For example, the spatial and temporal organization of Ca2+ signaling in the central nervous system is fundamental for neurotransmission, where local changes in cytosolic Ca2+ concentration are needed to transmit information from neuron to neuron, between neurons and glia, and even regulating local blood flow according to the required activity. However, under pathological conditions, Ca2+ homeostasis is altered, with increased cytoplasmic Ca2+ concentrations leading to the activation of proteases, lipases, and nucleases. This review aimed to highlight the role of Ca2+ signaling in neurodegenerative disease-related apoptosis, where the regulation of intracellular Ca2+ homeostasis depends on coordinated interactions between the endoplasmic reticulum, mitochondria, and lysosomes, as well as specific transport mechanisms. In neurodegenerative diseases, alterations-increased oxidative stress, energy metabolism alterations, and protein aggregation have been identified. The aggregation of α-synuclein, β-amyloid peptide (Aβ), and huntingtin all adversely affect Ca2+ homeostasis. Due to the mounting evidence for the relevance of Ca2+ signaling in neuroprotection, we would focus on the expression and function of Ca2+ signaling-related proteins, in terms of the effects on autophagy regulation and the onset and progression of neurodegenerative diseases.Calcium (Ca Signaling pathways utilizing Ca2+ are present during all stages of life, from egg fertilization to death, and have also been implicated in several pathologies and aging. Indeed, Ca2+ plays a central role in muscle contraction, cell proliferation, differentiation, metabolism, and death, as well as autophagy and other processes. However, the connection between Ca2+ signaling and central nervous system (CNS) protection remains elusive.As a key second messenger, Ca2+ mediates essential signaling processes and plays an important role in synaptic plasticity. In fact, the regulation of Ca2+ concentrations in each compartment is a prerequisite for normal neuronal function. The neuronal Ca2+ signaling machinery is able to simultaneously activate different Ca2+-dependent processes, even from a distance. Due to the fact that neurons are susceptible to slight perturbations in intracellular Ca2+ levels, regulating these levels can be viewed as neuroprotective.In neurons, Ca2+ concentrations, in an acute or chronic manner, lead to catastrophic cellular consequences, thus demonstrating the role this key second messenger has in controlling cell fate. In the present review, we discussed the role of Ca2+ signaling in neurodegenerative diseases, highlighting the interplay between the proteins altered in those pathological conditions and the subsequent alterations in Ca2+ regulation. A better understanding of these processes is directly relevant to the onset and progression of neuronal cell death.There are several circumstances where altered Ca2+ homeostasis and subsequent changes in the Ca2+ levels of cellular compartments can promote survival or induce the activation of cell death pathways [Altered Capathways ,2. IncreThe extrinsic apoptotic pathway is induced by the activation of death receptors in the cellular membrane. For example, tumor necrosis factor receptor 1 (TNFR1) promotes the activation of initiator caspases  and activates effector caspases  , as well as other molecules stored in the intermembrane space (IMS) [c binds to apoptotic protease activating factor 1 (APAF1), ATP/dADP, and procaspase 9, forming an apoptosome that subsequently activates effector caspases, with caspase 3 being especially susceptible to activation [During activation of the intrinsic pathway, Bax translocates from the cytosol to the outer mitochondrial membrane (OMM) , where ice (IMS) . In the tivation . The exttivation .2+, nicotinamide adenine dinucleotide (NAD+), proteins, glutathione, and other metabolites into the cytosol  opening. Described by Hunter et al. (1976), the mPTP is a voltage-operated channel, located in the inner mitochondrial membrane (IMM) . These piewed by ). In addiewed by ). These iewed by ,16. As wiewed by ), which Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) are among the most prevalent neurodegenerative diseases. In the elderly population, AD is perhaps the most frequently diagnosed neurodegenerative disorder, progressively impairing the memory and learning processes. Most cases of AD and PD are sporadic and characterized by late-onset, mostly affecting people with 60 years of age or more; however, about 10% corresponds to familial cases, having an early onset and commonly observed in individuals that are around 50 years of age or younger. On the other hand, HD is an inherited monogenic autosomal dominant disease, with symptoms often appearing at 40–50 years of age.2+ signaling include:Components associated with familial cases of neurodegenerative diseases that have been found to interfere with Ca(1) AD: mutations in genes codifying amyloid precursor protein (APP) or Presenilins 1 or 2. Presenilins are part of the catalytic subunit of the γ-secretase complex. The γ- and β-secretase enzymes together cleave APP, consequently generating β-amyloid peptides (Aβ), subsequently forming protein aggregates.(2) PD: the presence of intraneuronal protein aggregates called Lewy bodies, mainly composed of α-synuclein. Mutations in leucine-rich repeat kinase 2 (LRRK2) may stimulate protein activity.(3) HD: mutations, present as an expansion of CAG trinucleotides (polyglutamine repeats) close to the N-terminus, of the protein huntingtin (mHtt), which are prone to aggregation.c), known as PrPSc, resulting in neuroinflammation and neurodegeneration. A more thorough discussion related to these protein aggregation events and Ca2+ signaling would be discussed later in this review.Another inherited neurodegenerative disease involving protein aggregation includes frontotemporal dementia (FTD), which is caused by mutations in either the microtubule-associated protein Tau (MAPT:FTDP - 17MAPT) or the progranulin (PGRN:FTDP - 17PGRN) genes. Additionally, Creutzfeldt–Jakob disease (CJD) is associated with the accumulation and aggregation of a misfolded/unfolded isoform of brain cellular prion protein  stress. For example, PrPSc accumulation activates an adaptive response, known as the unfolded protein response (UPR), to correct improperly folded ER proteins [2+ concentrations are tightly regulated and are essential for neuronal function and survival, with Ca2+ overload being related to excitotoxic neuronal cell death. In the following sections, the mechanisms by which Ca2+ promotes cell death and the effect of neurodegenerative disease-related proteins on ionic buffering would be discussed.Moreover, it is well known that disruptions in Caproteins . Thus, i2+ levels is responsible for the selective activation of Ca2+-mediated signal transduction pathways. Neurons can modulate the activity of pumps, as well as voltage and/or ligand-gated ion channels, to generate Ca2+ fluxes through the plasma membrane. The activation of voltage-gated Ca2+ channels (VGCC) promotes Ca2+ influx with the electrochemical gradient, resulting in increased cytoplasmic Ca2+ concentration-dependent enzyme activation, neurotransmitter release, neurite outgrowth, hormone secretion, and other Ca2+-dependent processes.The regulation of Ca2+ channel subtype. The L-type Ca2+ channels participate in Ca2+-induced long-term potentiation in dendritic spines and postsynaptic dendrites, while P- and Q-type Ca2+ channels, present in the presynapse, generate inward Ca2+ currents and initiate neurotransmitter release and presynaptic plasticity. The opening of T-type Ca2+ channels results in currents that induce the rhythmic action potential production in the sinoatrial node of the heart  and low voltage-activated (T-type channels), with the α1 subunit determining the Caiewed by ).2+ influx through L-type VGCCs in a variety of cellular models , an effiewed by , and Aβ1activity . Interesactivity . In FTD,athology .pars compacta (SNpc) neurons produce action potentials without the need of synaptic input, setting them apart from most other types of neurons [V1.3 channel activity made SNpc cells more susceptible to Ca2+-mediated excitotoxicity, caused by subjacent mitochondrial dysfunction [2+ influx and low levels of intracellular Ca2+-buffering proteins, such as calbindin; thus, slight increases in Ca2+ levels could have potentially damaging results [Substantia Nigra  neurons . A previfunction . Further results .2+ channels, which contributes to the increased susceptibility of SNpc neurons to cell death [2+ channel activity is increased in SNpc neurons. Remarkably, in an in vivo model of PD, the pharmacological inhibition of Cav1.3 L-type Ca2+ channels, with isradipine, restores the normal activity of dopaminergic cells, protecting SNpc neurons from cell death [In the α-synuclein A53T transgenic mouse model for PD, the aggregate-prone mutant protein has been associated with the upregulation and hyperactivation of N-type Call death . During ll death .The VGCCs have also been shown to be sensitive to dihydropyridines, which are commonly used to treat hypertension. Additionally, amlodipine, an L-type VGCC blocker, is being tested in a clinical trial (NCT02913664), as a drug candidate that can reduce the risks for developing AD, and nilvadipine, an L-type VGCC antagonist, has recently completed a phase III trial as a treatment for AD (NCT02017340). In PD, some dihydropyridine compounds have been shown to reduce the overall population risk for developing PD ,31, and c has been associated with VGCC dysfunctions. A synthetic PrP fragment (PrP106–126) has been found to inhibit L-type Ca2+ channel activity in rat cerebellar granule cells, decreasing Ca2+ currents, and leading to apoptosis [Sc has presented N-type Ca2+ channel dysfunction, thus attenuating Ca2+ responses [2+ influx via VGCC, which could potentially impair Ca2+ homeostasis  receptors [3 levels, group II (mGluR2 and mGluR3) and group III (mGluR4 and mGluR6–8) are coupled to Gi protein and results in decreased cyclic AMP (cAMP) levels. Pathologically, Aβ42 oligomers, but not monomers, alter Ca2+ release from ER stores, via mGluR5. It was also previously reported that Aβ42 oligomers reduced Ca2+ release induced by IP3 and increased caffeine-induced ryanodine receptor (RyR) Ca2+ release without altering the total Ca2+ content in the intracellular stores [Physiologically, many stimuli can cause intracellular Caeceptors . Glutamar stores .2+ peak, primarily due to contributions from the AMPA and kainate receptors, which exhibit fast kinetics and rapid (millisecond) desensitization [2+ levels by activating, through the N2B subunit, NMDAR [2+ dysregulation and increases oxidative stress, consequently impairing synaptic plasticity [Glutamate primarily acts on ionotropic membrane receptors, such as N-methyl-D-aspartic acid (NMDA), β-amino-3-hydroxy-5-methylsoxazole (AMPA), and kainate receptors. The NMDA receptors (NMDAR) are composed of NR1 and NR2A-D subunits, and during action potential generation, there is a rapid depolarization resulting in a high, transient Catization . It is wtization . Howevert, NMDAR . Additiot, NMDAR . It has asticity . 2+ signaling in the spine, the coordinated enzymatic activities of Ca2+calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN)—a Ca2+-dependent phosphatase—are involved. In synapses, CaMKII and CaN are expressed at high levels, with slight alterations in Ca2+ concentrations, resulting in reduced CaMKII autophosphorylation and increased CaN activity, thus shifting the balance of these enzyme activities. Synaptic CaMKII is essential for LTP, being centrally involved in memory formation, whereas CaN activation is necessary for LTD processes in the hippocampus. In AD, alterations in Ca2+ signaling have been suggested to shift the synaptic activities of CaMKII and CaN, favoring CaN and disrupting the equilibrium between LTP and LTD mechanisms. Due to the subsequent LTD stimulation and LTP inhibition, synapses are lost in AD brains, resulting in memory impairment  and long-term depression (LTD) . This syiewed by ).2+ influx leads to apoptotic cell death, induced by increased ROS generation, sustained mPTP opening, and the release of pro-apoptotic signals [2+ and an essential component of excitatory synapses [Caenorhabditis elegans expressing a mutant form of Tau, 2N4R-TauA152T (TauAT). In this model, TauAT expression increased cytosolic Ca2+ concentrations, due to release from ER stores, hyperactivated CaN, and initiated necrosis [2+ signaling. Furthermore, in FTD, the involvement of AMPA receptors (AMPAR) has been associated with disease-related presenile onset due to the presence of anti-GluA3 (AMPAR) antibodies in the sera of patients, which promote dendritic spine loss in culture [2+ signaling could contribute to neuronal damage [The hyperactivation of glutamate receptors, mainly NMDAR, can inflict cell injury and, in some cases, result in cell death , a proce signals . It is psynapses . More renecrosis , thus pr culture . Anotherl damage .2+ homeostasis is altered when challenged with this excitatory amino acid [With regards to PD, in an experimental dopaminergic denervation model, increased cortico-striatal glutamatergic activity was observed . In addiino acid .2+ signaling [2+ currents have been associated with fast receptor translocation to the cell membrane [There is evidence that indicates there is a striatal synaptic dysfunction in HD that is related to alterations in Caignaling . For exaignaling . It is lignaling  since inmembrane . As a remembrane ). In extmembrane . Consistmembrane , as wellmembrane ,63. Howemembrane .1-42 [1-42/40 in a transgenic mouse model [1-40 promotes α7 nAChR activation in a neuroblastoma cell line [1-42/42 induced the activation of both α7 and non-α7, and increased Ca2+ influx [Besides glutamatergic signaling, cholinergic neurons are also associated with the cognitive impairments observed in AD patients , where A1-42 ,69. Addise model , and thaell line . Anotherell line . Finally+ influx .Sc initiated a synaptotoxic signaling cascade that activates NMDA and AMPA receptors in hippocampal neurons, increases intracellular Ca2+ concentrations, stimulates p38 mitogen-activated protein kinase (MAPK), and depolymerizes actin filaments in dendritic spines, leading to the collapse of the cytoskeleton [c protected neurons from Ca2+ overload via the modulation of ionotropic glutamatergic receptors function [c was reported to promote axonal growth in a Ca2+-dependent manner [c was shown to form a complex with mGluR5, and this complex could be activated by ligands, such as β-amyloid oligomers and laminin, promoting Ca2+ influx into the neurons [c induced abnormal Ca2+ currents in cultured neurons and cerebellar slices, resulting in glutamate-induced excitotoxicity and neuronal death [In prion disease, Fang et al. (2018) demonstrated that PrPskeleton . Synaptifunction . In addit manner . PrPc wa neurons . In factal death .2+ storage, lipid and protein biosynthesis, secretion, and transport [2+ concentrations in response to increased levels of the ion in the cytosol [The ER is the largest organelle in a cell and is responsible for a variety of functions, ranging from Caransport . Numerou cytosol . In addi cytosol . Notably2+ homeostasis in the ER, extracellular Ca2+ concentrations range from 1–2 mM, cytosolic is about 100 nM, and the ER lumen is 100–800 μM [3 receptor (IP3R), in the ER membrane, promotes Ca2+ release from the ER to cytosol, but other Ca2+ homeostasis mechanisms also exist in the ER (reviewed by [2+-mediated ryanodine receptor (RyR) activation leads to Ca2+-induced Ca2+ release (CICR) [2+ ATPase (SERCA), a pump that mediates the transport of Ca2+ back to the lumen of ER, represents a Ca2+ reuptake mechanism  and plasma membrane protein ORAI1 assemble to form the selective pore of store-operated Ca2+ channel (SOCC) and increase cytoplasmic Ca2+ concentrations when the ER stores are depleted . For exe (CICR) , and actiewed by ). Furtheiewed by ). 2+ signaling in AD. Additionally, mutant presenilins have also been implicated in AD-related Ca2+ dysregulation, exerting similar effects through different mechanisms. In the ER, presenilins have been proposed to function as Ca2+-leak pores, and several familial AD mutations may compromise the pore function, leading to Ca2+ accumulation in the ER [3R activity, consequently augmenting channel-mediated Ca2+ release [2+ due to physical interactions between these proteins and SERCA [2+ influx via SOCC and low levels of STIM1, indicating that mutant presenilins could dysregulate Ca2+ homeostasis [Previously, we discussed the impact of Aβ and Tau alterations on Can the ER ,87. Thes release ,89. Pres release . Previound SERCA . Recentleostasis .2+ release from the ER into the cytoplasm [3ryanodine binding, which is indicative of upregulated RyR protein expression [2+ release is increased [2+ contributions, mediated through RyR and IP3R, ultimately leading to ER stress and apoptosis [There is evidence that supports the role of dysregulated RyR function in AD, caused by excessive Caytoplasm . For exapression . In the ncreased . In priopoptosis .2+ influx, consequently resulting in SH-SY5Y cell degeneration in vitro. Conversely, the overexpression or activation of TRPC1 protected neurons against the effects of the neurotoxin [2+-independent phospholipase A2 group 6 (PLA2g6) protein, have also been associated with altered Ca2+ homeostasis. Interestingly, skin fibroblasts obtained from idiopathic and familial PD patients with the PLA2g6R747W mutation displayed reduced Ca2+ entry via SOCC and depleted Ca2+ stores in the ER. Inhibition of PLA2g6-dependent Ca2+ signaling in a mouse model, using genetic tools, triggered autophagic dysfunction in and death of dopaminergic SNpc neurons [Neurotoxins, such as 1-methyl, 4-phenyl pyridinium (MPP+) and salsolinol, used for pharmacological PD models, were found to downregulate transient receptor potential canonical l (TRPC1) expression and induce thapsigargin-mediated Caurotoxin ,98. Stud neurons .2+ stores are also indirectly influenced by mHtt. For example, mHtt has been shown to bind to IP3R1, increasing the affinity of the receptor for IP3, and leaking Ca2+ ions from the ER [2+ leakage through the receptor and depleting internal ER Ca2+ stores [2+ influx due to sensitization of IP3R to IP3 agonists in the ER, consequently leading to continuous ER Ca2+ store depletion. In an attempt to regulate Ca2+ influx via SOCC, there is an upregulation of STIM2 [2+ depletion.The ER Cam the ER . Additio+ stores . In HD, of STIM2  and TRPCof STIM2  expressi2+ homeostasis, with intense ion exchange occurring through mitochondria-associated ER membranes (MAM). This ER-specific domain is enriched with cholesterol and anionic phospholipids, allowing the ER and OMM to become very close (10–30 nm)  is the primary channel for transporting Ca2+ from the OMM to the IMM, and subsequently taken up into the mitochondrial matrix by mitochondrial Ca2+ uniporter (MCU) [3R activation, Ca2+ stored in the ER is released and travels across the MAM, and taken up by VDAC in the OMM of the mitochondria [2+ homeostasis and cell function. Furthermore, the protein deglycase DJ-1 and parkin play a role in the maintenance of ER-mitochondria contact sites, and parkin may also directly stimulate phospholipase Cγ1 (PLCγ1) in the cell membrane, ultimately leading to increased cytosolic Ca2+ concentrations (reviewed by [The mitochondria and ER play important roles in cellular Caiewed by ), and fuer (MCU) . Followichondria . In termiewed by ).2+ regulation (reviewed by [+), through the coordinated activities of enzyme complexes of the electron transport chain (ETC), located in the IMM . Many oiewed by ).2+ storage and flux in the organelle, which can influence the expression and activity of several enzymes and carriers. In the IMS, FAD-glycerol phosphate dehydrogenase (GPDH) has been shown to be regulated by changes in Ca2+ levels [2+ concentrations stimulate enzymes that participate in the tricarboxylic acid cycle (TCA). In addition, components of the ETC are also modulated in response to mitochondrial Ca2+ levels. In fact, it has been shown that increased Ca2+ levels stimulate, by about two-fold, the activities of ETC complexes I, III, and IV [2+ has also been shown to modulate the activity of F0F1-ATP synthase [Interestingly, many functions of the mitochondria are directly linked to Ca+ levels . While i, and IV . Moreovesynthase .Mitochondria gained notoriety in HD pathogenesis when it was discovered that systemically administering a mitochondrial complex II inhibitor, 3-nitropropionic acid (3-NP), to rats caused preferential striatal degeneration and impaired energy metabolism, resembling characteristics of the neurodegenerative disease . Additio2+ across the OMM is mediated by VDAC1, a channel permeable to ions and small hydrophilic molecules, of which some are related to metabolic functions, such as succinate, malate, pyruvate, NADH, ATP, ADP, and phosphate [2+ concentration changes in the IMS can impact the activity of some metabolic enzymes. Finally, to reach the mitochondrial matrix, Ca2+ must cross the IMM, which is impermeable to the ion. This transport step is achieved using the ΔΨm to create a driving force for Ca2+ accumulation in the matrix. However, any factor that can cause the ΔΨm to collapse will consequently reduce and/or suppress mitochondrial Ca2+ uptake [2+ entry into the mitochondria, the movement of Ca2+ through the IMM is more complex and requires the specialized MCU. This IMM protein modulates mitochondrial Ca2+ uptake when concentrations of this ion are elevated in the cytosol [2+ channel, which strongly binds to the ion and transports it against the electrochemical gradient and into the mitochondrial matrix [2+ levels in the mitochondrial matrix and that the overexpression of this protein caused a notable accumulation of this ion [The transport of Cahosphate ,118. As + uptake . Compare cytosol . The MCUl matrix . In vitrthis ion .It is possible that Aβ inhibits the transport of nuclear-encoded proteins into the mitochondria since interactions with the translocase of the outer mitochondrial membrane 40 (TOM40) complex reduce the import of complex IV subunits. Moreover, Aβ interferes with the function of ETC complexes IV and V, while Aβ and Tau act synergistically to impair complex I function. The accumulation of Aβ in the IMS has also been shown to compromise the permeability of both the IMM and OMM (reviewed by ).2+ homeostasis can contribute to neuronal degeneration in an in vitro model of PD [Neurotoxins, such as MPP+, induce SH-SY5Y cell death by activating the intrinsic apoptosis pathway, which is highly dependent on mitochondria. Additionally, it appears that MCU plays a role in MPP+-induced cell death since MCU overexpression partially attenuates neuronal cell death, and downregulation results in augmented autophagy and cell death due to AMPK activation, thus demonstrating that disruptions in mitochondrial Cael of PD . Furtherel of PD .2+ dysregulation when compared with normal fibroblasts. For example, fibroblasts from AD patients exhibited long-lasting mPTP activation, and pharmacological blockage with Cyclosporine A (CsA) improved mitochondrial and cytosolic Ca2+ dysregulation. Additionally, the AD fibroblasts displayed a pattern of mitochondrial dysfunction that resembled what is observed in AD brains, thus highlighting the potential utility in employing fibroblasts as peripheral biomarkers for early AD detection [Previous studies have shown that Aβ can trigger the opening of the mPTP by interacting with and translocating cyclophilin D (CypD), a key component of the mPTP, adenine nucleotide translocase (ANT), and VDAC to the IMM (reviewed by ). In AD,etection . The opeetection  and, in etection .2+ [c release, even at lower Ca2+ concentrations, and triggers neuronal apoptosis [Several findings suggest that mHtt interacts with the IMM and OMM through its cytosolic N-terminus. This interaction might have a negative impact on protein import  and coul2+ . Additio2+ , and stupoptosis . Althoug2+ is also stored in a variety of acidic organelles, including lysosomes, lysosome-related organelles, endosomes, secretory vesicles, vacuoles, and Golgi complex [2+-stores ranges from pH 4–5, with the stored Ca2+ playing a central role in intracellular Ca2+ signaling processes, hydrolase activity, and autophagy regulation [2+-permeable ion channels, including members of the transient receptor potential channel (TRP) and two-pore channel (TPCs) families [Besides the ER and mitochondria, Ca complex ,134, wit complex . Dependigulation . These ofamilies .2+ can vary considerably among TRP channels. While most display low selectivity for Ca2+ (PCa/PNa 0.3–10), TRPV5 and TRPV6 exhibit high Ca2+ selectivity (PCa/PNa > 100), and TRPM4 and TRPM5 are Ca2+ impermeable [The TRP family is divided into two groups, which are further divided into seven subfamilies of channels. Group 1 encompasses the TRPC , TRPV (vanilloid), TRPM (melastatin), TRPN , and TRPA (ankyrin) subfamilies, while Group 2 contains TRPP (polycystin) and TRPML (mucolipin) (reviewed by ). These ermeable .2+-mobilizing second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) [2+ release from acidic Ca2+ stores that subsequently triggers amplified Ca2+ release from the ER, via IP3R and RyRs [2+ release from acidic organelles, dysfunctions in these channels have been related to PD [2+ handling of acidic stores [2+ signals with BAPTA-AM -ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester), which is a Ca2+ chelator. Additionally, another study demonstrated that TPC-mediated lysosomal Ca2+ release stimulated autophagy via Ca2+/calmodulin-dependent kinase kinase β (CaMKKβ) in cells over-expressing an LRRK2 mutation that causes an autosomal dominant form of PD and activates autophagy [2+ concentrations due to increased TPC-mediated Ca2+ efflux from the acidic organelles  ,141. Iniand RyRs . Due to ed to PD . For exac stores , and alsc stores . Hockey c stores . Interesutophagy . Upreguliewed by ). The ro2+ release can be mediated by the activation of transient receptor potential cation channel, mucolipin subfamily, member 1 (TRPML1), TRPML3, and transient receptor potential cation channel, melastatin subfamily, member 2 (TRPM2) , a member of the MiT-TFE helix–loop–helix leucine-zipper (bHLH-Zip) family of transcription factors, that, once in the nucleus, binds to the coordinated lysosomal expression and regulation (CLEAR) sequence, transcriptionally regulating the expression of TRPML1, as well as other autophagic and lysosomal genes [2+ release activates CaN, a phosphatase that binds and dephosphorylates TFEB. Furthermore, TRPML1 overexpression was shown to induce autophagy, whereas TRPML1 silencing attenuated autophagy in HeLa cells [Previous work has shown that lysosomal Caiewed by ). Each oiewed by . More spal genes ,149. TRPLa cells . 2+ release is necessary for the activity of mTORC1 [Under nutrient-rich conditions, serines  of TFEB are phosphorylated by mammalian/mechanistic target of rapamycin (mTOR) , and S14f mTORC1 ,153 thatf mTORC1 .Located in early and late endosomes/lysosomes, TRPML3 has also been associated with autophagy regulation. In HeLa cells, it was demonstrated that TRPML3 overexpression potentiated, and channel silencing inhibited autophagy, and expression levels of this channel also influenced endocytosis and membrane trafficking .2+ release is stimulated by adenosine diphosphoribose [2+ release is much higher than the values reported for other channels stimulated by this second messenger. Currently, the physiological relevance of NAADP-mediated TRPM2 activation is still unclear [2+ release [2+ release was shown to be responsible for autophagy activation via CaMKKβ . 2+ release from late endosomes and lysosomes [The physiological importance of the TRPML1-mediated Caysosomes  is perhaysosomes .2+ homeostasis [2+ concentrations were reduced [In cases of early-onset AD, presenilin 1 mutations alter lysosomal activity, impairing V-ATPase-mediated lysosomal acidification, and perturbing Caeostasis . In caseeostasis , and, in reduced . TherefoWith regards to the familial α-synuclein PD models, it was recently demonstrated that TRPML1 agonists promoted lysosomal exocytosis, reduced α-synuclein secretion, and prevented α-synuclein accumulation in a human neuroglioma cell line. Thus, demonstrating the promising therapeutic potential of specific channel activating compounds could contribute to the treatment of PD .2+ release, lysosomal storage, vesicle trafficking, and lysosomal acid homeostasis, and the dysregulation of these processes has been associated with several degenerative disorders [2+ release, Coen et al. (2012) demonstrated that the content of lysosomal Ca2+ stores was significantly reduced in presenilin gene deleted cells and neurons [Previous studies have shown that TRPML1 is an important component of lysosomal Caisorders ,170,171.isorders . Using G neurons . This re neurons . Autophagy has evolved to become a lysosomal catabolic process, allowing cells to degrade and recycle deleterious components or organelles by autophagosomes and preserve cell homeostasis. Under normal physiological conditions, autophagy activation is maintained at basal levels but can be induced in response to nutrient deprivation, hypoxia, ER stress, and DNA. Autophagy supplies the cells with the essential amino acids, nucleotides, and fatty acids, needed for the synthesis of new molecules. However, post-mitotic cells, such as neurons, have a low proliferative rate and depend heavily on autophagy for cellular maintenance, making them extremely vulnerable to changes in this pathway damage (reviewed by ).2+-sensitive manner—which is in contrast to microautophagy or CMA, where cargos are sequestered and internalized with or without the aid of lysosomal membrane receptors  ,177. Maciewed by ).2+ levels and Ca2+ dependent kinases, such as Ca2+/calmodulin-dependent kinase 2 (CaMKK2), and blocks mTORC1, ultimately inducing autophagy [Autophagy is directly related to anabolic and catabolic processes, participating in a variety of regulatory pathways involved in cell growth and proliferation. In fact, the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and MAPK pathways are typically activated by the mechanistic target of rapamycin complex 1 (mTORC1) (reviewed by ), the mautophagy .2+ release leads to ATG1 protein Unc-51 like autophagy activating kinase (ULK1) activation and autophagy induction [The ATG proteins have also been shown to participate in the activation of autophagy, modulating the machinery responsible for autophagosome formation. Previous studies have shown that AMPK-mediated Canduction ,182. Tognduction ). It hasnduction .2+ in autophagy was performed by Gordon et al. (1993) and demonstrated that extracellular and intracellular Ca2+ chelators (BAPTA-AM) reduced the levels of autophagy in isolated rat hepatocytes [2+ mobilizing agents  induced autophagosome accumulation, which was accompanied by mTORC1 inhibition and CAMKK2 and AMPK activation [2+ can exert both inhibitory and stimulatory effects on autophagy [2+-mediated autophagy response depends on both the amplitude of the response and the specific channels involved  demonstrated that deleting Fkbp1b resulted in RyR leakage and mTOR induction . Furthertivation . In C2C1tivation . 2+ levels with a concomitant loss of ER Ca2+ stores, blocked autophagic flux and inhibited autophagosome and lysosome fusion [2+ levels. This compound has been shown to inhibit nutrient starvation-induced autophagy [The role of SERCA in autophagy induction is poorly understood. Studies showed that thapsigargin treatment, which increases cytosolic Cae fusion . Indeed,utophagy . Additioutophagy .2+ uptake increased AMP:ATP ratios, activated AMPK, and induced mTORC1 independent autophagy [With regards to MCU, data suggested that it negatively regulated basal autophagy rates since the pharmacological agent Ru360 and genetic inhibition of the uniporter abrogated mitochondrial Cautophagy ,201,202.2+ channel antagonists  increase autophagy independent of mTORC1, while L-type Ca2+ channel agonist BAYK8644 activates calpains and increases cAMP levels, which, in turn, positively regulates IP3 levels and results in increased cytosolic Ca2+ levels. As a result of continuous calpain activation, a cyclic pathway of autophagy inhibition is created [It is well known that VGCC has a regulatory role in autophagy; however, the specific function of these channels in autophagy is still unclear and likely depends on the type of channel activated or inhibited. For example, L-type Ca created .2+ entry by SKF-96365 was shown to stimulate autophagy through AKT/mTORC1 pathway inhibition [Several intracellular processes, including apoptosis, cellular proliferation, and gene expression, involve SOCC, and the available data in the literature suggests that these channels can differentially influence autophagy depending on cell type and autophagy inducer (reviewed by ). Moreovhibition ,206.2+ entry via SOCC positively regulates autophagy. In endothelial progenitor cells, increased Ca2+ transport through SOCC activated autophagy via the CAMKK2/mTOR pathway, and both Ca2+ chelators (EGTA or BAPTA-AM) and STIM1-silencing inhibited autophagy, thus confirming the involvement of SOCC in this process [On the other hand, studies have also demonstrated that Ca process . Under h process , which i process . Therefo2+ signaling has on autophagy regulation, with evidence suggesting that cellular Ca2+ signals are involved at different stages of the autophagy process, and playing roles in the initiation of phagophore formation and elongation and autophagosome/endosome fusion with the lysosome. Ca2+ signaling at ER-mitochondria contact sites would likely influence autophagy initiation, whereas Ca2+ channel activity at ER-lysosome contact sites would modulate lysosomal function, autophagosome fusion, and cargo degradation. For instance, with respect to AD, the deletion of presenilin-1 leads to a loss of lysosomal vATPase activity, which raises lysosomal pH, perturbs TRPML-mediated lysosomes, and blocks autophagy [The study results presented up to this point clearly demonstrates channel diversity and emphasizes the importance Cautophagy .2+ signaling and Ca2+ sources, the role this ion plays in autophagy is extremely complex and relies on multiple factors, such as cell type, status, including nutritional, along with ion channel type, localization, expression levels, isoforms, and regulatory mechanisms. Indeed, ion channel dysfunctions and altered autophagy regulation have been implicated in various diseases, suggesting that Ca2+ channel expression and/or activity could be exploited as potential therapeutic targets for the modulation of autophagic processes.Given the diversity of cellular Ca2+ release involving activated TPC channels. Following the release of Ca2+ from the lysosome, Ca2+ is also released from the ER, resulting in Ca2+/CaMKK/AMPK pathway activation and autophagy induction. The increase in LRRK2 activity and Ca2+ release leads to a pathological increase in autophagy induction and defective lysosomal acidification [2+-induced cell death, representing yet another potential cause of PD pathogenesis [In PD, the presence of Lewy bodies formed by aggregated α-synuclein is probably the result of protein aggregates that are resistant to autophagic degradation . Accordification . The ovefication ,216. Besogenesis .2+ homeostasis; however, the exact role that Ca2+ plays in apoptosis and autophagy regulation, especially in neurodegenerative diseases, remains unknown. In the CNS, Ca2+ controls cell fate and metabolism, accounting for most of the energy expenditure of the body. Altered Ca2+ homeostasis has been linked to numerous pathological processes and can lead to the activation of death pathways. Therefore, Ca2+ levels must be well regulated under these conditions since neurons are particularly susceptible to damage. The ER, mitochondria, and lysosomes mainly control Ca2+ buffering, and the interplay between these organelles, along with Ca2+ transport mechanisms involved in shuttling this ion across membranes, are essential to cellular homeostasis, as well as the modulation of autophagy. This catabolic process emerges as a neuroprotective strategy since the basis of neurodegenerative diseases lies in the accumulation of unfolded proteins that consequently impair cell function, alter metabolism, and upregulate the unfolded protein response, to the demise of the cytoskeleton. In addition, it has been reported that during aging and in age-related neurodegenerative pathologies, there is an impairment of autophagy, accompanied by elevated oxidative stress and unbalanced energy. Understanding the role of Ca2+ regulation buffering in apoptosis and autophagy could reveal novel therapeutic targets for combating neurodegenerative diseases.Based on the results from a variety of experimental models, it is clear that specific AD, PD, and HD-related proteins can disrupt Ca


The importance of zinc for male fertility only emerged recently, being propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review the properties, biological roles and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss the recently discovered signaling pathways involving zinc in sperm maturation and fertilization. Emphasis is on the zinc-interacting sperm proteome and its involvement in the regulation of sperm structure and function, from spermatogenesis and epididymal sperm maturation to sperm interactions with the female reproductive tract, capacitation, fertilization, and embryo development. Merits of dietary zinc supplementation and zinc inclusion into semen processing media are considered with livestock artificial insemination (AI) and human assisted reproductive therapy (ART) in mind. Collectively, the currently available data underline the importance of zinc ions for male fertility, which could be harnessed to improve human reproductive health and reproductive efficiency in agriculturally important livestock species. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize semen processing procedures for human infertility therapy and livestock AI. Zinc (Zn) is one of the most highly abundant elements on earth, an essential micronutrient to all things living, typically occurring as a divalent cation metal with moderate reactivity and reducing properties. Essential biological roles of zinc include signaling, enzymatic activities, regulation of normal growth and sexual maturation, digestion, homeostasis of central nervous system, and mitochondrial oxidative stress ,2. ConveE. coli to mammals, tightly regulate free zinc ion (Zn2+) distribution, even though its toxicity is relatively low [2+ is found in the muscle and bone [2+ are the prostate, liver, gastrointestinal tract, kidney, skin, lung, brain, heart, and pancreas [2+ is important for survival and fitness; thus, when Zn2+ is consumed in excess, it is important for the body to handle its surplus [2+ occurs via the serum, where Zn2+ is bound predominantly to albumin (major binding protein for up to 60% of Zn2+); the remaining Zn2+ is bound predominantly to 12 other proteins, including α2-macroglobulin, transferrin, ceruloplasmin, IgG, IgA, IgM, complement C4, haptoglobin, and prealbumin [2+ accounts for only ~0.1% of bodily Zn2+ [2+ storage system in the body, and therefore only the daily intake of Zn2+ will ensure steady availability [Cells of all organisms, ranging from vely low ,10. In hand bone . Other opancreas ,13,14. H surplus . Upon inealbumin ,17. Seruily Zn2+ . Further2+ localizes in the nucleus, while 50% is stored in the cytoplasm and the rest is associated with membranes [2+ through biological membranes, thus exercising sustained homeostatic control. These include zinc-importer  family proteins that transport Zn2+ into the cytosol and the zinc transporter (ZnT) family proteins transporting Zn2+ out of the cytosol [2+ enters a cell by ZnTs and ZIPs, it becomes sequestered within the endoplasmic reticulum, mitochondria, and Golgi, or other cell type-specific membrane bound vesicular structures, also called zincomsomes [2+ complexing with cytosolic proteins maintains the concentration of free cytosolic Zn2+ within range between picomoles and nanomoles, depending on the cell type [2+ is bound by the apoprotein thionein, to form metallothionein (MT). MTs are small ubiquitous proteins (6–7 kDa) that are rich in cysteine that can complex transition metal ions [2+, buff excess Zn2+, and supply such cation under Zn2+ deficiency states [On the cellular level, 30–40% of Znembranes . There a cytosol . Complet cytosol  families cytosol , and the cytosol . Once Zncomsomes ,24. Cytoell type ,25,26,27tal ions ,29. One y states . 2+ homeostasis in reproductive system. In female gametes, Zn2+ plays a gatekeeping role in regulating meiotic resumption [2+ release from the mammalian oocyte at fertilization was recently reported [2+ for male fertility only emerged recently, propelled in part by consumer interest in nutritional supplements containing ionic trace minerals. Here, we review properties, biological roles, and cellular mechanisms that are relevant to zinc function in the male reproductive system, survey available peer-reviewed data on nutritional zinc supplementation for fertility improvement in livestock animals and infertility therapy in men, and discuss recently discovered signaling pathways involving zinc in sperm maturation and fertilization.Limited information exists on the regulation of Znsumption ,33,34. Areported ; inspiriN,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN)-induced Zn deficiency causing germ cell apoptosis [2+ transporter ZIP9 serves as a membrane-associated receptor interacting with G-protein Gnα11 to mediate the non-classical testosterone signaling cascade in murine spermatogenic GC-2 cells [2+ oscillations that were observed in spermatogenic cells seem to be modulated by Zn2+ [2+ is incorporated into the nucleus at ejaculation [2+ associates with protamines and forms zinc bridges, most likely through imidazole groups of histidine and thiols of cysteine [2+ chelation with 2,2′,2′′,2′′′- tetraacetic acid (EDTA), causing the disruption of the protamine zinc bridges [2+ is bound to sulfhydryl groups of ODF protein cysteine groups, to protect the nascent flagellum from premature oxidation [2+ is selectively removed from the flagellum by a 160 kDa protein, enabling the oxidation of sulfhydryl groups and stiffening the ODF to support progressive motility [2+ have been found in the acrosome [2+ [2+ is therefore a prerequisite for the completion of sperm capacitation [2+ (100 µM) reduce sperm motility in a reversible manner [In eel, zinc is necessary for the sustenance of germ cells and spermatogenesis progression, with poptosis . Zinc trpoptosis  as well poptosis . Furtherpoptosis . Zn2+ tr-2 cells . Further by Zn2+ . Zinc io by Zn2+  and nasc by Zn2+ ,44. Addiculation . Nuclearcysteine , as propcysteine ,48. Thes bridges ,50,51. Ixidation . During motility . High coacrosome  and protsome [2+ ,55, as isome [2+ ,57. The citation , a comple manner . Initiate manner  and the 2+ becomes incorporated into spermatozoa during ejaculation [2+ becomes incorporated into spermatozoa upon mixing with seminal fluid, there are also seminal fluid Zn-interacting proteins competitively binding free Zn2+. In humans, a bulk of seminal fluid Zn2+ is bound to high and low molecular weight ligands derived from prostatic and vesicular secretions [Additional Znculation ,45 whereculation ,48, sperculation ,61, membculation , and antculation ,63. As Zcretions ,65,66,67cretions ,69. Zinccretions  and dog cretions ,72, and 2+, usually as a requirement for their biological activity. Human genome sequencing and combined proteomic approaches independently identified 1684 proteins in the human proteome as zinc-containing proteins [Zinc-containing proteins, commonly known as metalloproteins, are capable of binding one or more Znproteins . Metalloproteins ,74. Metaproteins . Spermatproteins . The maj2+ for their function [2+ is the only metal that is encountered in all six classes of enzymes, . This can be attributed to two properties of Zn2+: (i) relatively low toxicity when compared to other transition metals [2+ fulfills three functions in the Zn-enzymes: (i) catalytic, (ii) co-active , and (iii) structural [2+ takes part directly in enzyme catalysis. Co-active Zn2+ enhances or diminishes catalytic function in conjunction with catalytic Zn2+, but it is not indispensable for catalytic function [2+ is required for stabilization of the quaternary structure of oligomeric enzymes.More than 300 enzymes have been identified that require Znfunction , represen metals  and (ii)n metals . Zn2+ fuructural . Catalytfunction . Structu2+  and up to three calcium ions (Ca2+), which stabilize the structure. The cysteine rich region in propeptide chelates the catalytic Zn2+, keeping MMPs in an inactive zymogen form [Matrix metalloproteinases (MMPs) belong to a family of zinc-dependent endopeptidases, which are involved in the degradation of extracellular matrix proteins. Since the first discovery of MMPs in the early 1960s, MMPs have grown in number and at least 28 species have been identified to this date; for subtype categorization, distribution, and substrate specificities, see review by Cui et al. . Structugen form . MMP2 angen form ,83 and cgen form ,85. Furtgen form . High legen form  demonstrgen form  demonstrgen form  noticed 2−), to hydrogen peroxide (H2O2), and oxygen (O2) [Superoxide dismutases (SOD) are metalloenzymes that are responsible for dismutating the superoxide anion (Ogen (O2) . Three igen (O2) . It was gen (O2) ,97,98,99gen (O2)  suggestegen (O2) .2+ containing proteins of spermatozoa are sorbitol dehydrogenases that convert sorbitol to fructose, and endow spermatozoa with and have been correlated to motility [4) also has been reported to have relationship with sperm motility [4 blocked sperm capacitation [2+ containing protein, has been reported in testis, epididymis, and spermatozoa of stallion, boar, and man [2+ and one Mg2+ is present in mammalian seminal fluid [2+ containing proteins that are found in the spermatozoa include fructose-bisphosphate aldolases [2+ [2+ [Another significant group of Znmotility . Lactatemotility ,103,104.citation . We prevcitation , and impcitation . Angiote and man ,113,114. and man , sperm c and man ,117, and and man . Alkalinal fluid ,120 and al fluid . The preal fluid . Additioldolases , of whicases [2+ , and alcases [2+ ,126. Theases [2+ ). Notewo [2+ [2+ . The met [2+ [2+ . Altoget2+ influx was understood as key for capacitation, today it is understood that the Zn2+ efflux is the gatekeeper to this important Ca2+ influx [2+ entry . Prior + influx , there w+ influx . Much of+ influx , thus rexchanger  and possger sNHE  in murinn humans ), as wel2+ content, and sperm global and progressive motility in humans [2+ by -2,3-Bis(sulfanyl)butanedioic acid (DMSA), 2,3-dimercaptopropane-1-sulfonate (DMPS), or dl-penicillamine leads to increased average straight line velocity and progressive sperm motility while decreasing the percentage of nonlinear motile spermatozoa [2+ removal to be solely associated with the stiffening of the ODF. Voltage-gated proton channel, Hv1 localizes to the sperm flagellum and it is responsible for sperm cytoplasmic alkalinization through transmembrane proton extrusion [There is a moderate negative correlation between flagellar Znn humans . Chelatirmatozoa . Though xtrusion . Hv1 is xtrusion . 2+ localization patterns that are indicative of the sperm capacitation state [2+ establishes these sequential subpopulations, and thereby is the previously unknown regulatory time clock of sperm capacitation. The decrease in Zn2+ concentration from the ejaculation/deposition site to the site of fertilization could remove sperm Zn2+ simply via concentration gradient differences and the filtering out of seminal fluid, thereby promoting sperm capacitation. Further, it is well understood that the ubiquitin-dependent protease holoenzyme, the 26S proteasome regulates sperm capacitation  inhibiting Hv1, lower concentrations (20 and 50 µM) have been implicated in promoting acrosomal exocytosis in sea urchin [2+ interacts with Zn-sensing receptor (ZnR) GPR39 of the G-protein-coupled receptor (GPCR) family that is found in the sperm acrosome. Such interaction stimulates acrosomal exocytosis through epidermal growth factor receptor (EGFR) transactivation and the phosphorylation of phosphoinositide 3-kinase (PI3K) causing acrosomal Ca2+ mobilization [2+ in sperm capacitation and therefore more research will be needed to fully comprehend these contrasting pathways . The sperm Zn signature is a collective term for four distinct Znon state . These z (review ), as welrmatozoa , and parrmatozoa , bull [1rmatozoa , and humrmatozoa  spermatormatozoa , and boarmatozoa ). The ren2+ site . AdditioLa cells . Contrara urchin  and bovia urchin  spermatolization . This im2+ release from the oocyte cortex, nicknamed the Zn2+ spark, has recently been discovered in mammals [2+ spark [2+ increase in the ZP matrix observed when the fertilized oocyte zona becomes refractory to sperm binding in the mouse [2+ is chemorepulsive, possibly overriding the chemoattraction of oocyte-secreted progesterone in capacitated human, mouse, and rabbit spermatozoa [2+ has been shown to inhibit fertilization when added to bovine in vitro fertilization (IVF) media [2+ regulates the activity of the proposed sperm-borne ZP lysins, the proteinases [Successful embryo development in mammals depends upon efficient anti-polyspermy defense, preventing the entry of more than one spermatozoon in the oocyte cytoplasm at fertilization, and thus alleviating an embryo-lethal polyploidy. While membrane depolarization and cortical granule exocytosis are regarded as the main barriers to polyspermy, a sperm-induced Zn mammals ,35. Besi2+ spark , there irmatozoa . In lighrmatozoa , could iF) media . In furtteinases  implicatteinases ,55, 26S teinases ,144, andteinases , therefoteinases .2+ has been reported in cases of male infertility that are associated with accidental Chernobyl radiation in Ukraine [2+ and male fertility. Indeed, seminal fluid Zn2+ concentration is positively correlated with sperm count and normal sperm morphology [2+ supplementation improves sexual dysfunction in rats [2+ to increase serum testosterone levels [2+ supplementation [2+ supplementation results in increased sperm counts in ram [2+ intake and action on intraprostatic Zn2+ levels remain unknown; however, if such supplementation increases prostatic levels, it could perceivably increase the percent of non-capacitated signature 1 spermatozoa at the time of ejaculation. Such would be beneficial for inhibiting Hv1, warding off premature sperm capacitation. Goat dietary Zn2+ supplementation increases sperm plasma membrane and acrosome integrity, and percent of viable spermatozoa, also increasing seminal fluid SOD, catalase, and glutathione peroxidase activities [2+ and Zn2+ dietary co-supplementation to bucks of the Osmanabadi goat breed allowed for them to reach puberty 28–35 days earlier [2+ supplementation has a positive influence on multiple male reproductive measures and can serve as male infertility therapy under certain circumstances, one study suggests that supplementation over 100 mg/day is associated with a 2.29 relative risk of advanced prostate cancer [2+ is not generally considered a carcinogen [Reduced seminal fluid Zn Ukraine , signifyrphology . Consequ in rats  and urem in rats , which ie levels . The negentation . Oral Zns in ram  and humas in ram . Zinc sus in ram . Dietarytivities . Additio earlier . While Ze cancer . The exarcinogen ; yet, in2+ in media/semen extenders in agriculturally important livestock species propagated by artificial insemination (AI). A highly desirable property of such media is to reduce reactive oxygen species (ROS) that effect sperm function by the oxidation of lipids, proteins, and DNA . Notable anions , though entation  and imprentation . Spermat loading , to the 2+ is reprotoxic to the terrestrial worm Enchutraeus crypticus [2+ reprotoxicity [2 dietary supplementation to both male and female rats at 30 mg/kg/day but not 15 or 7.5 mg/kg/day showed significant reduction in fertility, offspring viability, and body weight of F1 pups; however, it had no effect on litter size  [r = −0.426) has been found between total flagellar Zn2+ content and the percentage of morphologically normal spermatozoa in men [2+ (as reported by fluorescent Zn-probe) [2+ toxicity or simply a product of defective spermatozoa failing to regulate their ion fluxes is unknown. Dietary Zn2+ deficiency studies in rat have shown reduced relative weight of the testes, epididymides, and seminal vesicles (organ by percentage of body weight) and reduced prostate weight . In the same study, the length of the flagellum was reduced on average by 24% in Zn2+ deficient rats, accompanied by a substantial increase in morphologically abnormal spermatozoa . Some of these defects could be due to abnormal testicular function from modulated fatty acid composition, interrupting essential fatty acid metabolism, where ω-6 polyunsaturated fatty acids were highly enriched [2+ deficiency is known to trigger autophagy in yeast [2+ deficiency. In the absence of fertilization, sperm capacitation is a terminal event leading down a rapid path of apoptosis [2+. No studies have been performed to observe whether micromolar levels of Zn2+ under capacitation-inducing conditions can prolong sperm lifespan during a fertilization competent state  . Nanosizbserved) . A modera in men . Morpholn-probe) . Whetherenriched . Zn2+ dein yeast , and elepoptosis , possiblpoptosis  in an enn2+ flux ). If suc2+ plays a gatekeeping role in male gametes just as it does in those of the female. Prostatic seminal fluid with the highest concentration of Zn2+ found in any bodily fluids plays a crucial role in fending off premature sperm capacitation and it provides antioxidant activity, while lower concentrations of Zn2+ may be a prerequisite for successful acrosomal exocytosis. A list of reviewed Zn-containing and interacting proteins that are involved from spermatogenesis through the final preparatory steps of fertilization is summarized in 2+ in male fertility, further research needs to be pursued, especially to fully disclose the Zn-interacting sperm proteome and its place in various cellular pathways controlling male reproductive function. Additionally, dietary and semen media Zn2+ supplementation has been found to be beneficial for male fertility. Collectively, the currently available data already hint at the importance of zinc ions for male fertility, which could be harnessed to improve the reproductive performance of livestock and increase the success rate of human assisted reproductive therapy. Further research will advance the field of sperm and fertilization biology, provide new research tools, and ultimately optimize the semen processing procedures for human infertility therapy and livestock artificial insemination.Through a variety of pathways, Zn


S. hollandi using RNA-seq. A total of 4.73 × 108 raw reads were obtained from nine brain samples. De novo transcriptome assembly identified 387,085 unigenes with 2.1×109 nucleotides. A total of 936 annotated unigenes showed significantly differential expression among the control, fasting, and fasting-refeeding groups. The down-regulated differentially expressed genes (DEGs) during fasting were mainly associated with cell cycle, DNA replication, and mitosis. The up-regulated DEGs were mainly related to glucose and lipid metabolism, material transportation, and transcription factors. Most decreased DEGs during fasting were restored to normal levels after refeeding. Comparing with the control group, genes associated with protein synthesis, stimulus response, and carbohydrate metabolism were significantly over-expressed and pro-opio melanocortin (POMC) was down-regulated during the refeeding period. In conclusion, fish mobilized stored energetic materials and reduced energy consumption to prolong survival during fasting. After refeeding, the down-regulation of DEGs, e.g., POMC may be associated with compensatory growth. Up-regulation of DEGs related to ribosomal protein, stimulus response, and carbohydrate metabolism may contribute to eliminate negative effect of starvation on brain. This study provided the first transcriptome data related with impact of short-time starvation and refeeding in S. hollandi brains.Starvation is a common stress in fish. The underlying molecular mechanisms associated with growth depression caused by feeding restriction and compensatory growth are not well understood. We investigated the effect of fasting and refeeding on the transcriptome profiles of brain in juvenile  Larimichthys crocea)[Oncorhynchus mykiss)[Ictalurus punctatus)[Starvation is a common stress in fish. Extreme aquatic conditions, such as unsuitable temperature, hypoxia, and high population density, can affect the feeding behavior of aquatic animals. The starvation condition is strongly associated with the mobilization of the body’s energy reserves, which are mainly carbohydrates, fats, and proteins. During s crocea), rainbows mykiss), and chaunctatus); howeverunctatus),4.Phoxinus phoxinus) [Carassius auratus gibelio)[Lates calcarifer)[Ictalurus punctatus)[Cynoglossus semilaevis)[Salmo salar)[Paralichthys adspersus)[Ctenopharyngodon idella)[Compensatory growth is defined as the physiological process of accelerated growth when food intake is restored following a period of food deprivation. The phehoxinus) , gibel c gibelio), barramulcarifer), channelunctatus), tongue milaevis), and Atlmo salar). Informadspersus), grass cn idella), and rain idella), have ren idella).Spinibarbus hollandi, an endemic Cyprinidae species in southeastern China, is mainly distributed in the provinces of Anhui, Hubei, Hunan, Fujian, Guangdong, and Guangxi. S. hollandi is an easy-to-raise omnivore, and has already attracted increasing attention owing to its high nutritional and medicinal value. However, the low growth rate has severely limited its farming[S. hollandi by improving the rearing condition and via cross-breeding. We have developed molecular markers associated with growth traits in S. hollandi[S. hollandi are rare. The underlying molecular mechanisms associated with growth depression caused by feeding restriction and compensatory growth are not well understood.s farming. In rece hollandi and receS. hollandi brain in response to fasting and refeeding. Our study provides an important dataset for understanding the molecular mechanism of starvation response and compensatory growth in fish.The next-generation sequencing technology has revolutionized the field of genomics. RNA-Seq can be used to analyze the transcriptome of non-model species without requiring their genomic information –20. BraiS. hollandi fish at about one-month-old were obtained from Shaoguan Fisheries Research Institute, Guangdong Province, China. The fish were sexually immature and had an approximate weight of 3–5 cm. 210 fish were equally distributed to seven aquariums containing 100 L of continuous flow, filtered water with aeration. The fish were housed at 26°C with a 14:10 h light/dark photoperiod. The fish were fed with a commercial pellet containing 35% crude protein, 4% crude fat, 14% crude fiber, 14% crude ashes, and 12% humidity , twice daily to apparent satiation at 7:00 am and 5:00 pm, for a 2-week acclimation. Of the seven aquariums, four were served as control groups, one was used as the fasting group and the remaining two aquariums were used as the refeeding groups.Fish in the control groups were fed to apparent satiation everyday as mentioned above. The fish were sequentially sampled on day 0 (C0), day 7 (C7), day 14 (C14), and day 40 (C40). Fish in the fasting group were deprived of feed for 7 days, and then sampled (F7). The fish in the refeeding groups were fed twice per day to satiation after 7 days of fasting. The fish were sampled after 7 (R7) and 33 days refeeding (R33). All seven groups were then anesthetized by an overdose (100 ppm) of eugenol (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). The body weights of each group were measured prior to sampling (n = 8). Brain tissues were rapidly isolated, immediately frozen in liquid nitrogen, and stored at -80°C.Total RNA of brain tissues was extracted using RNAiso reagents , following the manufacturer’s instructions. The quantity and quality of RNA samples were determined using Epoch Microplate Spectrophotometer , Agilent 2100 instruments and electrophoresis using 1% agarose gel. RNA samples with 28S/18S ratio >1.0, optical density (OD) 260/280 values >1.8 and <2.0, and RNA yield > 5 μg were used for subsequent transcriptome analysis.RNA samples of the C7, F7, and R7 groups were used for RNA-seq analysis. To minimize the variation among individuals, equal quantities of RNA from 5 samples belonging to the same group were pooled, and 3 replicates of mixed RNA pools were prepared for each group. A total of 9 RNA pools were used for cDNA library preparation. mRNA was purified from total RNA using oligo (dT) magnetic beads and fragmented using fragmentation buffer. First-strand cDNA was synthesized using SuperScript II Reverse Transcriptase  followed by second strand generation using the components dNTPs, buffer, DNA polymerase I, and RNase H. After purification, adapters were ligated to the cDNA, and then enriched using PCR. The nine cDNA libraries were sequenced on an Illumina-Hiseq 2000 platform using the 150 bp paired-end approach.de novo assembled using Trinity software version 2.5.1 [-5), Clusters of Orthologous Groups of proteins , Kyoto Encyclopedia of Genes and Genomes , and Swiss-Prot databases . Gene ontology (GO) annotation was performed using Blast2GO with an e-value threshold of 1e-6.Clean reads were generated by removing low-quality reads reads containing adapter and ploy-N from raw data. Clean reads were on 2.5.1 . The assp-value (p-adj) with a cut-off of 0.05 was used to identify significant differentially expressed genes (DEGs). Pathway enrichments of DEGs were analyzed using the KEGG database.Total number of mapped reads for each transcript was determined and normalized, to calculate the expected number of Fragment Per Kilobase of transcript sequence per million base pairs sequenced (FPKM) by using RSEM V1.2.15 . Differe−ΔΔCt method) [Six DEGs  belonging to different functional classification were selected and analyzed using the software FasParser  to ident method) . All datp < 0.05). After 7-day refeeding, the body weight of C14 and R7 were 1.94 g and 1.63 g. After 40 days, the body weights for C40 and R33 was 3.34g and 3.28 g, respectively. However, no significant difference between groups was detected at the same time points.The body weights of fish in the control groups  and the test groups  were measured. The growth curves for these groups were shown in 8 raw reads (150 bp) were yielded from nine brain samples. After removing low-quality sequences, 4.73 × 108 clean reads with 71.01 Gb were generated. The Q20 and Q30 of all samples were above 96.27% and 90.97%, respectively. The sequence data were deposited in the NCBI SRA database, under the accession number, SRP156354.Through high-throughput paired-end sequencing, 4.93 × 10de novo assembly, 864,488 transcripts  were generated. The longest transcript of a gene was defined as a unigene for further analysis. A total of 387,085 unigenes with 2.1×109 nucleotides were obtained, with an average length of 568 bp. The N50 and N90 were 752 and 254 bp, respectively.After Danio rerio, followed by those from Astyanax mexicanus (5.5%), Clupea harengus (2.8%), Mus musculus (2.4%), and Oncorhynchus mykiss (2.3%).A total of 349,336 (90.24%) unigenes could be annotated in at least one of the following database: Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO. A total of 10,438 (6.65%) unigenes were annotated in all these databases. Among of all unigenes, 76,988 (19.88%) unigenes were matched in the Nr database. Of which, nearly 58.6% could be annotated with sequences from Functional prediction and classification of all unigenes were performed using the GO database. A total of 71,164 unigenes were classified into 56 GO terms, including 10 molecular function terms, 20 cellular component terms, and 26 biological process terms. Cellular process , binding , single-organism process , metabolic process , and cell , were the five most abundant functional terms at the second GO level .Furthermore, all unigenes were aligned to COG database for phylogenetic classification. In total, 22,886 unigenes were divided into 26 functional categories . The larTo interpret the biological pathways, we annotated all assembled unigenes in the KEGG database, and 39,265 unigenes were matched in level 2 of the KEGG pathway. Signal transduction (6744), endocrine system (3302), cellular community (2976), immune system (2643), and nervous system (2432) were the five most significant matched catalogs in the KEGG pathway .A total of 936 annotated unigenes showed significantly differential expression among the C7, F7, and R7 groups. Of the DEGs, 464 were significantly expressed between C7 and F7 (fasting period), 491 were significantly expressed between F7 and R7 (refeeding period), and 230 were significantly expressed between C7 and R7.CCNA2, CDC20, CDC25A, CDC6, CDK1, CDK2, CHEK2, E2F3, PLK1, and PTTG1), DNA replication  and mitosis .After fasting, of the 464 DEGs, 169 and 295 genes were up-regulated and down-regulated, respectively. Most of the genes associated with cell division were significantly down-regulated . These gEscherichia coli infection, phagosome, gap junction, and sesquiterpenoid and triterpenoid backbone biosynthesis , material transportation , atrophy (FBXO32), protein synthesis , and autophagy   .E. coli infection, and terpenoid backbone biosynthesis , IGFBP1, IRS2, JARID2, ACACB, CHKA, PDK2, PPARA were also down-regulated.The down-regulated DEGs were identified and annotated . These gRPS11, RPS12, RPS13, RPS15, RPLP0, RPLP2, RPL10A, RPL12, RPL13, and RPL13A), the heat shock protein (HSP) family  and enzymes associated with glycolysis and gluconeogenesis  were all up-regulated  were down-regulated, and the expression of GTF2IRD2, JARID2, JARID2B, POMCA, STMN2, and UCHL1 were also significantly decreased.DEGs between the control and refeeding group were identified and annotated . DEGs enegulated . The expCDC6 and CDC20), DNA replication (MCM2), steroid biosynthesis (DHCR7), fat metabolism (FABP7), and stimulus response (HSP70) were analyzed using qRT-PCR. All of these genes  were significantly down-regulated after 7-day fasting. CDC20 and HSP70 were sharply increased and higher than the control group after 7-day refeeding. On day 33 of refeeding, HSP70 remained significantly higher in the refeeding group than the control group. CDC20, however, was restored to normal level, and other genes were also restored and stable after refeeding. All genes had stable expressions under the continuous feeding condition , Cyclin-dependent kinase 1 (CDK1), and Cyclin-dependent kinase 2 (CDK2) were down-regulated during fasting. CHEK2, E2F3, and ESPL1 play crucial roles in the control of cell cycle[MCM2, MCM3, MCM4, MCM5-B, and MCM7-A were all down-regulated. Mitosis is a part of the cell cycle wherein replicated sister chromatids are separated into two chromosomes. Several genes related to mitosis  were down-regulated. NCAPD2, NCAPG, SMC2, and SMC4 were involved in chromosome condensation [S. hollandi.The down-regulated DEGs were mainly enriched in the pathway of cell cycle, DNA replication, mitosis, steroid biosynthesis, immunity, gap junction, and sesquiterpenoid and triterpenoid backbone biosynthesis. Cyclins are important factors in the control of cell cycle, and can be activated by cyclin-dependent kinase enzymes . Cyclin ell cycle–29. CDC2ell cycle. CDC6 isell cycle. MCM, a ensation ,33. Secuensation . It has PDK4, ADIPOR1, ARRDC3, PMM1, and BBOX1) were markedly up-regulated. PDK4 decreases glucose utilization and increases fat metabolism through inhibiting pyruvate dehydrogenase. This plays an important role in regulating the shift in fuel economy to prolonged fasting and starvation [ADIPOR1 is essential in the regulation of normal glucose and fat homeostasis, and for maintaining normal body weight during the fasting period[ARRDC3 can decrease energy expenditure through decrease in the thermogenesis of adipose tissues [PMM1 has a phosphoglucomutase activity, which converts glucose-1-P into glucose-6-P[Several genes associated with regulating glucose and lipid metabolism  were up-regulated. These trans-membrane proteins are substrate-specific and their increased expression may aid in the mobilization of nutrients. Several genes associated with ketone body metabolism (SLC16A3 and PDK4) were up-regulated, and brain energy metabolism usually changes toward oxidation of ketone bodies during starvation [FBXO32 is associated with muscle atrophy during the fasting period [GABARAPL1 and TP53INP2 are essential genes for autophagosome maturation [Three nutrition transport genes  is an important precursor polypeptide, which can be enzymatically cleaved into many peptides, including melanotropins , adrenocorticotropin (ACTH), lipotropins, and endorphins . These lCDC6, CDC20, MCM2, DHCR7, and FABP7 were down-regulated during fasting and restored after refeeding. DHCR7 plays a vital role in the cholesterol biosynthesis pathway, which is part of the steroid biosynthesis pathway [CDC20, CDC6, and MCM2 were all down-regulated because of the blockage of cell division by starvation. After refeeding, the expression of CDC20 was higher than in the control group, despite there was no significant difference. HSP70 is important for protein folding, and protects cells from stress. Thus, the over-expression of HSP70, after refeeding, can protect organisms from undergoing stress.Expression profiles of six key DEGs were investigated using qRT-PCR.  pathway . StarvatS. hollandi. During fasting, the fish significantly reduced gene expression in the cell cycle, DNA replication, mitosis, steroid synthesis, and other physiological activities. After 7-day refeeding, the up-regulated DEGs were remarkably enriched in antigen processing and presentation, cell cycle, steroid biosynthesis, protein processing in endoplasmic reticulum, DNA replication, mitosis, pathogenic infection, and terpenoid backbone biosynthesis.A transcriptome analysis was carried out to reveal the effects of fasting and refeeding on brains of juvenile The study indicated that growth was inhibited during starvation since the fish mobilized the stored energy to prolong survival. After refeeding, the energy was stored again, and compensatory growth occurred, the body weight was recovered. In response to the starvation and feeding, a great deal of related genes and pathways were changed. This study provided the first transcriptome data on impacts of short-time starvation and refeeding to fish brains.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Depending on the levels of the generated H2O2, high or low, respectively, either programmed cell death (PCD) occurs or H2O2 is efficiently scavenged by enzymatic/nonenzymatic antioxidant factors that help plants coping with abiotic stress, recruiting different defense mechanisms, as compared to biotic stress. Amine and PA oxidases act further as PA back-converters in peroxisomes, also generating H2O2, possibly by activating Ca2+ permeable channels. Here, the new research data are discussed on the interconnection of PA catabolism with the derived H2O2, together with their signaling roles in developmental processes, such as fruit ripening, senescence, and biotic/abiotic stress reactions, in an effort to elucidate the mechanisms involved in crop adaptation/survival to adverse environmental conditions and to pathogenic infections.Polyamine (PA) catabolic processes are performed by copper-containing amine oxidases (CuAOs) and flavin-containing PA oxidases (PAOs). So far, several CuAOs and PAOs have been identified in many plant species. These enzymes exhibit different subcellular localization, substrate specificity, and functional diversity. Since PAs are involved in numerous physiological processes, considerable efforts have been made to explore the functions of plant CuAOs and PAOs during the recent decades. The stress signal transduction pathways usually lead to increase of the intracellular PA levels, which are apoplastically secreted and oxidized by CuAOs and PAOs, with parallel production of hydrogen peroxide (H Polyamines (PAs) are small aliphatic amines present in all living cells. For more than 100 years in biology, they were misunderstood as “ptomaine or food poisoning” substances by toxicologists . The larin planta, such as Alternaria alternata  inhibitor  exhibited different substrate preferences, with MdAO1 displaying elevated catalytic efficiency for 1,3-diaminopropane, Put, and Cad, whereas MdAO2 consumed only aliphatic and aromatic monoamines, comprising 2-phenylethylamine and tyramine  have been characterized at protein level (CuAO genes with two of them (MdAO1 and MdAO2) being identified at protein level , and mainly catalyze their oxidation at primary amino groups, thus generating 4-aminobutanal, H ammonia . Howevern of Spd . Recentltyramine . Plant Ctyramine . Their gbidopsis , chickpebidopsis , pea , apple (MdAO2), Arabidopsis (AtAO1 and AtCuAO1), and sweet orange  (Arabidopsis (AtCuAO2 and AtCuAO3), two from tobacco (NtMPO1 and NtCuAO1), one from apple CuAO (MdAO1), and two from sweet orange (CsCuAO2 and CsCuAO3)  . The secCsCuAO3) .2O2, 1,3-diaminopropane (DAP), and 4-aminobutanal (Spd catabolism) or N-(3-aminopropyl)-4-aminobutanal (Spm catabolism)  (PAO genes from barley (HvPAO1 and HvPAO2), rice (OsPAO7), sweet orange (CsPAO4), and Brachypodium (BdPAO2), which are proved to catalyze the PA terminal catabolism (PAO genes belong to the second group. All of the five existing PAO genes in Arabidopsis (AtPAO1–AtPAO5) catalyze the PA back-conversion reactions  out of seven (OsPAO1–OsPAO7) existing PAO genes execute the PA back-conversion reactions (PAO genes have been identified in sweet orange and only one of them (CsPAO3) is demonstrated to catalyze the PA back-conversion reactions (GhPAO1–GhPAO12) recognized in upland cotton, only one (GhPAO3) is verified to be implicated in the back-conversion pathway  . On the abolism) , in a ret plants , until nng maize , 2006, rng maize , barley ng maize , Arabidobidopsis , tobaccobidopsis , grapevibidopsis , poplar bidopsis , apple   and PAO tabolism . In conteactions . In the eactions . Similareactions . On the eactions  and, of  pathway . To dateBeyond their functional/subcellular localization, in terms of either the terminal or the back-conversion type, PAOs exhibit further individual substrate specificities. The AtPAO1 only catalyzed the oxidation of Spm, but not Spd , while AAtCuAO localization in root xylem tissues is preceding and overlays with the synthesis of lignin in Arabidopsis  gene via RNA interference, in tobacco, promotes vascular cell differentiation and induces PCD in root cap cells  are involved in programmed cell death (PCD) and xylem differentiation . As earlbidopsis , and theongation . In addiap cells . Recentlntiation , and to activity .2O2 controls pollen plasma membrane hyperpolarization-activated Ca(2+)-penetrable canals and pollen tube growth  has been further characterized functioning in PA terminal catabolism and playing an important role against salinity  activity increased 20-fold during the hypersensitive response (HR) to tobacco mosaic virus (TMV) infection; however, the levels of Put, Spd, and Spm were not greatly altered, as expected biotic stress conditions.Plant life and stress go hand-to-hand. During growth, in order to overcome abiotic stress conditions, plants develop a remarkable organ/tissue/age-specific PA-related phenotypic plasticity . Under f closure , 2010. I closure , 2010. T closure , 2017 an closure  stress h2O2  contents, that act as signaling molecules, inducing a ROS-dependent protective pathway, thus triggering abiotic stress tolerance reactions; (②) high apoplastic PAO, over a specific threshold, oxidizing Spd considerably faster, producing high (harmful) H2O2 levels, and resulting in down-regulation of pro-survival genes and execution of a specific PCD pathway in plants under abiotic stress conditions  are secreted in the apoplast and oxidized by PAOs  , resultinditions .2O2, higher PAs, and second messengers like Ca2+  that protects plants from phytopathogenic bacteria (Pseudomonas syringae pv tabaci and the hemibiotrophic oomycete Phytophthora parasitica var nicotianae (Arabidopsis resistance to Pseudomonas viridiflava (Pseudomonas syringae (Ralstonia solanacearum also produces abundant Put, acting as a virulence metabolite and accelerating wilt disease, possibly reducing ROS in the host (A biotic stress-induced increase in PAO gene/enzyme occurred in overexpressing PAO tobacco plants infected by v tabaci . Under bbacteria . In thiscotianae . PAO andcotianae . Oxidatiidiflava . This isidiflava . In addisyringae . This acsyringae . Ralstonthe host . The pre2O2 all have specific roles in sustaining plant developmental procedures, such as fruit ripening and senescence. Furthermore, in this work, the role for the concerted action of PA catabolism and its products, in reaction to both abiotic and biotic stress are discussed. The PA oxidation will surely remain a fascinating area for scientific examination, as its concerted action with the generated H2O2 is shown to classify specific stressful parameters and build an effective defense device.During development, several molecules exist inside common plant tissues in normal environmental and phytopathogenic-free states, concerting an orthodox plant behavior. However, as soon as normal conditions are substituted by stressful ones, these molecules begin to work as stress-relievers. In this work, PAs are suggested to work as such molecules, i.e., as “orthodox-concerters” under normal conditions and as “stress-relievers” under stressful ones. Polyamines have established duties inside plants; however, when they are found in adverse conditions, they may reveal novel functions, not expected until that time. Polyamines, PA oxidases, and the generated HArabidopsis. However, many key questions remain unanswered. Firstly, current studies show that the homeostasis regulation of PAs in plants is rather complex. So far, the information about specific regulatory mechanisms in PA biosynthesis and catabolism is very limited. Although it has been revealed, among others, that the transcription of PA biosynthetic genes is regulated by several transcription factors under stress (2O2 under abiotic and/or biotic plant stress conditions has been revealed, further study will enable researchers to better elucidate this role by using new era technology.To date, many attempts have been made to investigate the roles of PA catabolism in plant growth, development, fruit ripening, and responses to biotic and abiotic stresses. Therefore, the understanding of the roles played by CuAOs and PAOs in these processes has progressed significantly during the recent decades, especially in rice and r stress , relativWW, KP, J-CF, and JS wrote the paper. J-HL conceived the work and finalized the MS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: White adipose tissue (WAT) browning confers beneficial effects on metabolic diseases. However, visceral adipose tissue (VAT) is not as susceptible to browning as subcutaneous adipose tissue (SAT). Aim: Interpreting the heterogeneity of VAT and SAT in brown remodeling and provide promising lipid targets to promote WAT browning. Methods: We first investigated the effects of β3-adrenergic stimulation by CL316,243 on systemic metabolism. Then, high-coverage targeted lipidomics approach with multiple reaction monitoring (MRM) was utilized to provide extensive detection of lipid metabolites in VAT and SAT. Results: CL316,243 notably ameliorated the systemic metabolism and induced brown remodeling of SAT but browning resistance of VAT. Comprehensive lipidomics analysis revealed browning heterogeneity of VAT and SAT with more dramatic alteration of lipid classes and species in VAT rather than SAT, though VAT is resistant to browning. Adrenergic stimulation differentially affected glycerides content in VAT and SAT and boosted the abundance of more glycerophospholipids species in VAT than in SAT. Besides, CL316,243 increased sphingolipids in VAT without changes in SAT, meanwhile, elevated cardiolipin species more prominently in VAT than in SAT. Conclusions: We demonstrated the browning heterogeneity of WAT and identified potential lipid biomarkers which may provide lipid targets for overcoming VAT browning resistance.  White adipose tissue (WAT) and brown adipose tissue (BAT), two general types of adipose tissue, are critical for maintenance of metabolic homeostasis. As unilocular white adipocytes, WAT is typically classified as subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) . AccumulBrown remodeling of WAT, the emergence of brown-like adipocytes in WAT, switches WAT into an energy-disposal depot instead of an energy storage site and has become a promising strategy against obesity . SAT is Lipids are important biomolecules and serve numerous vital biological functions, which can be identified and quantified by lipidomics including lipid classes and molecular species . Over thIn the present study, we firstly examined the pharmacodynamics effects of β3-adrenergic agonist, CL316,243, on the whole-body metabolism and demonstrated browning resistance of VAT, while SAT was more susceptible to browning conversion. Further, we applied a high-coverage targeted lipidomics approach to determine the effect of CL316,243 on VAT and SAT lipids metabolism when transdifferentiating into BAT. Detection of lipids composition showed that the overall lipids of VAT were dramatically altered adapted to CL316,243 stimulation, including the reduction of triacylglycerol (TAG) and elevation of diacylglycerol (DAG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), lyso-PC (LPC), phosphatidic acid (PA), phosphatidylinositol (PI), cardiolipin, and unexpectedly, glucosylceramide (GluCer) and sphingomyelin (SM). However, CL316,243 just induced a reduction of free fatty acids (FFA) and increase of PC, PE, LPC, and cardiolipin in SAT. The current lipidomics analysis of individual molecular species also revealed that a more significant adaptation of molecular lipid species was present in VAT compared with SAT. Further study uncovered that the lipids remodeling heterogeneity of VAT and SAT may attribute to a more significant alteration of lipids metabolism genes in VAT rather than SAT in response to CL316,243 treatment. In summary, our datasets provide a comprehensive analysis of lipids metabolic heterogeneity between VAT and SAT browning and may provide promising lipid targets to motivate WAT browning.CL316,243 (C5976) was purchased from Sigma-Aldrich . Antibody against UCP1 was obtained from Abcam  and Proteintech , respectively. Antibody against β Tubulin (TUBB4) was purchased from ZSGB-BIO .The animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Chicago. Animal care was given in accordance with institutional guidelines. C57BL/6J male mice were purchased from Jackson Laboratory. The animals were housed in a temperature-controlled and humidity-controlled barrier system with a 12-h light/dark cycle and had free access to standard rodent pellet food and water.wt/vol) buffered formalin for histological diagnosis.Eight-week-old male mice were injected intraperitoneally with CL316,243  ) for 10 days, with PBS-injected mice serving as controls. Bodyweight, 24-h food intake, and water intake were recorded. After the end of 10-day treatment, mice were humanely sacrificed and blood samples were collected. BAT from interscapular region, SAT from inguinal region, and VAT from epididymal region were quickly excised to calculate the fat coefficient with fat weight/body weight, then were immediately frozen in liquid nitrogen for further lipids extraction or fixed in 10% .After 10-day treatment, blood levels of triglyceride (TG) and free fatty acids (FFA) were detected. The concentration of TG was detected with an automatic analyzer  using the Triglycerides Kit . The level of FFA was measured according to the manufacturer’s instruction of the Non-Esterified Fatty Acid Kit .At the end of CL316,243 treatment, the dorsal shell temperature was spotted by a thermal imaging camera purchased from FLIR  after the mice were anesthetized.Total RNA was isolated from adipose tissues using Trizol reagent (Invitrogen) and Direct-zol kit (Zymo) following the manufacturer’s recommendations. cDNA was prepared from 2 μg of total RNA according to the instructions of High-Capacity cDNA Reverse Transcription Kit (Invitrogen) with random hexamer primers. The resulting cDNA was diluted ten folds, and a 1.5 μL aliquot was used in a 6 μL PCR reaction  in the presence of primers at a concentration of 300 nM each. PCR reactions were run in triplicate and quantitated using the Applied Biosystems ViiA 7 Real-Time PCR system. Relative amounts of mRNA were normalized to TATA box-binding protein (TBP) expression and expressed as arbitrary units  were subjected to SDS-PAGE and immunoblotting was performed as described with anti-UCP1  or anti-β Tubulin  antibodies .wt/vol) buffered formalin, and paraffin-embedded sections were subjected to H&E staining. After paraffin embedding, bones were sectioned and stained with hematoxylin and eosin.Tissues were fixed in 10% (wt/vol) buffered formalin and paraffin embedded. 4 μm sections were made and stained with rabbit anti-UCP1 antibody  overnight at 4 ℃. After PBS washing, slides were incubated with goat anti-rabbit IgG  for 1 h at room temperature. Finally, staining was completed using DAB Chromogen . Images were observed under an optical microscope (Olympus BX51) and five randomly selected images per section were digitally captured (400× magnification). The adipose tissues were fixed in 10%  using an optimized program and incubated for 1 h at 4 °C with shaking at 1200 rpm. At the end of the incubation, 300 µL of chloroform and 400 µL of deionized H2O were added and vortexed. Lower organic phase was collected. A second extraction was carried out via addition of chloroform (500 µL) and 50 µL of 1 M hydrochloric acid. The two organic extracts were pooled into the same tube and dried using a Speed-Vac . Samples were stored at −80 °C before high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis.Lipids were extracted as described before . BrieflyAn ultra-performance liquid chromatography (UPLC) coupled with Sciex QTRAP 6500 Plus was used for UPLC-MS/MS analyses. Individual lipid classes of polar lipids including phospholipids and sphingolipids, as well as free fatty acids, were separated by normal phase HPLC using a Phenomenex Luna 3 µm silica column (150 × 2.0 mm) with mobile phase A  and mobile phase B . Multiple reaction monitoring (MRM) transitions specific to both head groups and fatty acid chains were set up for quantitative analysis of various phospholipids. Individual lipid species were quantified by comparison with spiked internal standards PC-14:0/14:0, PC34:1-d31, LPC-d4-26:0, PE-14:0/14:0, PE34:1-d31, phosphatidylserines (PS)-14:0/14:0, PS-16:0/18:1-d31, lyso-PS (LPS)-17:1, PA34:1-d31, PA-17:0/17:0, PI34:1-d31, CL-22:1(3)/14:1, ceramides (Cer)-d18:1/17:0, GluCer-d18:1/8:0, galactosylceramide -d18:1/8:0, SM-d18:1/12:0, which were purchased from Avanti Polar Lipids  and LIPIDS MAPS. Dioctanoyl phosphatidylinositol PI-8:0/8:0  was used for phosphatidylinositol quantitation. d31-palmitic acid  and d8-arachidonic acid  were used for quantitation of saturated and polyunsaturated fatty acids, respectively. For all LC/MS analyses, individual peaks were examined and only those above the limit of quantitation and within the linearity range were used for quantitation. The absolute amounts of all qualitative lipid standards were precorrected against quantitative standards before quantitative usage. Neutral lipids such as TAG, DAG, and cholesteryl ester (CE) were determined by a modified version of reverse phase HPLC/ESI/MS/MS . HPLC/ESIn addition, HPLC/APCI/MS/MS was used for the quantitation of free cholesterol (Cho), with corresponding d6-Cho (CDN isotopes) as internal standard.A schematic diagram of the experimental design is shown in P < 0.05 was considered significant.Data are presented as mean ± SEM (standard error of mean). Data analysis was performed using Prism software (GraphPad Software Inc.). Statistical analyses were performed by unpaired two-tailed Student’s t-test between two groups after examination of the normal distribution of experimental data by Kolmogorov-Smirnov Test. Independent experiments were performed at least thrice with similar results and studies were performed on five mice per group unless specified. A value of It is well-established that a highly selective β3-adrenergic agonist, CL316,243, plays an important role in regulating energy balance as well as cellular and metabolic remodeling of adipose tissue ,23,24. HTo further verify the vital response of adrenergic stimulation on heat production, we spotted dorsal shell temperature by a thermal imaging camera. As expected, we found significant yellow heat signature especially in scapular region as well as shell temperature rise, indicating an augmented thermogenic effect of CL316,243 H,I. AparTo deeply understand the browning transdifferentiation heterogeneity induced by β3-adrenergic agonist, we utilized the MRM lipidomics identification for all lipid metabolites of VAT and SAT in response to CL316,243 challenge. Subsequently, comprehensive bioinformatics analysis was performed to characterize the content and composition of total lipids and species, including 17 lipid classes and 334 lipid species. Firstly, a multivariate principal component analysis (PCA) was used to identify different lipids change patterns between VAT and SAT using the concentration data of 17 lipid classes, including TAG, DAG, Cho, CE, FFA, SM, ceramide (Cer), GluCer, galactosylceramide , PC, LPC, PS, lyso-PS (LPS), PE, PI, PA, and cardiolipin. Consistent with the phenotype of WAT browning, we observed saliently different lipid profiling between VAT and SAT. Furthermore, a distinct separation of lipid composition exhibited in both VAT and SAT after CL316,243 challenge with more significant changes in VAT A. In addTAG has been described as an important lipid for excessive energy storage which may cause obesity and associated comorbidities. In the present study, TAG content was measured in VAT and SAT exposed to CL316,243 treatment. As manifested in To gain further insight into metabolic profiling, we analyzed the detailed alteration patterns of molecular species in TAG, DAG, FFA, and CE. The majority of TAG species, which were dominated by chain lengths of 46–56 carbons, were significantly decreased in VAT following CL316,243 stimulation E, in agrGlycerophospholipids serve as major constituents of cellular and organellar membranes and participate in diverse biological processes. To elucidate the change patterns of glycerophospholipids in VAT and SAT induced by browning stimulation, seven main glycerophospholipids classes were analyzed. All in all, our analysis revealed that PC was the predominate class in both VAT and SAT, followed by PE, PS, and LPC. CL316,243 elevated the total amount of PC, PE, PA, PI, LPC in VAT and PC, PE, LPC in SAT without any change in the overall abundance of PS and LPS. More importantly, higher fold changes in elevated lipid classes were found in VAT than in SAT A.To gain a more informative perspective of glycerophospholipids profiling, molecular species of each glycerophospholipids class were quantified. In VAT, 22 out of 34 PC species were markedly elevated, including highly abundant species with side chains of 34:2, 34:1, 36:4, 36:3, 36:2, and 36:1 B. PE speInterestingly, CL316,243 elevated several PC species of SAT including the highly abundant 34:2, 34:1, 36:3, 36:2, 36:1 species and less abundant 36:1p species D with loAs relatively minor constituents of lipids, sphingolipids can be broadly divided into ceramide, sphingomyelin, and glycosphingolipid. Ceramide is the precursor for most sphingolipids and is synthesized from FFA and serine . In our To determine the species contributing to the elevation, 52 sphingolipids species were identified respectively. Although there were no significant differences in total concentrations of Cer and GalCer in VAT, significant augment in the content of Cer d18:1/20:0, d18:0/20:0, d18:1/22:0, d18:1/24:1, d18:1/24:0 and GalCer d18:1/22:0, d18:1/24:1 were detected B,C. FourAs the signature phospholipid of mitochondrial membranes, cardiolipin is closely associated with mitochondrial function and structural integrity ,29,30,31More interestingly, C72:8 (18:2) and C72:7 (18:2) are the most abundant cardiolipin species in VAT and SAT after CL316,243 treatment. In VAT, C72:8 (18:2) and C72:7 (18:2) dramatically enhanced by 29.73 and 30.46 folds, and constituted 45.76% and 27.17% of total cardiolipin, respectively. However, C72:8 (18:2) and C72:7 (18:2) increased by 2.44 and 2.87 folds, and made up 36.22% and 25.01% of overall cardiolipin in SAT B. This cObesity is a major risk factor for the development of prediabetes and type 2 diabetes mellitus (DM) . WAT broVisceral fat accumulation is associated with serum triglyceride levels. Moreover, VAT adipocytes are also more sensitive to lipolysis and more prone to develop insulin resistance than SAT adipocytes ,36. ActiGlycerophospholipids are major constituents of cellular and organellar membranes which participate in diverse biological processes. Among them, PC, the most abundant mitochondrial membrane phospholipid, is a critical constituent of both inner and outer mitochondrial membranes. In addition, PE accounts for a large portion in the inner mitochondrial membrane of mammalian cells. PC and PE are representative of nearly 80% of total mitochondrial phospholipids . Our lipPA is an essential precursor for the biosynthesis of most phospholipids and glycerolipids . PI is eSphingolipids have been regarded as a principal lipid class connecting obesity to its complications, such as type 2 diabetes and cardiovascular disease. As a precursor of all complex sphingolipids, ceramide is composed of a sphingosine backbone coupled to acyl-chains containing 14–30 carbons. Recently, Chaurasia et al. have reported that cold exposure and CL316,243 treatment decreased the content of ceramide in SAT . Quite uSM is the most abundant sphingolipids and sphingosine  is the most common long-chain base present in SM . The higAs a specific phospholipid of mitochondrial membranes, cardiolipin extensively interacts with mitochondrial proteins, including ADP/ATP carrier, complex I, III, and V, thus has been considered as an integral part of oxidative phosphorylation complexes to regulate mitochondrial function in particular oxidative phosphorylation ,41. It hIn summary, our current study mainly observed the lipid profiling of VAT and SAT under adrenergic stimulation of CL316,243 for 10 days and found lipids remodeling heterogeneity of VAT and SAT which may attribute to a more significant alteration of lipids metabolism genes in VAT rather than SAT. Nonetheless, further studies are needed to identify more biologically active lipid species to elucidate the browning heterogeneity between VAT and SAT, especially VAT browning resistance under different browning conditions and promote WAT browning.The present study has demonstrated the browning heterogeneity of WAT induced by adrenergic stimulation by lipidomics signature. Though VAT is not as susceptible to browning as SAT, lipidomics analysis reveals a more significant alteration of lipids remodeling of VAT than SAT in response to CL316,243 treatment. Our study provides lipidomics insight into VAT and SAT browning heterogeneity which may provide promising lipid targets for promoting WAT browning.