Ca2+ tends to be the catalyst for mitochondrial dysfunction, contributing to the synaptic deficiency seen in brain injury. Additionally, emerging data have shown that PP2A-activating drugs (PADs) suppress inflammatory responses by inhibiting different signaling pathways, indicating that PADs may be beneficial for the management of neuronal damage. In addition, a few bioactive compounds have also triggered the activation of PP2A-targeted drugs for this treatment, and clinical studies will help in the authentication of these compounds. If the safety profiles of PADs are proven to be satisfactory, there is a case to be made for starting clinical studies in the setting of neurological diseases as quickly as possible. The complex etiopathogenesis of brain injury associated with neurodegeneration has sparked a lot of studies in the last century. These clinical situations are incurable, and the currently available therapies merely act on symptoms or slow down the course of the diseases. Effective methods are being sought with an intent to modify the disease, directly acting on the properly studied targets, as well as to contribute to the development of effective therapeutic strategies, opening the possibility of refocusing on drug development for disease management. In this sense, this review discusses the available evidence for mitochondrial dysfunction induced by Ca Neuronal damage associated with brain injury and neurodegenerative diseases (ND) have an increasing incidence at worldwide level, and are currently conceived as having a permanent, incurable, and progressive nature, linked to a marked loss in neuronal activity ,2. The rIn the past two decades, extensive research has been conducted to understand the main triggering factors of the various and complicated pathways of neuronal damage associated with ND, which despite the advances stated so far are not too significant. Aging is conceived as the primary risk factor, despite of its multifactorial nature, involving protein misfolding, neurotoxic oligomers, mitochondrial dysfunction, protein modifications, protein deposition, oxidative stress, environmental factors, metal accumulation, and genetic factors. Over time, the list of triggering factors for these clinical situations is increasing, but the exact cascade of events that culminate with neuronal death has not yet been well understood. For almost every type, diverse hypotheses on the mechanisms responsible for neuronal dysfunction have been offered . NonetheThe classification of these pathologies is usually based on: (1) clinical symptoms which are determined by the evaluation of the anatomic regions where neuronal dysfunction is stated; and (2) by detecting the biochemical modification and deposition of intracellular or extracellular proteins in glial cells or neurons. This clinical classification, besides other benefits, is very helpful for estimating the early symptoms and for clinical identification . RegardiStrokes cause high mortality rates around the world, being one of the main factors of disability in adults. That is why it is a priority to seek new therapeutic strategies to treat these situations since the existing ones are very limited. A cerebrovascular accident is produced by an abrupt reduction in the supply of oxygen and nutrients, either due to hemodynamic failure due to a lack of irrigation or due to cerebral hemorrhage. It is estimated that if the blood flow decreases by more than 75% for a period of more than 30 min, the death of neurons and the glia occurs. This ischemic nucleus is surrounded by a zone of penumbra (twilight zone), with reduced blood flow, but enough to be able to recover if normal perfusion is re-established in less than six hours . If not,There are numerous mechanisms that participate in the cellular damage produced during cerebral ischemia. Initially, the energy failure triggered by the deficit of oxygen and glucose alters the maintenance of the membrane potential, producing a depolarization that causes the opening of ion channels, the release of neurotransmitters and the alteration of the activity of the transporters . Many obStudies carried out in patients affected by cerebral ischemia have made it clear that the increase in glutamate in the brain is critical for causing neuronal damage . HoweverThe loss of cell membrane integrity that occurs during cell death not only causes the uncontrolled release of glutamate into the extracellular space, but also of other substances that can have detrimental effects on still viable cells. There is evidence that shows that the volume of ischemic damage is proportional to the amount of amino acids, not exclusively neurotoxic, released during ischemia, although to date the underlying mechanism of action has not been demonstrated ,25. One Presently, the most common ND in seniors is AD, the major cause of dementia at a worldwide level and accounting for approximately 60–80% of all dementias . AD is gParkinson’s disease (PD) is another progressive ND after AD, which is caused by a loss of nigrostriatal dopamine neurons and an accumulation of the intracellular protein α-synuclein. The lack of dopamine in the basal ganglia results in classical Parkinson’s motor symptoms, e.g., rigidity, bradykinesia, tremor, and lateral postural instability, along with non-motor symptoms that are also present in PD, which become troublesome in the later stages of PD . Huntington’s disease (HD) is characterized by motor, psychiatric, and cognitive disturbance. It is triggered by an autosomal dominantly inherited trinucleotide (CAG) repeat expansion in the gene huntingtin (HTT) on chromosome 4, and as a result, abnormal HTT protein or mutant HTT (mHTT) protein is produced . It is eAmyotrophic lateral sclerosis (ALS), is characterized as a multisystem ND, widely known as a pure motor neuron disease ,38,39. AThe effect of socioeconomic status on old-age dementia was also evaluated by Haselgren and colleagues. More specifically, they investigated the exposure of a few work environments, closely linked to socioeconomic status, on dementia with APOEε4 allele . Data ob2+) concentration regulates different cellular functions, such as metabolism, secretion, fertilization, gene expression, proliferation and neural excitation c) can cause the dysregulation of intracellular Ca2+ homeostasis c as a signal. The concept of Car signal . After anjection . The pro2+ signaling, although, in excitable cells, the initial chemical stimulus can result in membrane excitability, which in turn also stimulates the Ca2+-signaling pathway. Specialized channels and pumps are necessary for the proper functioning and mobilization of Ca2+ in cytoplasmic and extracellular spaces. It must be stressed that the likelihood of a high Ca2+ micro-domain increases drastically with concomitance in space and time of the activation of several Ca2+ channels on action potential firing. This localized Ca2+ is favored by the Ca2+-induced Ca2+ release (CICR) from the ER, through both inositol triphosphate (InsP3R) and ryanodine (RyR) receptors c micro-domains that enable the regulation of various functions, using the same triggering signal but different time patterns and subcellular locations. In normal excitable cells, Ca2+ homeostasis is attained by fluxes occurring between three compartments, namely extracellular space, cytosol, and Ca2+ storing organelles. At the resting phase, the exchange rate of Ca2+ at the plasmalemma and ER membrane ranges from 1–10 µmol/l cells/s, whereas Ca2+ uptake through the mitochondrial Ca2+ uniporter (MCU) is extremely slow due to its exponential kinetics and poor Ca2+ affinity. However, at homeostasis, the [Ca2+]c content is around 10−7 M in the mitochondrial matrix and cytosol, and 10−3 M at the ER lumen and extracellular space, so a very high gradient (104-fold) is required for the diffusion of Ca2+ in the cytosolic region , they regulate Ca2+ homeostasis in cells [2+]c level increases, activating Ca2+ transport through the MCU, and, in that case, most of Ca2+ load is taken up by subcellular organelle mitochondria [2+ stored in mitochondria is effluxed to the cytosol within a few seconds or minutes [2+]c level remains very high during this process, helping to mobilize secretory vesicles from the reserve pool to the membrane and thus to be ready for the next exocytotic events . A. A113]. 2+ accumulative capacity of mitochondria upon histamine stimulation, monitoring maximal calcium peak, the area under the curve, and clearance rate, due to the mNCX blockade m. Hence, compound ITH16662 was selected as the best NCLX blocker of the family, with a similar potency to the hit CGP37157, slightly neuroprotectant, but with much better solubility than previous NCLX ligands. As was observed with ITH12575, it scarcely affected depolarizing-stimulated Ca2+ currents, thus confirming a lesser off-target behavior. Being a more promising therapeutic candidate, it has been extensively studied in several pharmacological models. For instance, ITH12662 was used to assess the role of mitochondria in the secretory catecholamine release mediated by cytosolic Ca2+ levels. The C57BL6J mice chromaffin cells stimulated with short ACh pulses manifested a secretory catecholamine release that, as expected, slowly decayed after repeated pulses [2+ affinity, mitochondria-targeted aequorin that ITH12662 diminished mitochondrial Ca2+ efflux, but to a lesser extent than CGP37157. However, ITH12662 was proved to have the opposite regulatory actions on ACh-mediated catecholamine release to those found with CGP37157. It was hypothesized that ITH12662, by slightly reducing the release of Ca2+ from the mitochondria to cytosol without affecting other calcium transports, would be more efficiently buffering the Ca2+ levels necessary to refill and prepare ACh vesicles to pool, ready to be released by exocytosis. By contrast, the blockade of both nicotinic currents and K+ pulses performed by CGP37157 seemed to be detrimental for catecholamine release. This differential behavior underlines the high contribution of the VGCC blockade evoked by CGP37157 in its whole pharmacological effect on the cell physiology of excitable cells, which overpasses any intracellular action, such as its activity as an NCLX blocker. , which subsequently form the so-called neurofibrillary tangles (NFT), one of the chief morphological hallmarks of AD in patients’ brains . The relIn addition, PP2A dephosphorylated key amino acids are implicated in dissociation from microtubules and self-aggregation. Interestingly, the phosphorylating activity of PP2A in AD patients’ brains is compromised . These fHowever, the reality is that phosphatase enzymes are not biological targets selected for drug discovery projects, as happens for kinases. The rich therapeutic battery of compounds able to inhibit kinase enzymes has given rise to the so-called “kinoma”, where the hundreds of phosphorylating enzymes described are studied and shown in phylogenetic trees together with their pharmacology, and the therapeutic implications. There is a simplified belief that views phosphatase enzymes as very unspecific enzymes, so a therapeutic intervention would lead to many non-desired secondary effects . IncreasFor instance, the entire active PP2A is a trimer formed by: (a) a catalytic scaffold subunit called PP2A-A, having two possible polymorphisms, the Aα and the Aβ; (b) a subunit in charge of the enzymatic hydrolysis of phosphate esters from amino acids, called PP2A-C, that is also present in two possible polymorphisms, i.e., C′ and C″; and (c) a subunit with a regulatory role, called PP2A-B, which can be found in about 30 different polymorphisms, divided into four big subgroups  ,128. ThuRight after the synthesis of the catalytic PP2A-C subunit, it is bound to α4 and PP2A-methylesterase-1 (PME-1) enzymes that prevent PP2A activation . This laRegulatory mechanisms for PP2A are reinforced by the action of several peptides that operate as endogenous inhibitors, such as I1PP2A and I2PP2A  . This imThe most extended family is that of SET inhibitors , among wSeveral drugs have been claimed to inhibit CIP2A . The penThe second family of PP2A activators comprises drugs that affect the post-translational regulatory mechanisms of PP2A. The phosphorylation of PP2A, mainly at Thr304 or Tyr307, hinders the formation of the complete PP2A trimer. In this context, forskoline , indepenOtherwise, there are many other drugs described to exert an up-regulatory action on PP2A through unknown action mechanisms. Such is the case of melatonin, whose pharmacodynamic actions include counteracting the glutamate-elicited excitotoxicity by mitigating the under-expression of the PP2A-B subunit . In a reThe marine toxin OA represents a reliable model of neurodegeneration with its origin in tau hyperphosphorylation, due to it producing a huge and highly selective inhibition of PP2A . ConsideWe used OA to induce tauopathy and therefore neurodegeneration in SH-SY5Y neuroblastoma cells. Among the family of tacrine derivatives disclosed, only ITH12246 dissipated the OA-associated loss of cell viability in a statistically significant manner. Moreover, ITH12246 reduced neurotoxicity exacerbated by OA in the same experimental model and abolished the loss of tissue viability of rat hippocampal slices challenged with oxygen and glucose deprivation followed by reoxygenation, or by the excitotoxicity developed by high glutamate . RealiziOverall, this new PAD offers neuroprotection against toxic stimuli related to PP2A depression, whose principal hallmarks are tau hyperphosphorylation and extended excitotoxicity. Using ITH12246 as an innovative drug to treat neurodegeneration, we probed its ability to mitigate cerebral damage in two in vivo models that resemble neurodegeneration, specifically in mice suffering a scopolamine-induced loss of memory, as a model of AD, and mice subjected to photothrombosis, as a model of focal cerebral ischemia . The ant2+-sensitive probe Fluo-4, we appreciate an important blocking effect on high K+-evoked depolarization, whereby we hypothesized these compounds were acting as VGCC antagonists, too. In most cases, those compounds capable of performing such a dual pharmacological, pro-PP2A and VGCC blockade, illustrated a very noticeable neuroprotective profile in the experimental in vitro model of the SH-SY5Y cells exposed to either OA or depolarizing stimuli such as veratridine. The best compounds of the series confirmed their neuroprotective capability in hippocampal slices challenged with glutamate. Molecular docking predicted that the new gramine derivatives posed in a binding pocket of PP2A-C defined by the amino acids His191, Ser120, Trp200, and Ile123, residues far from the catalytic site, but that it contributes to drug affinity. In addition, Western blot analyses proved that the most highlighted compounds such as ITH12657 or the chlorinated derivative ITH12691 reduced the OA-induced tau phosphorylation rate [According to the computational prediction based on molecular docking, the presence of an ionizable tertiary amine was preserved to favor interaction with PP2A, backed by the PP2A interactive behavior of some basic amines such as protamine. The aromatic indole scaffold presented in gramine would be highly stabilized (reducing the rate of the retro-Mannich reaction) if the aromatic nitrogen was alkylated. Thus, several substitutions were challenged, for instance, benzyl, n-butyl, propargyl, and so on, which in turn could generate an additional hydrophobic interaction with neutral amino acids present in the binding site of PP2A-C, far from the phosphate ester hydrolysis catalytic triad . Finallyion rate ,166 (FigNevertheless, the fact that this new family of real PAD possessed another significant pharmacological activity may be relevant from a therapeutic point of view, where the multitarget approach for the treatment of neurodegenerative diseases is a highly reliable strategy. However, from a pharmacological point of view, it does not clarify how the recovery of the PP2A activity contributed to the whole neuroprotective profile . For thiThe most recent step forward has been the incorporation of 7-azaindoles into hybrid systems based on clay minerals, to facilitate oral administration and their controlled release. We tested several nanocarriers, such as montmorillonite and halloysite nanotubes, but the real controlled release in systems simulating the gastro-intestinal tract was achieved by encapsulating the hybrid material into a biopolymer matrix composed of alginate and zein. Thus, the biononcomposite complexes encapsulating the 7-azaindole/clay hybrids enhance the pharmacokinetic features of this series of gramine azaderivates . OtherwiHence, small analogs of the C19–C27 fragment should interact similarly to this allosteric pocket. As proof of concept of this new hypothesis, we have recently tested an earlier intermediate, which we have named ITH12680, that already possesses structural similarity to the central fragment of OA . Despite its simplicity, ITH12680 generated an appreciable recovery of PP2A activity, measured by a more accurate malachite green method, using several polymorphisms of PP2A isolated by pull-down protocols . The PADMany cellular homeostatic functions, ranging from energy generation to cell death and necrosis, are regulated by calcium influx into the mitochondria. The new molecular identification of the NCLX, mitochondrial calcium uniporter complex has offered vital insight into the function mitochondrial calcium influx plays in energy production under an elevating workload, and in the progression of diseases such as neurological disorders. The present review discussed the necessity of understanding the processes that regulate mitochondrial calcium influx and activity in neurodegeneration. PP2A activity is downregulated in tumors as a proven tumor suppressor, and reactivation of the protein can cause cancer cells to die. Reduced PP2A activity promotes tau hyperphosphorylation and Aβ production in the brains of AD patients. Upstream mechanisms that inactivate PP2A in illnesses have not been fully explained, and more research will aid in the refinement and development of new PP2A-targeted drugs for the treatment of ND . There i


KMT2A rearrangement, DEK-NUP214 fusion, and NPM1 mutation are associated with the upregulation of HOX genes. The dominant tyrosine kinase activity of the mutant FLT3 or BCR-ABL1 fusion proteins is transduced by the AKT-mTOR, MAPK-ERK, and STAT5 pathways. Concurrent mutations of ASXL1 and RUNX1 are associated with activated AKT. Both TP53 mutation and mis-expressed MECOM are related to impaired apoptosis. Clinical data suggest that adverse genetic factors can be found in at least one in eight AML patients and appear to accumulate in relapsed/refractory cases. TP53 mutation is associated with particularly poor prognosis. Molecular-targeted therapies focusing on specific genomic abnormalities, such as FLT3, KMT2A, and TP53, have been developed and have demonstrated promising results.The European LeukemiaNet (ELN) criteria define the adverse genetic factors of acute myeloid leukemia (AML). AML with adverse genetic factors uniformly shows resistance to standard chemotherapy and is associated with poor prognosis. Here, we focus on the biological background and real-world etiology of these adverse genetic factors and then describe a strategy to overcome the clinical disadvantages in terms of targeting pivotal molecular mechanisms. Different adverse genetic factors often rely on common pathways.  FLT3) gene is described in association with nucleophosmin 1 (NPM1) mutation status, with only a high allelic ratio (>0.5) of FLT3-ITD considered a poor prognostic factor. Mutations in the runt-related transcription factor 1 (RUNX1) and additional sex combs such as the 1 (ASXL1) and tumor antigen p53 (TP53) genes were recently added as adverse genetic abnormalities in the 2017 edition. Many of the genetic factors described in this recommendation are closely related to one another. For example, the NPM1 mutation (NPM1mt) is considered to be a favorable factor only if FLT3-ITD is absent or has a low allelic ratio (<0.5) [ASXL1 and RUNX1 exert poor prognosis, particularly in younger adults [KIT) gene mutation is associated with worse outcome [KIT mutation because the negative effect can be negated if a three-log or deeper reduction in minimal residual disease is achieved after induction therapy [Patient-related factors  and treatment modality are unarguably essential in predicting the prognosis of acute myeloid leukemia (AML). Nevertheless, tumor-related genetic factors still have a major impact on prognosis ,2. The ro <0.5) . Concurr . Concur outcome ; althougAML with adverse genetic factors, as defined in the ELN classification, uniformly shows resistance to standard chemotherapy and is associated with poor prognosis. Here, we focus on the biological background and real-world etiology of the adverse genetic factors and then describe a strategy to overcome the clinical disadvantages in terms of targeting pivotal molecular mechanisms. In a comprehensive survey of the available preclinical literature, we searched the literature using the key words “ELN”, “FLT3”, “NPM1”, “DEK-NUP214”, “KMT2A”, “BCR-ABL1”, “GATA2”, “MECOM”, “EVI1”, “RUNX1”, “ASXL1”, “TP53”, “complex karyotype”, “menin”, and “anti-cd47 antibody” with the necessary key word “AML”. A systematic literature search was performed using PubMed and the Tip Medical Database, identifying a total of 1839 records including 92 controlled trials, 90 systematic reviews, and 1228 ongoing clinical trials. We chose 169 records according to the theme of this review article.NUP214) gene or t is a distinct WHO classification category of AML [DEK gene has been clinically noted in many human cancers [HOX) family genes, such as homeobox protein A9 (HOXA9), HOXA10, HOXB3, HOXB4, and PBX homeobox 3 (PBX3), is highly upregulated. Other studies have suggested that DEK-NUP214 is associated with the upregulation of mechanistic targets of rapamycin (mTOR) [Normal AML with the fusion of the DEK proto-oncogene (DEK) with the nucleoporin 214 y of AML . DEK proy of AML . The upr cancers . The NUP cancers ,14. Xeno cancers . In the n (mTOR)  and the n (mTOR) .KMT2A) gene, previously known as mixed lineage leukemia 1 (MLL), is a proto-oncogene that encodes a histone methyltransferase essential in hematopoiesis [HOXA9 gene transcription [KMT2A gene forms oncogenic fusions with a variety of partners, including mixed-lineage leukemia translocated to chromosome 3 (MLLT3), AF4/FMR2 family member 1 (AFF1), and the afadin adherens junction formation factor (AFDN) [KMT2A are commonly found in AML (approximately 5–10%) and are associated with poor prognosis [HOXA9 and myeloid ecotropic viral insertion site 1 (MEIS1) [MEN1) tumor suppressor gene, is critically involved in leukemogenic mechanisms by linking KMT2A protein with the lens epithelium-derived growth factor (LEDGF) on target genes [The lysine methyltransferase 2A  . Rearranrognosis ,22. Recerognosis ,24 throu (MEIS1) ,26. Meniet genes . ABL gene location, and chromosome 22q11, the breakpoint cluster region protein (BCR) gene location [BCR-ABL1-encoded fusion protein  is associated with chronic myeloid leukemia (CML), while the small molecular weight fusion protein  is associated with acute lymphoblastic leukemia (ALL). The oncogenic BCR-ABL1 fusion protein is located in the cytoplasm and phosphorylates a variety of proteins [BCR-ABL1-dependent proliferation of CML cells [BCR-ABL1-positive cell lines [ABL proto-oncogene 1 (ABL1) is a non-receptor tyrosine kinase involved in cell growth and survival. Normal ABL is principally located in the nucleus and regulates DNA damage-induced apoptosis ,29, as wlocation . The larproteins . Major BML cells . Major BML cells , which tML cells . Major BML cells  and the ML cells . Recent ll lines ,38.BCR-ABL1 fusion gene is a rare entity, accounting for approximately 1% of all AML subtypes [BCR-ABL1-positive AML. A summary of published BCR-ABL1-positive AML cases showed that slightly less than 40% of cases belonged to AML not otherwise specified (AML-NOS) and approximately a quarter of the cases were found to be AML with recurrent genetic aberrations such as CBF-AML and AML with an NPM1 mutation [BCR-ABL1 status, more than three quarters of the BCR-ABL1-positive AML cases were classified as belonging to the non-favorable risk group [BCR-ABL1 itself has not been confirmed.Normal AML with the subtypes , and is subtypes ,41. WhilMDS1 and EVI1 complex locus (MECOM) gene, formerly known as ecotropic virus integration site 1 protein homolog (EVI1), is located in 3q26 and encodes the transcription regulator protein EVI1 which binds to the promoter region of target genes. EVI1 protein regulates apoptosis through Jun N-terminal kinase (JNK) and tumor growth factor (TGF)-beta signaling [MECOM gene has been observed in AML with 3q-rearrangement [MECOM gene in AML with 3q-rearrangement was induced by the recruitment of a GATA2-distal hematopoietic enhancer (G2DHE) to the proximity of the MECOM gene, which subsequently disturbed the function of an allele of the endothelial transcription factor GATA-2 (GATA2) gene [GATA2 is known to be associated with bone marrow failure, immunodeficiency, lymphedema, and organ dysfunction as well as MDS and AML [GATA2 gene, which is caused by the mis-expression of EVI1 protein, is expected to play a key role in the leukemogenesis of AML with 3q-rearrangement.The chromosomal translocation and inversion between 3q21 and 3q26 (3q-rearrangement) are well-known recurrent genetic abnormalities in myelodysplastic syndrome (MDS) and AML . The MDSignaling ,44. Incrangement . KatayamA2) gene . The GATA2) gene . The ger and AML ,48,49. CNRAS mutation.3q-rearrangements, such as t or inv, are rare cytogenetic abnormalities that are found in approximately 4% of AML  and are In the ELN classification, a complex karyotype is defined as three or more chromosomal abnormalities in the absence of the WHO-designated recurring translocations or inversions, such as t, inv(16) or t, t9;11), t, t, inv(3), or t, whereas the UK National Cancer Research Institute Adult Leukaemia Working Group requires four or more chromosomal abnormalities as an adverse risk factor [;11, t transcription [NPM1 mutations are concentrated in the nuclear export signal (NES) domain, which enables the export of the protein from the nucleus to the cytoplasm, mutant NPM1 protein inappropriately accumulates in the cytoplasm [NPM1 mutation leads to the cytoplasmic localization of several nuclear proteins associated with DNA repair and apoptosis [HOX genes has been reported in AML with NPM1 mutation [NPM1-mutated AML through upregulating HOXA, HOXB, and MEIS1 [Nucleophosmin 1 (NPM1) protein is involved in a variety of cellular mechanisms including ribosome nuclear export , the chacription . Overallytoplasm . Indeed,poptosis , as wellpoptosis ,74. The mutation . Recent nd MEIS1 ,77,78.FLT3-ITD—a common pattern of activating mutation that leads to the extension of the juxta-membrane domain of FLT3 tyrosine kinase—and NPM1 mutation have opposite prognostic impacts in AML: NPM1 mutation can be a favorable factor only in the absence of FLT3-ITD, and the presence of NPM1 mutation attenuates the adverse prognostic impact of FLT3-ITD [FLT3 from FLT3-ITD with a low allelic ratio (FLT3-ITDlow), some reports have suggested that concurrent NPM1 mutation and FLT3-ITDlow is not related to favorable outcomes [FLT3-mutated AML—indicated that the first-generation FLT3 inhibitor was equally effective for all ELN risk groups regardless of NPM1 mutation status [FLT3-ITD . Althougoutcomes . In termn status . RUNX1 gene silencing results in the upregulation of TP53, which stabilizes the RUNX1 protein via the enhanced expression of the CBFB gene [The runt-related transcription factor 1 (RUNX1), previously known as acute myeloid leukemia 1 (AML1) or core-binding factor subunit alpha-2 (CBFA2), protein forms a heterodimeric complex, called CBF, with the core-binding factor B (CBFB) protein. The RUNX1 protein recognizes certain DNA sequence patterns, such as 5′-TGTGGT-3′, in the target domain and CBFB allosterically enhances RUNX1 function. The CBF complex interacts with promoters and enhancers of a variety of genes and plays an essential role in normal hematopoiesis . A recenBFB gene . RUNX1T1), previously known as eight twenty one (ETO), plays a critical role in the leukemogenesis of CBF-AML via the aberrant transcription factors that contain oncogenic RUNX1-RUNX1T1 or AML1-ETO, fusion proteins [RUNX1-RUNX1T1 fusion gene, or t translocation, is generally considered a favorable prognostic factor in AML [RUNX1 mutation, accounting for 10% of newly diagnosed AML, was independently associated with poor prognosis and co-mutations with epigenetic modifiers ) and/or spliceosome-related genes  and plant homeodomain-like finger (PHF6)) predicted notably worse outcomes [RUNX1 and ASXL1 promoted cell renewal in bone marrow and leukemic transformation, which was induced by the augmented production of hypoxia-inducible factor 1 subunit alpha (HIF1-α) and the consequent upregulation of the inhibitor of DNA binding 1 (ID1) and AKT1 signaling [The pathogenic fusion of RUNX1 with its partner transcriptional co-repressor 1  and is a component of the Polycomb repressive deubiquitinase (PR-DUB) complex. PR-DUB removes monoubiquitylation of the 119th lysine residue in histone H2A (H2A-K119Ub) and is degraded following 6 mA formation . H2A-K11risk MDS . OverallASXL1 gene, mainly in exon 11 or 12, is found in approximately 10% of AML and is more prevalent in MDS (10–25%) and CMML (40–50%). ASXL1 mutation is known to be mutually exclusive with DNA-methyltransferase 3 alpha (DNMT3A), NPM1, and splicing factor 3b subunit 1 (SF3B1) mutations [Asxl1 along with Tet methylcytosine dioxygenase 2 (Tet2) mutation initiated MDS-like hematopoietic impairment [Kras and Nf1 accelerated the development of myeloid leukemia [ASXL1 mutation is known as an independent adverse prognostic factor of AML [RUNX1 is particularly associated with worse prognosis [Nonsense mutation or the frame-shift of the utations . In mouspairment ,96, and leukemia . ASXL1 mr of AML  and concrognosis .ARF, an isoform product of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene that is upregulated in response to cell proliferation signals, inhibits MDM2 and thereby activates TP53 [TP53 protein acts as a major tumor suppressor in many tumor types. TP53 regulates cell division by activating inhibitors of cyclin-dependent kinases   and indutes TP53 . TP53 mutation is found in less than 10% of normal AML and 30–40% in secondary AML [TP53 mutation in AML is generally associated with complex karyotypes and universally poor prognosis [DNMT3A, TET2, and ASXL1 [TP53-mutated secondary AML and MDS are associated with the increased expression of programmed death ligand 1 (PD-L1), a major immune checkpoint molecule that leads to the anergy of effector T cells on hematopoietic stem cells and decreased numbers of cytotoxic/helper T cells in the bone marrow [dary AML . TP53 murognosis . TP53-mund ASXL1 . Interese marrow . HOXA9 is a common phenomenon in AML with KMT2A rearrangement, NPM1 mutation, and DEX-NUP214 fusion. The aberrant tyrosine kinase activity of FLT3-ITD and BCR-ABL1 fusion affects three major signaling pathways: the AKT-mTOR, MAPK-ERK, and STAT5 pathways. Suppressed apoptosis is caused by TP53 mutation and MECOM mis-expression.The pro-leukemic mechanisms of adverse genetic factors are schematically summarized in RUNX1 mutation (5.1%) and the rearrangement of the KMT2A gene (3.0%). Five-year OS was 59.1% in the favorable risk group, 32.6% in the intermediate risk group, and 22.6% in the adverse risk group.JALSG AML201 was a Japanese multi-center phase 3 randomized study evaluating the equality of standard-dose idarubicin and high-dose daunorubicin as induction therapies, as well as high-dose cytarabine and standard-dose chemotherapies as consolidation therapies in previously untreated AML ,110. TheThis section is composed of a detailed analysis of published data from the Japanese genomic sequencing study HM-SCREEN-Japan 01 ,112. Alt® was performed for patients with AML. The inclusion criteria were patients with refractory/relapsed AML (R/R) or with previously untreated AML who were ineligible for standard therapy (ND unfit). The available specimens were paraffin-embedded bone marrow clots. The submission of archival specimens was allowed. After submission, annotated genomic reports were returned to the participants. Patients with the FLT3 mutation were allowed to submit specimens multiple times at different opportunities. Clinical information was gathered, including age, sex, treatment modality, response, stem cell transplantation status, and survival information. HM-SCREEN-JAPAN 01 was a multicenter genomic profiling study in which next-generation sequencing by FoundationOne HemeFLT3-ITD, and NPM1 mutation. However, these PCR data were not available in the HM-SCREEN-JAPAN 01 study. To supplement the lack of data on FLT3-ITD and NPM1, “classification by conventional cytogenetic tests” was defined as that with G-band karyotyping plus FLT3-ITD and NPM1 information obtained from NGS analysis. Due to the lack of FLT3-ITD allelic ratio assessment, we considered all patients with FLT3-ITD and NPM1 wild type as belonging to the adverse risk group. NGS was performed on bone marrow samples either at the time of diagnosis or relapse.Using conventional cytogenetic tests and next-generation sequencing (NGS), the patients were classified as belonging to non-adverse (favorable or intermediate) or adverse risk groups according to the 2017 ELN recommendation. In Japan, commercially available cytogenetic tests include G-band karyotyping, polymerase chain reaction (PCR) for recurrent rearrangements, FLT3-ITD in the absence of mutated NPM1 , and KMT2A gene rearrangement . Next, we performed NGS-based classification. The timing of NGS analysis was at diagnosis in approximately half of the patients (48.8%). Contrary to the conventional cytogenetic tests, NGS detected an additional 88 adverse gene aberrations, resulting in the re-classification of 39 non-adverse risk patients as adverse risk. Consequently, 66 (39.3%) patients were grouped as non-adverse risk and 102 (60.7%) as adverse risk  were detected in the samples at diagnosis. Notably, one re-classified patient harbored the sole TP53 mutation in the specimen at diagnosis without adverse chromosomal abnormalities. Co-mutation was most frequently observed with ASXL1 and RUNX1 in 5/19 (26.3%) patients. Among the 63 patients in the adverse risk group who were not re-classified, NGS detected 49 adverse gene aberrations, of which TP53 mutation was the most frequently found in 32 (50.8%) patients. The clinical and cytogenetic characteristics are summarized in rse risk . Of the ASXL1  and RUNX1  mutations were found less frequently in the 63 patients in the adverse risk group based on the conventional cytogenetic tests. Moreover, the majority of TP53 mutations (72%) co-occurred with a complex karyotype or a specific aneuploidy involving chromosomes 5 and 7.Contrary to the re-classified patients, p = 0.01) and a 2-year PFS of 32.3% . With NGS analysis, prognosis was similarly discriminated between the non-adverse and adverse groups; a 2-year OS of 68.1%  and a 2-year PFS of 47.9%  in the non-adverse group versus significantly worse outcome in the adverse group, with a 2-year OS of 43.5%  and a 2-year PFS of 36.5% . Of note, patients with mutated TP53 had particularly unfavorable outcomes with a 2-year OS of 24.1%  and a 2-year PFS of 17.1% . Patients with mutated TP53 who received HSCT exhibited significantly improved OS compared to those who did not .After enrollment, many of the patients were treated with non-intensive treatment (64.9%) or best supportive care (9.5%), while 24.4% were treated with intensive chemotherapy. Fifty-nine patients (35.1%) underwent subsequent allogeneic hematopoietic stem cell transplantation (HSCT). When classified based only on the conventional cytogenetic tests, 2-year OS and 2-year progression free survival (PFS) in the non-adverse group was 61.6% ; 49.3–71.8%) and 46.1% , respectively, while significantly worse outcomes were observed in the adverse group, with a 2-year OS of 39.7% , followed by consolidation with high-dose cytarabine) in combination with midostaurin showed longer OS and event-free intervals than chemotherapy alone in patients newly diagnosed with FLT3-mutated AML . The comAL trial . SimilarAL trial . The novAL trial ,118,119.ated AML . A phaseated AML . Front-lated AML , and itsated AML .FLT3-ITD and BCR-ABL1 fusion described above, the amplification of AKT signaling can be caused by KIT activation [The PI3K-AKT-mTOR, RAS-MAPK-ERK, and JAK2-STAT5 pathways are major downstream signals of a variety of tyrosine kinases, including FLT3 and ABL1. The amplification of the PI3K-AKT-mTOR pathway is seen in at least 60% of patents with AML . In additivation , RAS muttivation , and thetivation . Althougtivation ,129, duativation ,131,132.FLT3 and JAK2 , as well as the functional loss of the suppressor of cytokine signaling-1 (SOCS1) leads to the constitutive activation of JAK2-STAT5 signaling in AML [FLT3-mutated AML in their first relapse in a phase 3 trial [Increased levels of phosphorylated JAK2 and STAT5 were noted in AML patient’s bone marrow samples ,134. Actg in AML . To dateg in AML , the comg in AML . The seq 3 trial . Pacriti 3 trial . NRAS, KRAS, PTPN11, NF1, and KIT are commonly found in patients with AML, and RAS-MAPK-ERK signaling is often highly activated [Mutations in genes that regulate the RAS-MAPK-ERK pathway, such as ctivated . Howeverctivated ,142,143.ctivated ,145. Tamctivated . InteresKMT2A rearrangement (KMT2Ar) is pathologically characterized by upregulated HOXA9 and MEIS1 genes, which are dependent on the interaction of oncogenic KMT2A fusion proteins with other complex-forming proteins such as LEDGF, DOT1L, and menin. Strategies for disrupting the connection between KMT2A protein and menin have recently been investigated. A preclinical study in which PDX mice with KMT2Ar were treated with the orally bioavailable menin-KMT2A inhibitor VTP50469 demonstrated that KMT2A-target genes such as MEIS1 were uniformly suppressed in the bone marrow [MEIS1 gene in a PDX model experiment [KMT2Ar or NPMmt, which was accompanied by the attenuation of BCL-2 and cyclin-dependent kinase 6 (CDK6) levels. Interestingly, the concurrent administration of SNDX-50469 with a BCL-2 inhibitor or a CDK6 inhibitor led to synergistic lethality [KMT2A-r or NPM1mt and concurrent FLT3 mutation (KMT2Ar/NPM1mt-FLT3mt). Dzama and colleagues showed the synergistic inhibition of the proliferation and enhancement of apoptosis in leukemic cell lines with KMT2Ar/NPM1mt-FLT3mt when treated with FLT3 inhibitors and menin-KMT2A inhibitors [KMT2A rearrangements or NPM1 mutations. Among the 59 patients recruited, clinical responses were observed in 28 patients (47%), including 8 patients (14%) with complete remission (CR) for more than 6 months [As mentioned in the previous section, AML with e marrow . Anotherperiment . Fiskus ethality . Combinahibitors . Notably6 months . TP53 mutation [TP53-mutated high-risk MDS or AML [A number of small molecules that reactivate mutant TP53 have been identified. Bykov and colleagues demonstrated that one such molecule (PRIMA-1) and its methylated form induced apoptosis in human tumor cells with mutation ,153. APRmutation . In a phS or AML . HoweverS or AML . The nexS or AML .TP53 wild-type AML cell lines [TP53 mutation status [TP53 wild-type, achieved complete or partial remission. Thus, MDM2 inhibitors seem appropriate for TP53 wild-type cases. Novel MDM2 inhibitors  in combination with hypomethylating agents (HMAs) are now under evaluation in clinical trials  for patients with newly diagnosed and relapsed/refractory AML. Mouse double minute 2 (MDM2) protein is a TP53-specific E3 ubiquitin ligase and functions as a principal cellular antagonist of TP53 . Idasanull lines . Anothern status . Thirty-TP53-mutated AML and high-risk MDS, among 2200 participants in the Haemato-Oncology Foundation for Adults in the Netherlands and Swiss Group for Clinical Cancer Research (HOVON-SAKK) clinical trials [DNMT3A, TET2, and ASXL1 , accounting for 24.3% of all TP53-mutated patients. These DTA mutations are known to result in disturbed epigenetic modulation within the tumor cells and are associated with better survival when treated with HMAs such as azacitidine [TP53 mutation was associated with improved response to HMAs but also linked to worse prognosis [TP53 mutation, but represents a promising component of combination therapy with mutant TP53-specific agents.Grob and colleagues described the genetic background of 230 patients with l trials . The moscitidine . In a merognosis . These dTP53-mutated AML in this study [The transmembrane protein CD47, also known as the “don’t-eat-me signal”, is the ligand for signal regulatory protein alpha  on macrophages and dendritic cells, and confers the inhibition of phagocytosis . The binis study . A phaseis study .Molecular-targeted agents for AML with adverse genetic factors described to date are summarized in FLT3 mutations, with the clinical benefit of their front-line use in combination with standard chemotherapy being demonstrated in large-scale clinical trials. However, resistance to FLT3 inhibition is a relevant issue to overcome, and pathologic FLT3 mutations  are not detected in the majority of AML patients. Targeting common pathways essential to adverse genetic factor function, such as HOXA9-related effectors, tyrosine kinase downstream pathways , and the loss of TP53 function could be the key to overcoming this issue. Of note, HOXA9 plays a pivotal role in leukemogenesis in AML with KMT2A rearrangement, NPM1 mutation, and DEK-NUP214 fusion, as well as AML with NUP98-involving fusion genes. Given that the interaction of menin and KMT2A protein is essential in initiating the oncogenesis of HOXA9-driven leukemia, menin-KMT2A inhibitors are promising molecular-targeting agents applicable to a considerable proportion of AML patients. Apart from FLT3 inhibitors, the direct inhibition of the PI3K-AKT-mTOR, RAS-MAPK-ERK, and JAK2-STAT5 pathways has been evaluated in preclinical and clinical studies. JAK2 inhibitors may be beneficial in leukemic states arising from MPNs. Although MEK inhibitors did not demonstrate apparent effectiveness for AML, pan-RAF inhibitors, especially in combination with BCL-2 inhibitors, represent a promising strategy in AML therapy. Specific approaches to AML with TP53 mutation remain largely investigational. However, a next-generation TP53 stabilizer is under evaluation in an early-phase trial. An anti-CD47 antibody in combination with HMAs/chemotherapy is another potential strategy for dealing with this category of AML, since this agent seemed equally effective for TP53-mutant and wild-type AML. Although these targeted agents exhibited only a limited anti-leukemic effect in the form of monotherapy, combination therapy with other agents such as conventional chemotherapy, HMAs, and BCL-2 inhibitors represents a promising new strategy.Treating AML with adverse genetic factors remains challenging, especially in patients who are ineligible for and/or refractory to intensive chemotherapy. In our real-world study, adverse genetic factors were apparently accumulated in patients with relapsed/refractory AML and who were ineligible to intensive therapy, suggesting a relevant need for a new strategy specific for AML with adverse genetic factors. FLT3 inhibitors have been an archetypal example of a successful molecular-targeting strategy for AML with


Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite–1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime – 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite–1) or synthetized (oxime–1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel , and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime–1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime–1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite–1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite–1 and oxime–1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions. Senecio nutans Sch. Bip. (syn.: Senecio graveolens Wedd) (Asteraceae) is widely used as a medicinal plant by the Andean communities of northern Chile [S. nutans is commonly called “chachacoma” and its infusion is used to treat gastric ulcers, [S. nutans have shown significant antioxidant, hemolytic, cytotoxic [The species rn Chile . S. nuta ulcers, , cold, b ulcers, ,4. Moreo ulcers, . Extractytotoxic ,6,7, vasytotoxic ,9.S. nutans show that p-hydroxyacetophenone and its benzophenone derivatives are the most abundant compounds [S. nutans.From a phytochemical point of view, analysis of the aerial parts of ompounds ,10,11. Mompounds ,7, the sompounds  have beeS. nutans is the p-hydroxyacetophenone derivative, 4-hydroxy-3-(3-methyl-2-butenyl) acetophenone, which displays significant antitumor activity in breast cancer cell lines [Candida albicans [One of the most studied metabolites of ll lines , specifill lines , and higalbicans . Additioalbicans . On the albicans .2+ channels in plasma membranes and Ca2+ uptake by intracellular stores [2+ channel blockers or potassium channel activators [The vascular endothelium regulates the balance between vasodilator and vasoconstrictor tone under normal physiological conditions . This mer stores . The funr stores ,19. Regativators .Although somewhat forgotten, oximes have been reported to have a vasodilator effect in the presence and absence of vascular endothelium on isolated blood vessels . In rece4-hydroxy-3-(isopenten-2-yl)-acetophenone (Metabolite–1). This natural compound was isolated from S. nutans, identified in a previous study [m/z 204.150 ; IR (KBr) 3000–3500, 1750–2000, 1645, 1350–1550 cm−1. us study . Crystal4-hydroxy-3-(isopenten-2-yl)-acetophenoxime (Oxime–1) crystallizes from dichloromethane as green crystals, m.p. 118–120 °C molecular weight 219, which is assignable to C13H17NO2. The IR spectrum shows bands of low intensity between 1750–2000 cm−1 and 1350–1550 cm−1, assignable to the presence of a benzene ring; a wide band between 3000–3500 cm−1, associated with the presence of vibrations of a –C=N–OH group and hydroxyl group; and an intense band at 1602 cm−1, resulting from –C=N–OH interactions of oximes. These last two sets of signals confirm the modification of the carbonyl group in the reaction. The 1H and 13C NMR spectra show signals assignable to 15 hydrogen atoms and 13 carbon atoms, respectively. The chemical shifts and the DEPT spectrum indicate the presence of three CH3, one CH2 δ to the aromatic ring, three aromatic CHs, one olefinic CH, and 5 quaternary carbons of the sp2 type. A methyl group is unique to the ketoxime group (CH3–C=N). This methyl is displaced towards the higher field at δ 2.26 ppm, with respect to the precursor metabolite–1 δ 2.60 ppm. The remaining two methyls are vinyl  characteristic of oximes; this new signal replaces the signal that resonated at δ 198 ppm (C-12) and that corresponded to the carbonyl group of metabolite–1. All the above information confirms the formation of the reaction product. A signal can be observed in the In the Heteronuclear Multiple Quantum Coherence (HMQC) spectrum, the same couplings are observed as for the precursor (metabolite–1), which is reasonable as the structural modification has been made on the carbonyl group and does not affect the rest of the compound’s structure. The analysis of the spectroscopic information has allowed us to establish that the compound obtained after the reaction corresponds to 4-hydroxy-3-(isopenten-2-yl)-acetophenoxime Oxime–1; . The cou−5 M; p < 0.001). As shown in 50) to oxime–1 (10.14 ± 8.46 μM) was significantly lower (p < 0.001) compared to metabolite–1 (24.44 ± 7.98 μM). Both compounds caused vascular relaxation in PE-treated intact rat aorta: 41 ± 6% metabolite–1, 73 ± 2% oxime–1 10−5 M; A. Intere−5 M concentration for both molecules. Thus, the relaxation was approximately 20% in endothelium-denuded rat aorta: 22 ± 4% oxime–1 versus 20 ± 5% of metabolite–1,  . We foun < 0.001 A; oxime–p < 0.01 B.50 in the presence and absence of L-NAME for all molecules (Our observations were confirmed by the significant differences in the EColecules . 50 to oxime–1 was significantly lower (p < 0.01) in the presence of L-NAME (26.49 ± 7.97 μM) compared to endothelium-denuded aortic rings (81.88 ± 7.79 μM), although it was significantly higher than the EC50 obtained in intact aortas treated with oxime–1.On the other hand, the pre-incubation with L-NAME partially blunted the relaxation of metabolite–1 in endothelium-denuded aortas, whereas oxime–1 did not. As shown in −5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl compared to the intact aorta: 130 ± 9% control versus 62 ± 9% oxime–1 (50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) versus control  in Ca2+-free medium. Second, the vascular tissue was stimulated with KCl (40 mM) to induce a contractile response while adding a cumulative concentration of extracellular Ca2+ (0.1 to 1.0 mM). In a Ca2+-free medium, the results confirmed that metabolite–1 (72 ± 11%), and oxime–1 (71 ± 16%) significantly (p < 0.01) decreased the contractile response to a submaximal KCl concentration (40 mM) compared to intact aorta  reduction of the contractile response to different concentrations of PE (10−7 to 10−5 M).The next step was to determine if the relaxation induced by metabolite–1 and oxime–1 can be mimicked in a receptor-mediated contractile response. In this line, the contractile response to PE was tested and both compounds significantly reduced the contractile response to PE, although oxime–1 showed a more highly significant (−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 10−7 M PE (62 ± 3% oxime–1) compared to the intact aorta (108 ± 6% control), whereas metabolite–1 did not it (−7 to 10−5 M). Significantly increased EC50 to PE confirmed the adrenergic vascular response observed in the presence of oxime–1  versus control  significantly (p < 0.001) decreased the contractile response to PE (10−7 M) compared to intact aorta  significantly decreased the adrenergic contraction induced by releasing of intracellular Ca2+ in Ca2+-free medium compared to intact aorta without oxime–1 (36 ± 2% control). However, metabolite–1 did not decrease the contractile response by releasing intracellular Ca2+.2+ influx across the plasma membrane by opening CaV1.2 channels. The effect of oxime–1 was higher compared to that displayed by metabolite–1: 39 ± 3% control vs. 25 ± 5% metabolite–1 (p < 0.05) and 22 ± 4% oxime–1  reduced the relaxation induced by metabolite–1 and oxime–1, strongly suggesting that the nitric oxide (NO) pathway is involved in the vascular response promoted by metabolite–1 and oxime–1, in agreement with previous studies showing the relaxation effects of oximes on isolated blood vessels with denuded endothelium and endothelium . Oximes latation ,27.Interestingly, at a maximal concentration, oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubating the tissue with L-NAME. Vascular reactivity of the tissue was recovered after washing. Other studies demonstrated that L-NAME did not reduce the relaxation effect of oxime derivatives, but inhibitors of soluble guanylyl-cyclase did . This fiCynanchum wilfordii (Maxim.) Hemsl. ameliorates the vascular endothelial dysfunction by improvement of the NO/cGMP pathway in rat aorta [Metabolite–1 and oxime–1 present 4-hydroxyacetophenone moiety in their backbone see . Previouat aorta ,29. Anotat aorta . Therefoat aorta ,31.2+-free conditions decreased the contraction induced by KCl or voltage-dependent Ca2+ channel type-L opening induced by Bay K8644. In the contraction studies approach, the vascular contractile response was studied by membrane depolarization with KCl and by the activation of G-protein coupled receptor with PE. We found that oxime–1 counteracted the contraction in aortic rings induced by low and intermediate concentrations of KCl, but not at the maximal concentration of KCl. Conversely, although metabolite–1 did not decrease the contractile response to KCl in normal medium, both compounds under Ca2+-free medium, but not with metabolite–1. Reduction of the contractile response to KCl in the presence of oxime–1 can be considered moderate compared to PE. In fact, the attenuated contraction in the presence of oxime–1 was maintained even with maximal concentration of PE, but not with KCl. In a same line of the drug potency, the contractile response to PE significantly decreased with pre-incubation of oxime–1 in normal and Ca2+ release from intracellular stores as well as Ca2+ influx from extracellular space. This is important because the Ca2+ release from intracellular stores is the first step in triggering Ca2+ influx through Ca2+ channels of the plasma membrane and produces arterial vasocontraction [2+-free medium, before adding Ca2+ to the bath. Second, there was a small increase in contractile response after adding Ca2+.Our data suggests that oxime–1 reduces the vascular contraction because it decreases Catraction . First, 2+ influx through single Transient Receptor Potential Cation Channel (TRPV3) in endothelial cells of cerebral parenchymal arterioles and uterine radial arteries in rats [2+ influx via TRPV1, and the contraction is amplified by depolarization-induced Ca2+ influx via Cav1.2 in the plasma membrane of the coronary arteries [Metabolite–1 and oxime–1 present isopentenyl residue in their backbone. It is possible that isopentenyl derivatives affect the vascular response. In fact, isopentenyl derivative causes a selective inhibition of Ca in rats ,34. On tarteries . 2+ channels, blunted the relaxation caused by 4-hydroxyacetophenone in rat aorta pre-contracted with PE, suggesting that voltage-dependent Ca2+ channels are involving in the relaxation effect of acetophenone derivates, such as metabolite–1 and oxime–1 [Previous studies reported that nifedipine, an antagonist of voltage-dependent Ca oxime–1 .G-nitro-l-arginine methyl ester (L-NAME), and (±)-Bay K8644 were bought from Sigma-Aldrich . The metabolite and oxime were dissolved in DMSO . Hydroxylamine hydrochloride, pyridine, magnesium sulphate, L-phenylephrine hydrochloride (PE), acetylcholine chloride (ACh), NS. nutans according to a previous protocol [The natural product 4-hydroxy-3-(isopenten-2-yl)-acetophenone (Metabolite–1) was isolated from protocol . Briefly4. Concentration in vacuo provided the oxime as a 3:1 mixture of E:Z isomers as a colorless solid.Synthesis of oxime was performed as previously described with a few modifications . To a son = 12) weighing between 170 g and 200 g were used in this study. The investigation was conducted in accordance with the local animal research committee of Universidad de Antofagasta (CEIC #275/2020). The animals were housed in plastic cages at room temperature (22–25 °C) and a humidity of 45–51% and had full access to tap water and food (ad libitum). They were randomized and assigned into the different groups tested.Male Sprague Dawley rats  for 10 min. The passive tension on aorta was 1.0 g, which was determined to be the resting tension for obtaining maximum active tension induced by 60 mM KCl [This procedure was performed based on the method previously described . Animals0 mM KCl .−10 to 10−5 M) or KCl (10 to 60 mM), the tissue was pre-incubated in KRB for 20 min prior to contraction. In another applied protocol, the relaxation capacity of the extract or isolated metabolite was determined. In this case, the tissue was pre-contracted with 10−6 M PE, and increasing concentrations of bioactive molecules were added to the organ bath on the vascular plateau response. The integrity of the vascular endothelium was evaluated with 10−6 M acetylcholine (ACh) at the beginning of the experiment. To evaluate the contractile response to phenylephrine  was performed using nonlinear regression  via Graph Pad Prism software, version 5.0. . Statistical significance was set at p < 0.05.The results obtained from the experiments are expressed as mean ± standard error of the mean. Statistical analysis of the data was performed using analysis of variance (two-way ANOVA) followed by Bonferroni post hoc test. In addition, the determination of the sensitivity (EC2+ from intracellular stores and apparently blocking Ca2+ influx by channels [2+ influx from the extracellular space [S. nutans through chemical modification of natural products will provide new molecules for treating hypertension.As shown in channels . Metabolar space . Both bi


Exocytosis is a tightly regulated reaction that occurs within a millisecond of the arrival of an action potential. One crucial parameter in determining the characteristics of the transmitter release kinetics is the coupling distance between the release site and the Ca2+ channel. Still, the technical limitations have hindered detailed analysis from addressing how the coupling distance is regulated depending on the development or activity of the synapse. However, recent technical advances in electrophysiology and imaging are unveiling their different configurations in different conditions. Here, I will summarize developmental- and activity-dependent changes in the coupling distances revealed by recent studies.Synapses are junctions between a presynaptic neuron and a postsynaptic cell specialized for fast and precise information transfer. The presynaptic terminal secretes neurotransmitters  Because the Ca2+ needs to diffuse from the channel pore to vesicular Ca2+ sensors, the coupling distance between VGCCs and Ca2+ sensors of releasable vesicles is a critical determinant of the release probability of the synaptic vesicles  is a suitable model to investigate coupling distance before and after the long-term potentiation (LTP) since it is applicable for patch-clamp recording, and the LTP can be induced by a pharmacological manipulation (Drosophila neuromuscular junction (NMJ) that occurs in response to postsynaptic impairments and leads to a compensatory increase of presynaptic transmitter release  arrives at the presynaptic terminal, Cawnstroke , resultiwnstroke . Since t buffers , not onl buffers . VGCCs-r buffers .2+. Because a large fraction of the readily releasable vesicles is depleted with the first AP, “tight” coupling synapses often demonstrate short-term depression, characterized by progressive weakening of transmitter release upon repetitive stimulations , some synapses have “loose” couplings , while oulations . On the ulations .2+ channels and the coupling distance of VGCCs and release-ready vesicles as shown above, there still are other factors that could affect synaptic vesicles’ release probability. A major alternative possible factor is heterogeneous Ca2+-sensitivity for transmitter release. Ca2+-sensors for the synaptic vesicle fusion synchronized to APs at the CNS are mediated mainly by Synaptotagmin-1, 2, or -9   . At the rtening) . Developrtening) . These sDrosophila NMJ, where Unc13A and -B are simultaneously present at mature presynaptic boutons, but those two isoforms arrive at different timing during synaptic development  or homeostatic plasticity mode .Drosophila NMJ, which induces a compensatory increase of presynaptic transmitter release in response to postsynaptic receptor blockade  or phasic  activity of the synapse. Recently, the coupling distance has been estimated based on the kinetics of transmitter release measured mulation . The stumulation , simultamulation . The tecIt is interesting to ask what is shared and what is different between developmental and activity-dependent modulation of coupling distance. The initial triggering signal might be different since basal synaptogenesis mechanisms, and functional maturation of at least a subset of synapses are activity-independent . However2+ influx from VGCCs. Simultaneous measurement of exocytosis of individual synaptic vesicle together with Ca2+ influx (The ideal experiment will be to visualize the exocytosis of a single synaptic vesicle with Ca+ influx  or an ac+ influx  has beenMM wrote the manuscript and prepared the figures.


Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50–100 nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644–1665) and Ca2/CaM12’, a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12’ N-lobe does not interact with the IQ motif. The CaM12’ C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.The L-type Ca V1.2 is the main L-type channel in heart, blood vessels, and brain   (2+-free apoCaM has been suggested to be preassociated with CaV1.2 (V1.3 (V1.2 IQ-motif with a dissociation constant (KD) of ∼10 μM  , 9. CDI on (CDI)  that is  100 nM) . Ca2+-frh CaV1.2  and the .2  that is functionally preassociated with CaV1.2 under basal conditions. The NMR structure of Ca2/CaM bound to the CaV1.2 IQ-motif (residues 1644–1664) suggests that the Ca2+-bound CaM C-lobe  forms intermolecular interactions with the side chain atoms from CaV1.2 residues , whereas the Ca2+-free CaM N-lobe does not interact with the IQ motif. Electrophysiological data of key mutants of CaV1.2 (I1654A and Y1657E) contrasted with the earlier findings for the K1662E mutant along with the consequences of ectopic expression of CaM34 all suggest that Ca2/CaM, rather than apoCaM, preassociates with CaV1.2 under basal conditions to augment channel open probability (Po) and mediate rapid CDI.To fill a critical gap in our understanding of how CaM governs CaV1.2 IQ-motif with a dissociation constant (KD) of 10 μM  in neurons and cardiomyocytes .Isothermal titration calorimetry (ITC) studies have suggested that apoCaM binds to the IQ peptide with submicromolar affinity in the absence of salt . Howeverof 10 μM , 15. Ear segment . Collectmyocytes , implyineviously , 13, 17.2+ affinity, which allows Ca2+ to bind to the CaM C-lobe under basal conditions  in which the N-lobe is devoid of Ca2+ (2+ as well as the IQ motif with 10-fold higher affinity than the N-lobe (2/CaM), and Ca2+-saturated CaM (Ca4/CaM) each bound to the IQ as a function of free Ca2+ concentration are shown in Fig. S1A. The Ca2/CaM intermediate species  has a significant occupancy of ∼50% at 100 nM Ca2+ concentration . Since the apoCaM N-lobe (CaMN) does not bind to IQ under physiological conditions , whereas the N-lobe should be devoid of Ca2+. To test this prediction, we prepared a CaM mutant  that completely disabled Ca2+ binding to EF1 and EF2 but retained normal Ca2+ binding to EF3 and EF4. The apparent Ca2+ affinity of CaM12’ in the presence of saturating IQ peptide under physiological conditions (27 °C and 37 °C) was measured by ITC (A and B). The ITC isotherm at 27 °C is biphasic, suggesting possible sample heterogeneity. The major binding component  and may be an artifact of IQ partial self-association or other sample heterogeneity. Fitting the ITC isotherm with a two-site model reveals a Ca2+-binding apparent KD . This analysis predicts that slightly more than half of the CaV1.2 channels should be preassociated with the CaM intermediate, Ca2/CaM, under basal conditions.Thus, the CaM C-lobe is calculated to have a 10-fold higher apparent Cad by ITC , A and B, and N2 . The oth ± 20 nM , which aA show that half saturated CaM (Ca2/CaM) coexists in an equilibrium mixture with apoCaM and Ca2+-saturated CaM (Ca4/CaM). At a basal Ca2+ concentration of 100 nM, the fractional occupancy of Ca2/CaM is calculated to be 55% compared to 7% occupancy of Ca4/CaM and 38% occupancy of apoCaM. Therefore, under basal conditions, Ca2/CaM cannot be resolved from the other CaM species. To isolate the half Ca2+ saturated species, we performed structural studies on the CaM mutant  that completely disables Ca2+ binding to EF1 and EF2 but retains Ca2+ binding to EF3 and EF4. The NMR assignments of Ca2+-bound CaM12’ bound to the IQ peptide (Ca2/CaM12’-IQ) reveal two downfield NMR peaks assigned to G99 (EF3) and G135 (EF4) that indicate Ca2+ is bound to EF3 and EF4 . The chemical shifts assigned to the CaM12’ C-lobe (residues 80–149) of Ca2/CaM12’-IQ  are nearly identical to those of the isolated Ca2+-bound CaM C-lobe bound to IQ . NMR peaks assigned to CaM12’ N-lobe (residues 1–79) of Ca2/CaM12’-IQ are similar to those of apoCaM12’ in the absence of IQ , indicating that the CaM12’ N-lobe is Ca2+ free and does not interact with the IQ peptide. Thus, only the C-lobe, but not N-lobe, residues in Ca2/CaM12’ exhibit IQ-induced spectral shifts.The NMR spectrum of Cafree CaM , A and B2/CaM12’-IQ were reported previously (BMRB accession number 27692)  are separately folded and noninteracting, as was seen previously for the NMR structures of apoCaM  and 0.9 ± 0.1 Å from the main chain atoms in the N-lobe. The lowest energy NMR structure of Ca2/CaM12’ bound to the IQ peptide is shown in Figure 4D. The quality of the NMR structures of Ca2/CaM12’-IQ was assessed using PROCHECK-NMR (2+-bound CaM C-lobe (residues 80–149) of Ca2/CaM12’-IQ (dark red in D and E) looks similar to that observed in the crystal structure of Ca2+-saturated CaM bound to the IQ  (2+-free CaM N-lobe (residues 1–78) of Ca2/CaM12’-IQ  adopts a closed conformation and looks similar to that of apoCaM . In the Ca2/CaM12’-IQ structure , the IQ residues  point toward CaM and make extensive contacts with CaM C-lobe residues . The IQ peptide in the Ca2/CaM12’-IQ structure does not make any contacts with the Ca2+-free N-lobe, in contrast to the crystal structure of Ca4/CaM12’-IQ  . These pr 27692) . NMR strof NOESY  and longof NOESY  as descrf apoCaM , 25, 26.Fig. 4E) . The straM12’-IQ , 29, 30 2/CaM12’-IQ (D) looks quite different from the recent NMR structure of apoCaM bound to IQ (2/CaM12’-IQ structure and does not contact either E85 or E88 (D). This analysis predicts that the CaV1.2 mutation K1662E weakens binding to apoCaM more than it does to Ca2/CaM12’. Because the K1662E peptide (IQK1662E) was not soluble enough for ITC with Ca2/CaM12’, we used fluorescence polarization (FP) to measure binding affinity in the nanomolar range. As predicted, titration of the IQ peptides with Ca2/CaM12’ reached full saturation at 100 nM Ca2/CaM12’, indicating a KD < 100 nM for both, IQWT and IQK1662E (F). It was not possible to more accurately determine the actual KD because the IQ peptide concentration in Figure 4F had to be 100 nM due to limited detection sensitivity. This concentration is much larger than the KD for IQWT . During the second half of the titration, KD of both, IQWT and IQK1662E, consistent with the 16 nM KD for IQWT as seen by ITC  that is 6-fold higher than that of IQWT . Accordingly, the K1662E mutation can be used to selectively disable apoCaM binding to CaV1.2, while retaining CaV1.2 binding to Ca2/CaM.The NMR structure of CaaM12’-IQ D looks qnd to IQ . In the 5 or E88 D. This aIQK1662E F. It was16 nM in  and appan by ITC . As a re of IQWT . Thus, t2/CaM12’-IQ reveals intermolecular contacts with IQ residues, Y1649, I1654, Y1657, and F1658, that are each located on the same side of the IQ helix pointing toward the Ca2+-occupied C-lobe of Ca2/CaM12’ (D). As predicted by this analysis, the IQ peptide mutants IQY1649A, IQF1654A, IQY1657D, and IQF1658D each exhibited weaker binding to Ca2/CaM12’ compared to IQWT. The KD was 16 ± 5 nM for IQWT, 26 ± 5 nM for IQY1649A, 60 ± 10 nM for IQI1654A, 8000 ± 10 nM for IQY1657D, 4000 ± 10 nM for IQF1658D, and 32 ± 5 nM for IQF1658A  to 37 ± 10 nM at 37 °C. Also, the temperature dependence of ΔH  indicates a negative ΔCp value, which is consistent with the relatively large change in solvent accessible hydrophobic surface area that occurs when Ca2/CaM12’ binds to the IQ peptide.The highly exothermic binding of the IQ peptide to Ca2/CaM12’ but not apoCaM under physiological conditions . This differential effect informs interpretation of recently published data that showed that the K1662E mutation has no effect on Po . Strikingly, the K1662E mutation had no significant effect on CDI (nor on voltage-dependent inactivation), in contrast to the I1654A mutation, which reduced CDI by ∼75% . The small, remaining CDI seen for the I1654A mutant channel may be due to N-lobe effects such as its binding to the N terminus of the CaV1.2 α1 subunit . Single-channel recordings show a remarkably strong decrease in Po for Y1657D versus WT CaV1.2 (F and G). This loss in Po and CDI is comparable to similarly strong effects for the I1654A mutation on Po , which can explain some, but not all, of the 80% loss in Po.Our new Ca2/CaM12’ . In supptivation , A–E. SiT CaV1.2 , F and Gon on Po  and CDI on on Po  but the on on Po  or CDI  by immunoblotting extracts of 293T cells transfected with CaV1.2 expression constructs ± WT CaM or CaM34 plasmids (34 is ∼20 fold (A–D). To test whether ectopic expression of CaM affects levels of endogenous CaM, we expressed YFP-tagged WT CaM or CaM34, which migrate at an MR of ∼ 45 kDa . Probing immunoblots with anti-CaM identifies a prominent 45 kDa band and a weaker signal for the endogenous 17 kDa band. Comparison of the 17 kDa band in mock-transfected (no CaM vectors) cell lysate to the same MR immunoreactive band in the CaM plasmid-transfected samples did not indicate a significant effect of ectopic CaM on endogenous CaM levels (E and F).To further analyze the role of CaM in Po, we ectopically expressed CaM in HEK 293T cells. Although this approach has been used before to define the role of CaM in CDI, the level to which exogenous CaM was expressed in these CDI studies had not been thoroughly assessed . Thus, wribed by ) was sufplasmids . We foun∼20 fold , A–D. ToV1.3 by Adams et al.  implies that Ca2+ binding to EF3 and EF4  is essential for CaV1.2 channel function. Also, our binding analysis reveals that IQ binding to CaM increases the apparent Ca2+ affinity by at least 10-fold  has a closed conformation (2+-free N-lobe and a Ca2+-bound open conformation (2/CaM12’-IQ are separately folded and do not exhibit interdomain contacts (C). The two separate lobes in Ca2/CaM12’-IQ are dynamically independent, similar to apoCaM , whose structure is similar to the C-lobe of Ca4/CaM bound to the IQ (E) (2/CaM12’ is rotated 180° compared to the orientation of the IQ bound to apoCaM (2/CaM with at least 100-fold higher affinity (F) compared to that of apoCaM (V1.2 with Ca2/CaM (rather than with apoCaM) predisposes CaV1.2 for CDI. Since Ca2/CaM and Ca4/CaM both bind to CaV1.2 with the same orientation, CaM can remain bound to CaV1.2 upon Ca2+ influx to facilitate rapid CDI. By contrast, preassociated apoCaM would first need to dissociate from CaV1.2 upon Ca2+ influx and then subsequently rebind in the conformation adopted by Ca2+-saturated Ca4/CaM to engage CDI , did not affect Po , as well as Ca2/CaM  K1662E versus Y1657D on Po and CDI; and (3) WT CaM versus CaM34 or CaM1234 on Po collectively indicate that preassociated Ca2/CaM is an important factor in determining channel Po.Our functional analysis fully supports the relevance of prebinding of Caf apoCaM  but reta ∼100 nM F, did noffect Po , CDI, IB, or ICa . Further and ICa . We alsod CaV1.3 , overexpd CaV1.3 . The maid CaV1.3  was thater CaM34  nor CaM134  and low = 10 μM ) and fulD = 1 μM ). How thments Po . Thus, wentrated  or by Capression , perhapspression . On the echanism . At thisV1.2 and its role in controlling channel activity and CDI. These findings are not only of functional relevance for understanding the physiological effects of CaV1.2 but also inform the current understanding of pathological events such as arrhythmias due to impaired CDI  was introduced into Xenopus CaM complementary DNA by PCR QuickChange procedure (12’ protein was expressed from a pET11d vector in a BL21(DE3) Codon Plus Escherichia coli strain (Stratagene) and purified as described previously (V1.2 IQ peptide (residues 1644–1664) was purchased from ChinaPeptides. The peptide was dissolved in d6-dimethyl sulfoxide to give a peptide concentration of 7.8 mM. The peptide concentration was determined by measuring absorbance at 280 nm with ε280 = 2980 M−1 cm−1. An aliquot of peptide  was added to a dilute solution of CaM12’  with 95% H2O/5% D2O). The complex was then concentrated to a final concentration of 500 μM in a final volume of 500 μl for NMR experiments. The 1.5-fold excess of IQ peptide in the NMR sample of Ca2/CaM12’-IQ was necessary to minimize the occupancy of a 2:1 complex, in which two molecules of CaM12’ were bound to one IQ. The HSQC spectrum of a sample that contained an equal concentration of CaM12’ and IQ revealed two distinct peaks for each C-lobe residue of CaM12’ (D). The most intense peak represented a 1:1 complex (∼90% occupancy) and a weaker second peak  represented a second CaM12’ molecule bound to IQ in a 2:1 complex (∼10% occupancy). The relative occupancy of the 2:1 complex could approach nearly 100% when the CaM12 concentration is more than 10-fold higher than that of CaV1.2, like what exists inside HEK293 cells used in the CaV1.2 electrophysiological experiments (2+-bound C-lobe on one side of the IQ helix (CaM12’ C-lobe contacting I1654 and Y1657) as well as a second CaM12’ C-lobe that binds with lower affinity to the opposite side of the IQ helix (CaM12’ C-lobe contacting F1648 and F1652). The binding of a second C-lobe from CaM12’ mimics the binding of the Ca2+-bound N-lobe from WT CaM. Therefore, we suggest that the CDI observed for CaV1.2 in the presence of CaM12’ (12’ molecules bound to a single CaV1.2.The CaMrocedure . The muteviously . The CaVf CaM12’ D. The moeriments . The 2:1f CaM12’  is likelhttps://www.originlab.com) as described previously  and CaM12’–IQ complex (titrant) were prepared by exchanging each into buffer containing 20 mM Tris, pH 7.4, and 100 mM KCl. The CaM12’–IQ complex in the sample cell (10 μM at 27 °C or 8.0 μM at 37 °C in 1.5 ml) was titrated with aqueous CaCl2 (0.23 mM at 27 °C or 0.3 mM at 37 °C) using 35 injections of 10 μl each. For the ITC experiments in C, E–H, samples of Ca2/CaM12’ (injectant) and IQ peptide (titrant) were prepared by exchanging each into buffer containing 20 mM Tris, pH 7.4, 100 mM KCl, and 1 mM CaCl2. The concentrations of the IQ peptides  were each 10 μM in 1.5 ml in the sample cell for titration with 0.1 mM Ca2/CaM12’ and the concentrations of Y1657D and F1658D were each 50 μM in 1.5 ml for titration with 0.5 mM Ca2/CaM12’ using 35 injections of 10 μl each.ITC experiments were performed using a VP-ITC calorimeter  at 27 °C and 37 °C. The data were acquired and processed with MicroCal software (eviously . The fir2/CaM12’-IQ was described previously (15N-labeled Ca2/CaM12’ (0.5 mM) bound to unlabeled IQ (0.75 mM). Each sample was dissolved in 20 mM 2-Amino−2−hydroxymethyl-propane-1,3-diol-d11 (Tris-d11 at pH 7.5), 1.0 mM CaCl2, and 95% H2O/5% D2O. Three-dimensional NMR experiments for assigning backbone and side-chain resonances, and NOESY distance restraints were analyzed as described previously  was used as an orienting medium. Pf1 (12′ mg/ml) was added to an NMR sample that contained either 15N-labeled Ca2/CaM12’ bound to unlabeled IQ. 1H-15N residual dipolar coupling constants (DNH) were measured using a 2D IPAP (inphase/antiphase) 1H-15N HSQC experiment as described by /RMS(Dmeas), where Dmeas is the measured RDC, Dcalc is the calculated RDC, and RMS is the root mean square difference. A Q-factor of 30% corresponds to 2 Å resolution.All NMR measurements were performed at 303 K using a Bruker Avance III 600 MHz spectrometer equipped with a four-channel interface and triple-resonance cryoprobe. NMR sample preparation of Caeviously . Two-dimeviously . NMR dateviously  and analeviously  of Ca2/Cribed by . Represeby PALES . The Q-f2/CaM12’ bound to the IQ peptide were calculated using restrained molecular dynamics simulations within Xplor-NIH , and 13C-filtered NOESY-HSQC as described by  as input. Hydrogen bond restraints in helices and β-sheets were verified by measuring amide hydrogen-deuterium exchange rates as described by  or apoCaM in Ca2+-free buffer . FP was measured with a Synergy 2 plate reader (BioTek) as described (v - g∗Ih)/(Iv + g∗Ih); Iv and Ih are vertical and horizontal fluorescence intensity, respectively, and g is the correction factor for fluorescein. To obtain binding curves and KD values, data were fitted in GraphPad Prism 5 (GraphPad Software Inc) to the equation Y = B∗X/(Kd + X); B is maximal FP value that would be reached at saturation as determined by extrapolation of the fitted curve.Fluorescein-labeled peptides  were titrated with increasing concentrations of purified Caescribed . FP was 2/CaM-IQ, and Ca4/CaM-IQ as a function of the free Ca2+ concentration were calculated according to the following scheme in The concentration profiles of apoCaM-IQ, Ca11.2 subunit N-terminally tagged with eCFP (2A (2δ-1 (V1.2 channel subunits and later further optimized (JetPrime) for CaM (at ratio of 1:1:1:0.5 for α11.2:β2A:α2δ−1:CaM). Rat brain α11.2 (GenBank ID: M67515.1) N-terminally fused to eCFP was utilized as previously described  were maintained as previously described , 53. Forith eCFP , 53 or mith eCFP  plus pGWeCFP . For soAdelman, .2+- (IBa) and Ca2+ currents (ICa) of CaV1.2 L-type Ca2+ channels were obtained in the whole-cell configuration using external bath solution containing (in mM) 134 N-methyl-D-glucamine, 10 BaCl2 , 1 MgCl2, 10 Hepes, and 10 glucose with an adjusted pH of 7.4 (Cs-OH) and an osmolarity of 300 to 310 mOsm (sucrose). Intracellular pipette solution contained (in mM) 125 Cs-MeSO3, 5 CsCl, 10 EGTA, 10 Hepes, 1 MgCl2, 4 Mg-ATP, and pH 7.3 (CsOH), mOsm 290 to 300 (sucrose). Cells were clamped at a holding potential of -80 mV and depolarized for 900 ms to a series of activating potentials, from −60 mV to +50 mV (or +80 mV for Ca2+ currents), in increments of 10 mV at an interval of 0.033 Hz. The series resistance and the cell capacitance were directly taken from the Amplifier  and compensated to ∼40%. Data were sampled at 10 kHz and lowpass filtered at 2 kHz. Leak subtracted raw data were analyzed with Pclamp10 and GraphPad Prism IX software. All recordings were performed at room temperature (RT).Macroscopic Ba2, 1 CaCl2, 10 EGTA, 10 Hepes, and 2 Na2-ATP pH 7.4 (KOH). The intracellular pipette solution contained (in mM) 110 BaCl2 and 10 Hepes, adjusted to pH 7.4 (TEA-OH). Cells were depolarized for 2 s from a holding potential of -80 mV to 0 mV every 7 s. Event lists were created from raw Ba2+ currents after leak and capacity transients were digitally subtracted by pClamp 10. Unitary current events were then analyzed based on the half-height criterium . The number of channels in the patch was estimated based on the observed simultaneous openings and is a precise parameter for k < 4, as included in this article and originally described by R. Horn , respectively, the maximum of simultaneously open channels  and placed on ice. Cell were incubated with freshly prepared 0.4 mg/ml of EZ-Link-Sulfo-NHS-LC-biotin (Thermo Fisher Scientific) in PBS-CM for 30 min, followed by quenching of remaining NHS reactive groups with ice-cold 100 mM glycine in PBS-CM, four separate washes with quenching buffer, and a final rinse with PBS alone. Labeled and quenched cells were dislodged by scrapping and directly lysed into ice-cold radioimmunoprecipitation assay buffer  supplemented with protease inhibitors: 1 μg/ml leupeptin (Merck Millipore), 2 μg/ml aprotinin (Merck Millipore), 1 μg/ml pepstatin A (Merck Millipore), and 34 μg/ml PMSF (Sigma). Lysates were cleared of insoluble material via centrifugation at 200,000g for 30 min at 4 °C. The protein concentration of the solubilized material in the cleared lysate was determined by a standard bicinchoninic acid assay (Thermo Fisher Scientific). Biotinylated constituents in equal amount protein lysates  were affinity purified by incubation with 30 μl of NeutrAvidin-conjugated Sepharose beads (Thermo Fisher Scientific) for 2 h at 4 °C. Bead-bound material was sedimented by centrifugation, washed several times with ice-cold buffer, and bound proteins extracted in SDS sample buffer (with shaking at 65 °C for 15 min). Proteins from pull downs as well as directly loaded lysates were fractionated by 7.5% acrylamide SDS–PAGE and transferred onto polyvinylidene difluoride  membranes. For experiments used for analysis of CaM expression levels in directly loaded lysates  and the CNC2 epitope near the C terminus of α11.2 . Background signals in individual lanes were subtracted from the band signal prior to quantitative analysis. Differences in immunosignal strengths were corrected for potential immunoblotting and film exposures differences between experiments, as described  lysate immunosignals were used to correct for minor differences in protein amounts loaded in individual sample lanes. To correct for variation in test immunosignals  between experimental replicates, normalization was done according to the ‘sum of the replicates’ method as described  on one blot was divided by the sum of all immunosignals from the same immunoblot exposure for that experimental run to obtain the relative signal fraction for each band  from all experiments  and these means then divided by the mean value of the test control . All data were statistically analyzed (GraphPad Prism IX software) applying either a Student's t test (two-sample comparison) or ANOVA with Tukey post hoc test.Signal intensity for each band in scanned film images of immunoblots were assessed using ImageJ , and all other data are contained within the article.Atomic coordinates were deposited in the Protein Databank (accession no. This article contains The authors declare that they have no conflicts of interest with the contents of this article.


We studied Ca2+ entry pathways induced by Aβ oligomers in rat hippocampal and cerebellar neurons. Aβ oligomers induce Ca2+ entry in neurons. Ca2+ responses to Aβ oligomers are large after synaptic networking and prevented by blockers of synaptic transmission. In contrast, in neurons devoid of synaptic connections, Ca2+ responses to Aβ oligomers are small and prevented only by blockers of amyloid channels (NA7) and NMDA receptors (MK801). A combination of NA7 and MK801 nearly abolished Ca2+ responses. Non-neuronal cells bearing NMDA receptors showed Ca2+ responses to oligomers, whereas cells without NMDA receptors did not exhibit Ca2+ responses. The expression of subunits of the NMDA receptor NR1/ NR2A and NR1/NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca2+ responses to NMDA but not to Aβ oligomers. We conclude that Aβ oligomers promote Ca2+ entry via amyloid channels and NMDA receptors. This may recruit distant neurons intertwisted by synaptic connections, spreading excitation and recruiting further NMDA receptors and voltage-gated Ca2+ channels, leading to excitotoxicity and neuron degeneration in AD.The molecular basis of amyloid toxicity in Alzheimer’s disease (AD) remains controversial. Amyloid β (Aβ) oligomers promote Ca This has been studied in detail in cerebellar neurons but may also occur in hippocampal and/or cortical neurons  after stimuli presentation (% responsive cells) as revealed by the change of the slope of the recording F340/F380 ratio. Second, we also determined the maximum increase in the F340/F380 ratio during the stimulation period from the resting value of intracellular Ca2+ just before the addition of the stimuli (Δ Ratio). In the cases in which resting cytosolic Ca2+ showed oscillations of intracellular Ca2+, the resting value was the lowest Ca2+ value during the recording period before addition of stimuli. To analyze the effects of specific antagonists, we normalized calcium responses to values obtained in the absence of antagonist (control). As stimuli may induce a minor change in a small fraction of cells, whereas others may induce a similar or larger increase in most of the cells, we also quantified Ca2+ responses to obtain a single parameter corresponding to the product of the fraction of the responsive cells by the maximum increase in Ca2+ induced by stimuli relative to the value obtained in cells treated with vehicle (% activation).For analysis of Ca2+] oscillations in primary cultures of rat hippocampal and cerebellar neurons are quite variable. Accordingly, for the analysis of calcium oscillations, it is straightforward to measure the amplitude and/or frequency of those oscillations. To overcome this issue, we developed and reported previously [2+]  during a given period of time. The OI, therefore, is a parameter influenced by both the amplitude and the frequency of Ca2+ oscillations. Cells showing no oscillations  had OI values lower than 0.05. Cells exhibiting large and/or high frequency Ca2+ oscillations had OI values about 20-fold larger. Thus, OI values ranged from 0.05 (no Ca2+ oscillations) to 1 (high frequency and/or amplitude Ca2+ oscillations). Synchronous and spontaneous oscillations of intracellular  oscillations were blocked by TTX (96%), an antagonist of voltage-gated Na+ channels involved in action potential propagation and network activity. In addition, synchronous oscillations were also blocked by MK801 (90%) and ω-agatoxin (93%) but not by dihydropyridines. These data indicate that synchronous oscillations in cerebellar neurons are dependent on TTX-sensitive, voltage-gated Na+ channels, NMDA receptors and P/Q type Ca2+ channels. Similar findings were observed in hippocampal neurons , hippocampal neurons did not show synchronic oscillations, even in the absence of Mg2+. However, synchronous oscillations were frequently observed at longer time points (7–8 DIV). Furthermore, synchronic oscillations are promoted under special conditions, such as increased cell plating density. Specifically, we observed primary hippocampal neuronal cultures form neuronal circuits at a density of 70–90 × 103 cells per well. We previously demonstrated that cerebellar neurons in primary culture develop neuronal networks with synaptic connections starting at 6–7 days in vitro (DIV), with clear evidence of the occurrence of synchronous cytosolic Callations . The rem neurons . Interes2+ channels and voltage-gated Na+ channels, but not L-type Ca2+ channels, are also involved in generating the synchronous activity in hippocampal neurons. Quantitative analysis of Ca2+ oscillations was carried out by means of the oscillation index, a parameter that shows elevated values in cells showing high frequency and/or high amplitude of Ca2+ oscillations. However, the oscillation index values are low in cells showing no oscillations  in hippocampal neurons  composed of seven amino acids (EVHHQKL) [3 cells per well) to favor network activity, and Ca2+ responses to Aβ1–42 oligomers were tested at 1 and 7 DIV. 2+ recordings obtained before (1 DIV) and after (>7 DIV) network formation, respectively. As shown above, Ca2+ responses to Aβ1–42 oligomers were much larger after network formation. In this scenario, all the agonists tested, including TTX, significantly inhibited Ca2+ responses to Aβ1–42 oligomers. In contrast, before network formation, only MK801 and NA7 prevented Ca2+ responses. Similar results were also obtained in cerebellar neurons in culture (We tested whether network activity can modulate the inhibition of CaEVHHQKL) ,21 that  culture . 2+ responses to Aβ1–42 oligomers are enhanced by network formation. In the absence of network formation, only MK801 and NA7 blocked Ca2+ responses to oligomers, suggesting that NMDA receptors and amyloid channels can be the primary Ca2+ entry pathways induced by amyloid oligomers.These data suggest that Ca2+ responses to NMDA, 2+ responses to Aβ oligomers to a larger extent than each drug used alone  cells [2+ responses to NMDA as index. HEK293 and HT29 cells, which have no Ca2+ response to NMDA but respond to ATP, did not show Aβ oligomer-induced Ca2+ responses  cells  and humaP) cells  that expesponses . In contligomers . 1–42 oligomers and NMDA in GT1 cells, a hypothalamic neuron cell line also expressing NMDA receptors [2+ responses to oligomers  and the mechanism(s) by which Aβ peptide oligomers induce Ca2+ entry and/or Ca2+ mobilization into neurons. Different molecular targets have been proposed, including several Ca2+ channels, such as glutamate receptors, especially the NMDA receptor [2+ entry, mitochondrial Ca2+ overload and neuronal death in both cerebellar and hippocampal neuronal cultures [2+ responses to oligomers. This suggests that oligomers interact and activate multiple Ca2+ entry pathways or, alternatively, oligomers induce a single pathway in some neurons, and excitation potentially spreads throughout a network of interconnected neurons. This second possibility is supported by the observation that cerebellar or hippocampal cultures do develop actual neuronal circuits. The relevant question that immediately arises is whether it would be possible to stop disease progression and neuronal loss by inhibiting Careceptor ,23,24. Hreceptor , thus qucultures . We used2+ oscillations can be observed in resting conditions but are generally activated after removing Mg2+, which blocks the NMDA-type receptor in a voltage-dependent manner. Synchronous Ca2+ oscillations are blocked following treatment with MK801, P/Q-type Ca2+ channel antagonists and TTX. Our results support the mechanism of synchronous Ca2+ oscillations in rat hippocampal neurons, which are able to generate networks in primary culture even earlier than cerebellar neurons [2+, suggesting that these neurons are more excitable than cerebellar cells, and/or they involve other excitatory receptors not inhibited by extracellular Mg2+. Hippocampal neurons are also sensitive to MK801, P/Q channel antagonists and TTX but, in contrast to cerebellum, only TTX can block network activity. In addition, MK801 and ω-agatoxin were able to partially inhibit synchronous Ca2+ oscillations, thereby suggesting that other types of receptors (in addition to NMDA-type receptors) could play an important role in the synchronization of these cells and the formation of neural circuits. Moreover, synchronous Ca neurons . Synchro2+ responses to oligomers, we investigated the effects of oligomers on neurons before (1 DIV) and after (7 DIV) circuit formation. Our findings demonstrated that oligomers induce Ca2+ responses before and after circuit development. Nevertheless, the number of cells responding to oligomers and the rise in Ca2+ concentration were significantly higher at 7 DIV relative to 1 DIV. The data suggest that circuit formation amplifies the response to oligomers. An alternative explanation might be related to a change in the expression of receptors for the oligomers, but our results show that the Ca2+ responses to NMDA are similar before and after circuit development. Our results showed that primary cultures of cerebellar and hippocampal neurons display synchronous oscillations, which start to be evident after the 3–4 DIV and consistently after 7 DIV. To determine the potential contribution of circuit formation to Ca2+ oscillations. In the absence of a neuronal circuit (1 DIV), only MK-801 is capable of consistently preventing the effect of oligomers in the cerebellum and hippocampal neurons. These observations suggests that the NMDA receptor could act not only as an amplifier of the response but might also play an essential role as a direct target.Our findings also suggest that circuit formation could amplify the response to oligomers. Consistently, we found that TTX  is able to largely inhibit the effect of oligomers at 7 DIV, though it has no effect at 1 DIV. In addition, MK-801 and ω-agatoxin inhibit the effect of the oligomers and synchronous Ca2+ entry induced by oligomers. NA7 was able to inhibit oligomer-induced Ca2+ entry at 7 DIV, and this effect was independent of the inhibition of voltage-gated Ca2+ channels or NMDA receptors. Furthermore, NA7 was able to inhibit Ca2+ responses induced by the oligomers even in the absence of a neuronal circuit. Taken together, these findings suggest that oligomers could directly activate NMDA receptors and/or form amyloid channels, as well as excite some hippocampal neurons. This activation could be propagated and amplified through the neural network. In support of this notion, we demonstrated that the combination of NA7 and MK801 inhibits more efficiently Ca2+ responses compared to each compound alone. NA7 and MK801 practically blocked the Ca2+ response entirely, thus indicating that both act through independent pathways and that NMDA receptors and amyloid channels are primary targets of amyloid oligomers.Oligomers can also target the amyloid channels. It has been proposed that oligomers could insert themselves and form selective pores (channels) in the plasma membrane of some cell types ,21. Aris2+ responses to NMDA, such as anterior pituitary cells, Jurkat T cells and GT-1 neurons, were also responsive to amyloid oligomers. In contrast, cells without NMDA receptors that consistently lacked Ca2+ responses to NMDA were not responsive to amyloid oligomers. These data indicate that NMDA receptors are vital to generate a Ca2+ response to oligomers. However, the expression of NMDA receptors may not be sufficient, since the expression of NR1 and either NR2A or NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca2+ responses to NMDA but not to amyloid oligomers. Our study confirmed that NMDA receptors are the primary targets of amyloid oligomers. Cell lines bearing NMDA receptors and showing Ca2+ entry induced by the oligomers. The stimulation of Ca2+ influx into some neurons developing a neural network would induce activation of all interconnected neurons, depolarization of presynaptic neurons, action potential propagation, activation of voltage-dependent channels, exocytosis of neurotransmitters (mainly glutamate), activation of NMDA-type glutamatergic receptors and synchronous activity of the entire network. Therefore, the NMDA receptor and the amyloid channels seem to be the primary targets in the activation of a lethal Ca2+ influx to neurons. The controversial relationship between NMDA-like receptors and amyloid channel formation remains to be further clarified.In summary, we propose that a primary mechanism induced by oligomers in hippocampal neurons would be the formation of amyloid channels, a process that may require the expression of NMDA-type receptors. There is an additional unidentified component responsible for at least 20% of Ca