase activity, promoting phosphorylation of DVL that results in the signal propagation and downstream effects ,17,33. I of NFAT  or NF-κB of NFAT . AlthougOf note, all members of CK1 kinase groups  have been described to have distinct and non-redundant functions in vivo . For exaBecause of the abovementioned involvement of different CK1 isoforms in key biological processes, the CK1 kinases have been connected to the pathogenesis of diverse disorders, including cancer ,12. In p50s demonstrated efficacy in vitro and in preclinical in vivo models of breast cancer . Some C SR-3029 ), which  SR-3029 .Currently, hematological malignancies represent the only therapeutic area of cancer research where kinase inhibitors targeting the CK1 family have reached the clinical stage (see below). To orient the reader, we have summarized  the propSection 550 = 6 μM for CK1δ) [50 = 1.0 μM for CK1 δ/ε, IC50 = 16 μM for CK1α) [50 = 200 nM and 500 nM, respectively) [The first described CK1 inhibitors CKI-7 (ICor CK1δ)  or IC261or CK1α)  were notor CK1α) . Howeveror CK1α) . D4476, ctively)  has beenctively)  and in cctively) , and has50 = 32 nM and 711 nM respectively) [The newer and more selective generation of inhibitors—PF-670462 and PF-4800567—have helped to isolate and distinguish the biological effects of CK1δ and ε, mainly in the studies on circadian rhythm ,55. To dctively) . The lisOverall, no isoform-specific CK1α or CK1γ inhibitors have been produced so far . The potCLL is a lymphoproliferative disease characterized by clonal expansion of mature antigen-experienced CD5+ B cells in the peripheral blood (PB), lymphoid tissue and bone marrow (BM) . It is tThe multiple links between Wnt signaling pathways and B-cell leukemia and lymphoma have been reviewed recently . Here, wMany non-canonical Wnt pathway components, including CK1δ/ε and their substrates DVL proteins, are overexpressed in CLL ,32,62,63The ROR1-driven non-canonical Wnt pathway is involved in the communication of CLL cells with their microenvironment ,79. The In addition to gene expression and in vitro studies, the key role of the non-canonical Wnt pathway in CLL pathogenesis was confirmed by independent genetic experiments in the Eμ-TCL1 mouse model of CLL ,90: OverCK1ε and CK1δ act downstream of these receptors and represent a possible therapeutic approach for CLL and other B cell lymphomas. Indeed multiple CK1 inhibitors—D4476, PF-4800567 and PF-670462—were able to block efficiently CXCL12 and CCL19-induced chemotaxis of CLL cells ,30 and DIn the context of CLL, it is important that an orally bioavailable phosphoinositide-3-kinase delta (PI3Kδ) inhibitor, umbralisib , currently being studied in several hematologic cancer indications, was identified to also act, at higher concentrations, as a CK1ε inhibitor . It is cA FDA filing for umbralisib for patients with marginal zone lymphoma (MZL) and follicular lymphoma (FL) was recently accepted for review ,94. In cTwo recent studies have assessed the possible contribution of CK1ε inhibition to the clinical effects of umbralisib by comparing umbralisib to duvelisib and/or idelalisib, two approved PI3K inhibitors devoid of CK1 inhibitory activity ,96. In tIn the second study , authorsIn summary, umbralisib is a very selective dual inhibitor. Despite relatively lower potency as a CK1ε inhibitor, clinical data from umbralisib show that CK1ε inhibition is well tolerated and brings therapeutic benefit in CLL, opening the possibility that a more potent and selective CK1δ, CK1ε or CK1δ/ε inhibitor could demonstrate activity especially in combination with other compounds and become an important drug for the treatment of hematological cancers.+ B-cell malignancies such as MCL.The majority of cancer treatments today are comprised of combinations of multiple drugs with distinct mechanisms of action. In CLL and MCL, for example, both, BCR and non-canonical Wnt pathway mediated by WNT-5A/ROR1, represent intensively exploited therapeutic targets ,67,69. BInterestingly, umbralisib, the PI3Kδ and CK1ε dual inhibitor discussed in the previous section, has recently been tested in CLL and MCL relapsed or refractory patients, in combination with ibrutinib in presence  or absenThe therapeutic potential for CK1 inhibitors in hematologic malignancies is not limited to CLL and MCL. Another interesting clinical potential for CK1 inhibition comes from studies on the role of CK1α in MDS and AML. These diseases vary greatly from CLL or MCL that originate from mature B cells.MDS represents a diverse spectrum of disorders characterized by variable degrees of refractory cytopenia and morphological dysplasia of precursor and mature BM blood cells . It is aMDS can transform to AML in up to 30% cases . AML, siMDS and AML share a number of common features, including a set of recurrent cytogenetic aberrations, gene fusions and mutations  that havCSNK1A1) is located in the region commonly deleted in MDS and AML–del(5q32) [CK1α encoding gene (el(5q32) ,108. Chrel(5q32) ,110,111.el(5q32) .Csnk1a knockout in HSC resulted in rapid lethality (5–17 days) due to BM failure accompanied by the increased number of cells exiting quiescence and entering cell cycle, followed by apoptosis induction. Csnk1a haploinsufficient cells, however, recovered and managed to outgrow the control cells despite initially slower repopulation in secondary/tertiary transplants. Importantly, Csnk1a haploinsufficient cells were more sensitive to a small-molecule CK1 inhibitor D4476 [Csnk1a deficient cells in the BM.The effects of CK1α haploinsufficiency on adult hematopoiesis has been studied by Schneider et al.  using a or D4476  that cauCsnk1a revealed signatures of increased p53 activity and myeloid differentiation, well in line with the known role of CK1α as a regulator of p53 pathway [Csnk1a silencing. This is a key observation that pointed towards two important facts: (i) the role of CK1α in AML LSCs regulation is complex but likely more connected to p53 pathway than to Wnt/β-catenin pathway, a known driver of AML [Additional evidence came from the short hairpin RNA (shRNA) screen in a MLL-AF9 mouse model of AML  that ide pathway ,117,118. pathway  could inr of AML  that is It has been shown that Wnt/β-catenin signaling pathway is required for self-renewal and survival of AML LSCs ,121,122 TP53, encoding p53, are not common in AML/MDS patients and represent 5–20% de novo cases, however the proportion is increased in the older patients, in AML transformed from MDS, in cases carrying del(5q) or complex karyotypes or in the therapy-induced AML (chemotherapy/irradiation) [TP53 but can be also synergistic with the approaches focused on the restoration of mutant p53 function [Mutations in diation) ,125. Ovefunction ,127.CSNK1A1 mRNA expression was not affected. This mechanism seems to be rather universal as researchers confirmed their findings in multiple human cell lines and in the BM and PB of AML patients treated in vivo by lenalidomide. Importantly, lenalidomide shows therapeutic effect in del(5q) MDS but failed in AML clinical trials [CSNK1A1 [CSNK1A1 or in del(5q) AML that often carries TP53 mutations. In theory, this can be overcome, at least in TP53 wild type cases, by direct inhibition of CK1α.The importance of CK1α in del(5q) MDS was uncovered by the studies attempting to understand why lenalidomide is therapeutically active only within this patient subgroup ,128. Lenl trials . The rea[CSNK1A1  but failTargeting CK1α thus seems to be a promising approach for the treatment of MDS and AML that was, until recently, hampered by the lack of suitable CK1α inhibitors. This issue has been addressed by Minzel, Venkatachalam and colleagues , who recCK1 kinases are critically involved in multiple cellular processes that appear to be valuable therapeutic targets in leukemia. No specific CK1 inhibitors, however, have reached the clinical stage yet, although two compounds that among others target also CK1 are currently in the clinical trials. Umbralisib, a dual PI3Kδ and CK1ε inhibitor, is currently under review for approval by the FDA for indolent non-Hodgkin lymphoma. Second, BTX-A51 that targets CK1α, CK1δ, CK1ε, CK1γ and also multiple other kinases including CDK7 and CDK9  is in a Both the mechanism of action and the CK1 isoforms targeted by these two drugs are different. In CLL, the CK1δ/ε inhibition slows down disease progression in preclinical models, likely via inhibition of the non-canonical Wnt pathway and by disruption of the microenvironmental interactions . UmbraliSeveral pan-CK1 or CK1δ/ε dual inhibitors appear to be well tolerated in mouse ,30,134 aIt remains to be studied which molecular and cellular mechanisms dominantly contribute to the effects of CK1 inhibition in preclinical models and in clinical trials. Their actions can be cell intrinsic as well as more complex including interaction with the microenvironment and cell-cell communication. CK1 is a critical regulator of multiple signaling pathways including Wnt, Hedgehog and Yap/Taz . CK1Last but not least, the potential use of CK1 inhibitors is not limited to the hematological malignancies discussed in detail in the previous chapters. There is growing evidence for the critical role of CK1α in multiple myeloma (MM), which is a cancer of plasma cells. CK1α has been proposed, based on the shRNA-mediated gene knockdown and treatments with D4476 inhibitor as a critical regulator of MM that promotes plasma cell proliferation and survival . DepletiIn summary, individual CK1 kinases appear to be essential players in multiple hematological malignancies. They act by various mechanisms and provide therapeutic opportunities for several poorly manageable clinical conditions. However, further preclinical research and clinical trials are needed to assess the full potential of CK1 inhibition in hematologic cancers.