In this review, we also compare findings from studies using invertebrate eggs with what is known about the contributions made by the actin cytoskeleton in mammalian eggs. Since the cortical actin cytoskeleton affects microvillar morphology, movement, and positioning of organelles and vesicles, and the topography of the egg surface, these changes have impacts on the fertilization process, as has been suggested by recent morphological studies on starfish oocytes and eggs using scanning electron microscopy. Drawing the parallelism between vitelline layer of echinoderm eggs and the zona pellucida of mammalian eggs, we also discuss the importance of the egg surface in mediating monospermic fertilization.Much of the scientific knowledge on oocyte maturation, fertilization, and embryonic development has come from the experiments using gametes of marine organisms that reproduce by external fertilization. In particular, echinoderm eggs have enabled the study of structural and biochemical changes related to meiotic maturation and fertilization owing to the abundant availability of large and transparent oocytes and eggs. Thus, in vitro studies of oocyte maturation and sperm-induced egg activation in starfish are carried out under experimental conditions that resemble those occurring in nature. During the maturation process, immature oocytes of starfish are released from the prophase of the first meiotic division, and acquire the competence to be fertilized through a highly programmed sequence of morphological and physiological changes at the oocyte surface. In addition, the changes in the cortical and nuclear regions are essential for normal and monospermic fertilization. This review summarizes the current state of research on the cortical actin cytoskeleton in mediating structural and physiological changes during oocyte maturation and sperm and egg activation in starfish and sea urchin. The common denominator in these studies with echinoderms is that exquisite rearrangements of the egg cortical actin filaments play pivotal roles in gamete interactions, Ca With the onset of meiosis, these diploid cells are transformed into primary oocytes and spermatocytes. The meiotic (maturation) divisions that follow are markedly different in the two gametes. In male, each primary spermatocyte divides to produce four haploid spermatids, which differentiate into spermatozoa capable of fertilizing the eggs. In female, unequal partition of the cytoplasm results from two meiotic divisions that are asymmetric in size and lead to the formation of one large haploid oocyte and three polar bodies, the latter of which eventually degenerate. Another striking distinction between oocytes and spermatozoa is that the latter fertilize the eggs at the end of the meiotic divisions, whereas oocytes reach the period of fertilizability at different stages of the maturation process, depending on animal species. Indeed, marine invertebrate oocytes can be grouped into four different classes based on the meiotic stage at which fertilization takes place. Oocytes of various annelids and molluscs are fertilized at the GV stage, and the breakdown of the nuclear envelope is a sign that the oocytes have been activated. Eggs of nemerteans, ascidians, some annelids, and molluscs are blocked at the metaphase of the first meiotic division until fertilization triggers the completion of meiosis. The eggs of frogs and mammals are arrested and fertilized at metaphase II, i.e., after the second maturation spindle has formed and one polar body has been extruded –4. Thus,ucleus”  and Astropecten aranciacus (Mediterranean Sea) have made interesting observations about the time frame and other requirements for eggs’ optimal fertilizability and successful development [Although sperm can penetrate immature oocytes of starfish before GVBD, cortical events that block the entry of supernumerary spermatozoa and ensure normal egg activation and cleavage take place within a precise time frame only after 1-MA stimulation. Indeed, it is well known that starfish eggs lose their ability to prevent polyspermic fertilization when inseminated after being treated with 1-MA for several hours (overripe eggs). These results indicate that the competence of the egg cytoplasm to be successfully fertilized is achieved at a precise maturation stage but is lost soon after that. Studies of oocyte maturation using elopment –12.2+, it has been suggested that actin may act as an intracellular buffer storing and releasing Ca2+ [A. aranciacus mature eggs at their optimum period of fertilizability to actin-depolymerizing agents, such as latrunculin A (LAT-A) and mycalolide B, triggers intracellular increases of Ca2+ and plasma membrane depolarization following their activation [Recent studies have provided evidence that the cortical actin cytoskeleton is a key player in the development of mature and competent eggs manifesting normal fertilization responses. It is well established that actin, which is one the most abundant and highly conserved proteins in eukaryotic cells, participates in the maintenance of cell shape, as well as in many cellular functions such as cell migration, growth, motility, organelle movement, polarization, and exocytosis/endocytosis. Together with myosin, actin can drive not only muscle contraction, but also regulation of genes in the nucleus . Actin ming Ca2+ –17. Constivation –20. New A number of failures in assisted reproduction technology (ART) are linked to oocytes that are fertilized not in their optimum period of maturation, but after remaining in the oviduct (in vivo ageing) or culture (in vitro ageing) . It is wP. pectinifera dissected from the ovary  oocytes of ary Fig. a are surary Fig. b. Applicary Fig. c detacheary Fig. a, arrowsary Fig. c. These ary Fig. d, arrow ary Fig. b, arrowsary Fig. f, may reary Fig. e, which ary Fig. a and b. ary Fig. f, arrowsP. pectinifera before and after maturation by 1-MA that have been subsequently fractured during fixation to visualize the oocyte surface, microvilli, CG and organelles in the cortex and inner cytoplasm. Longer microvilli are seen in the immature oocytes beneath the VL .At variance with other animal species in which the complex signaling pathways regulating oocyte maturation have not been fully elucidated, those leading to meiosis resumption and their downstream effectors are relatively well documented in starfish. After 1-MA exposure, immature oocytes begin to form a cytoplasmic factor before GVBD occurs. The maturation promoting factor (MPF) containing cyclin-dependent kinase 1 (Cdk1) thus induces entry into meiotic M-phase by phosphorylating its substrate proteins needed for disassembly of the nuclear lamina, and thereby promotes GVBD . The med Markert  and late Markert –73. Cdk12+ signals in oocyte maturation has been established for many animal species. In starfish, 1-MA exposure could trigger a transient Ca2+ increase in the cytoplasm of immature oocytes and from the cell extract enriched with plasma membrane [2+ changes in several species of starfish has led to the conclusion that GVBD and the continuation of maturation (meiotic) cycle were not Ca2+-dependent [2+ transient, 1-MA induces a nuclear Ca2+ increase which was essential for the continuation of meiosis. Moreover, direct microinjection of the Ca2+ chelator BAPTA into the GV of the starfish oocyte prevented the disassembly of the nuclear envelope and the intermixing of the nucleoplasm with the cytoplasm [2+ increase in the nucleus, not in the cytoplasm, was essential for meiosis continuation. Calmodulin (CaM) was identified as a possible downstream target of the 1-MA Ca2+ signals, since microinjection of antibodies against CaM directly into the GV of immature oocytes blocked meiosis progression [2+ increase only a few minutes after 1-MA application. The increase of Ca2+ always starts at the vegetal hemisphere of the oocyte and is independent of its position in the chamber, as the oocyte surface was entirely exposed to the hormone. Furthermore, the Ca2+ transient induced by 1-MA failed to spread globally to the inner cytoplasm, but was localized to the oocyte cortex. As for the nature of the 1-MA-triggered Ca2+ signal, our laboratory has found that it was not linked to extracellular Ca2+, but strictly correlated to the structural organization of the cortical actin cytoskeleton [2+ release mediated by G-proteins has been provided by the microinjection of the non-hydrolyzable analogue of GTP and GDP. We have found that GTPγS induced a Ca2+ signal similar to that triggered by 1-MA, whereas GDPβS induced striking modifications of the cortical actin network and significantly inhibited the 1-MA Ca2+ signal [2+ increase, which runs in the oocyte cortex from the vegetal to the animal hemisphere, is followed by the subsequent increase of Ca2+ in the nucleus located in the latter hemisphere [The importance of Camembrane . Nonetheependent , 76. Howytoplasm , 78. Thegression . Later sskeleton . A link + signal . The F-amisphere .2+ following precise, species-specific, spatiotemporal patterns. The ability of an egg to produce appropriate Ca2+ signals at fertilization is acquired during oocyte maturation, encompassing resumption of meiosis and progression to meiosis II. In starfish, the same amount of the Ca2+-linked second messenger inositol 1,4,5-trisphosphate (IP3) microinjected into mature eggs induces a release of Ca2+ much higher than that elicited in immature oocytes. This higher Ca2+ increase does not coincide with the increased Ca2+ contents in the restructuring ER, but is due to the enhanced sensitivity of the Ca2+ stores to IP3 [3 in the maturing oocyte have revealed that the sensitivity of the Ca2+ stores to IP3 developed with a spatial pattern similar to that of MPF, i.e., it started at the animal hemisphere and propagated towards the vegetal side [3 in maturing oocytes is correlated with the sequential activation of MPF in the cytoplasm and nucleus. Since MPF did not directly phosphorylate IP3 receptors (IP3R) as shown in other systems, an alternative hypothesis was proposed in which some components of the actin cytoskeleton may be phosphorylated by MPF and thereby produce much higher release of Ca2+ in response to IP3 [3-dependent Ca2+ release mechanism is modulated by microfilaments, our laboratory had previously found that microinjection of actin-binding protein cofilin into starfish eggs significantly enhances the Ca2+ release in response to IP3 or fertilizing sperm [2+ increases that was reminiscent of the Ca2+ response in fertilized eggs [2+ response when applied to GV stage oocytes [3-sensitive Ca2+ response, it has been shown that the sensitization of the Ca2+ stores to IP3 during oocyte maturation coincided with the ability of LAT-A to release Ca2+ from maturing eggs [2+ release mechanisms activated by LAT-A have now shown that its effect on actin filaments occurs via actin-dependent production of IP3 following stimulation of PLC, and it is not linked to sensitization of the Ca2+ stores to IP3 [Paracentrotus lividus (sea urchin) eggs exposed to LAT-A induced an increased production of IP3, but it did not lead to an intracellular Ca2+ increase [2+ stores of the eggs of this species are less sensitive to the action of IP3, arguing against the previously suggested idea that IP3 is the Ca2+-linked second messenger produced in sea urchin eggs upon fertilization [2+ wave that occurs a few minutes after its application, we have detected a train of Ca2+ spikes at the time of GVBD in the maturing oocytes of P. pectinifera, which are strictly linked to Ca2+ influx because they are inhibited when the oocytes were exposed to 1-MA in Ca2+-free seawater [2+ increases requires the presence of GV and the rearrangement of the cortical actin cytoskeleton. This actin-dependent structural modification of the oocyte cortex, concomitant with Ca2+ influxes at the time of GVBD, may be related to the changes in microvilli length (shortening)  that occurs during oocyte maturation correlates with the higher release of Cailaments . A recenilaments . The altmpetence .2+ in the regulation of the AR in sea urchin sperm was first suggested by Dan [2+ ionophore A23187 or by simply increasing the external pH in the presence of Ca2+. Both treatments initiated the internal polymerization of actin in the acrosomal process [2+ entry and pH increase in the sperm. It has been known that at least three different molecules of the starfish jelly coat act in concert to induce the structural and biochemical changes in sperm, which lead to the formation of the acrosomal process [2+ influx into the periphery of the egg and the propagation of the Ca2+ wave , and the Ca2+ wave in the egg at fertilization [2+ influx and the onset of wave is much shorter, e.g., 2 s. The different kinetics of the electrical and Ca2+ changes in the two species probably reflects differences in the structural organization of their egg cortices [+ [+ is due to alterations in cortical actin filament dynamics, and it is not the result of the formation of multiple mitotic spindles following aberrant microtubules organization exerted by supernumerary sperm-centrosomes [Even if the first evidence for a sperm-induced Caish eggs , the con cortex” , which w cortex” , 114. Th cortex” . The cor cortex” . Because cortex”  has call cortex” . Activat cortex” . In sea tential’ –119. In lization . In starcortices . As for cortices  suggestecortices , 122, otcortices , 124. Intices [+ –127. As tices [+  on page trosomes , 129. Futrosomes .2+ signals activates the zygote to initiate embryonic development, one of the major problems in this field for decades has been to understand the mechanisms of the signal transduction leading to the Ca2+ influx and the intracellular Ca2+ release at fertilization [2+ entry prior to the exocytosis of the cortical granules has been visualized at the cell periphery in eggs loaded with Ca2+ sensitive dyes [2+ across the plasma membrane were able to undergo early activation and metabolic changes similar to those occurring during normal fertilization [2+ free seawater [2+ liberation at fertilization. However, in the experiments on the activation of sea urchin eggs by A23187, the release of Ca2+ from intracellular stores was judged only by the occurrence of the elevation of the VL in sea urchin eggs [2+ imaging between the patterns of Ca2+ release induced by activation of sea urchin eggs by sperm or by a A23187 had not been made, unlike in the fish eggs [2+ ionophore, ionomycin, to immature starfish oocytes have shown that ionomycin induced a dramatic rearrangement of the actin cytoskeleton in the cytoplasm and egg cortex of the oocytes that led to microvilli retraction and disruption and fusion of cortical granules with vesicles. When these ionomycin-pretreated oocytes were matured and fertilized, they gave rise to altered Ca2+ responses,  and inhibition of the CG exocytosis [Since the sperm-induced Calization . In sea ive dyes , 131, ralization , 133. Fuseawater  added wehin eggs . A compaish eggs . Apart focytosis . The resocytosis .2+ signals triggers inactivation of the two kinases, transition from metaphase to anaphase by the CaMKII-dependent activation of APC/C, inactivation of MPF, and initiation of embryo development [2+ oscillations is triggered upon delivery of a soluble sperm-specific factor identified as a PLCζ that enters the egg upon gamete fusion [3 formation through the hydrolysis of PIP2 and Ca2+ release upon IP3-gated opening of IP3Rs on the ER has been claimed by experiments in which the microinjection of anti-IP3Rs antibodies inhibited the sperm-induced Ca2+ release [2 substrates located in intracellular membranes rather than the plasma membrane [Plcz1-null mice have shown the absence of the typical Ca2+ oscillatory pattern required for oocyte activation. Nevertheless, a small number of zygotes fertilized by the sperm genetically lacking PLCζ were able to develop to blastocyst, and to produce live offspring [Plcz1 KO mice failed to induce Ca2+ changes following ICSI, but still elicited a Ca2+ increase following IVF. The altered pattern of Ca2+ release suppressed embryonic development, probably due to the failure of proper oocyte activation or to the polyspermy induced by the absence of adequate Ca2+-linked block mechanisms against the entry of supernumerary sperm [2+ release mechanism(s) at the oocyte surface are yet to be understood in mammalian oocytes, and raise some concerns in that, despite the essential role of PLCζ in inducing Ca2+ oscillations to ensure monospermic fertilization in mice, the ICSI protocol bypasses what normally occurs at the oocyte cortex following sperm-egg fusion. In this regard, it is noteworthy that the cortices of mouse and ascidians oocytes are highly reactive and structurally organized to facilitate the initiation of Ca2+ oscillations. This has been demonstrated by the visualization of the Ca2+ signal that originates from the periphery of these oocytes after ICSI and then propagates across the cytoplasm [2+ release in the regulation of a normal oscillatory Ca2+ response, the Ca2+ imaging analysis of intact Ciona intestinalis ascidian oocytes (with chorion and accessory cells attached) shows a CF, resulting from Ca2+ influx, which precedes the Ca2+ oscillations that is usually undetected if the chorion and neighbouring cells are removed prior to fertilization [In mammals, the fertilizable oocyte is arrested at metaphase of second meiotic division due to the high activity of MPF and of the MAPK kinase, (M-phase activating protein kinase), and inhibition of anaphase promoting complex or cyclosome (APC/C) . Followielopment . At ferte fusion , 139. Th release . At vari release  and thusmembrane . Analysiffspring . In anotry sperm . These rytoplasm . In linelization .2+ influx in the initiation of the sperm-induced Ca2+ signals and polar body emission, recent studies on ZP-free mammalian oocytes have shown that, STIM and Orai-linked store-operated Ca2+ entry mechanism is not required to sustain the persistent sperm-induced Ca2+ oscillations [2+ influx following fertilization of mouse oocytes [2+ entry via mechanosensitive activation of TRP-type cation channels during fertilization of mammalian oocytes may be heavily dependent on the structural integrity of the egg and appropriate tension controls by the cortical actin cytoskeleton [In line with a role of Callations . However oocytes . These rskeleton , 150.Drosophila, it has recently been reported that an intracellular Ca2+ increase following egg activation, which is independent of fertilization but triggered by mechanical pressure in the female reproductive tract, is initiated by entry of extracellular Ca2+. This cortical Ca2+ increase starts at one pole of the egg and initiates the Ca2+ wave that propagates to the opposite one. The initial Ca2+ influx is mediated by the opening of mechanosensitive ion channels indicating a conserved mechanism of calcium signaling in egg activation between protostome (Drosophila) and deuterostome (mouse) [2+ increase at egg activation has been demonstrated by showing that reorganization of the actin cytoskeleton that covers the cortex of the mature eggs is required for the Ca2+ wave upon egg activation [Interestingly, in  (mouse) . Furthertivation .2+-ionophore treatment, which is currently used to restore the lack of normal sperm-induced Ca2+ response in human oocytes [2+ oscillations that are critical for successful oocyte activation and the high quality blastocyst formation [2+-ionophore, which elicits a single Ca2+ increase, thus not mimicking the Ca2+ oscillation at natural fertilization [2+ released may not ensure that these events occur and persist. In human oocytes, about 10 to 20 Ca2+ transients occurring at intervals of 30–60 min are the physiological pattern associated with successful development. The oscillatory pattern (amplitude and frequencies) of Ca2+ increases appear to have no effects on early cleavages, implantation, and post-implantation development, but foetal morphology [2+ signals [2+ ionophores can induce disintegration of the cytoplasm with prolonged exposure and severe structural modifications of the egg cortex in several species [As to the clinical applications, Ca oocytes , 154, isormation , 156. Thlization , may stirphology  and weig signals . This, t species , 161, ra2+ signaling pathways in the fertilization process and development. These eggs have completed the meiotic cycle and await the sperm to activate their developmental program. Microinjection of Ca2+ dyes into the cytoplasm of intact unfertilized eggs allows the visualization of the sperm-induced Ca2+ increase, which occurs in the form of a subitaneous release of Ca2+ at the periphery of the egg [2+ channels following depolarization of the egg plasma membrane. A Ca2+ wave follows that starts at the sperm-egg interaction site and propagates to the opposite pole. The role of a Ca2+ influx from external seawater [2+ [2+ free seawater by acrosome-reacted sperm [2+ release in echinoderm eggs have pointed to the exclusive role of IP3 as the messenger that initiates the increase and spreading of Ca2+. This possibility emerged from experiments in which the measurement of phosphoinositide turnover and IP3 generation were found to coincide with the sperm-elicited Ca2+ wave [2+ increase by chemical inhibitors of PLC [2+ wave was found to be altered [3R antagonist heparin was found to abolish the sperm-induced Ca2+ wave only when it was co-injected with antagonists of ryanodine receptors (RyRs), while a massive amount of Ca2+ was mobilized by the microinjection of ryanodine and cyclic ADP-ribose (cADPr). These Ca2+ signals regard only the Ca2+ wave taking place at fertilization, and not the initial cortical Ca2+ influx. They were nevertheless used to propose an exclusive coupling mechanism between IP3 and RyRs in shaping the Ca2+ signaling at fertilization of sea urchin eggs [2+ transient cast doubt on the notion that IP3 alone initiates Ca2+ release at fertilization [2+-linked second messenger, NAADP, mobilizes Ca2+ from stores localized in acidic vesicles in the cytoplasm of intact eggs, which were insensitive to IP3 and cADPr [Sea urchin gametes have been widely used to study the role of Ca the egg , 162. Thseawater  has beenater [2+ , or evened sperm . Investia2+ wave , and by s of PLC , 166. Ad altered , 168. Hohin eggs , 170. Holization . Additiond cADPr . It was nd cADPr .2+ increase from Ca2+ stores confined to the cortex, which then spread to the cell centre. However, the NAADP-induced Ca2+ increase was inhibited when the uncaging was performed in Ca2+-free seawater [2+ permeable ion channels on the plasma membrane. NAADP was shown to trigger plasma membrane depolarization and was thus suggested to initiate sperm-induced Ca2+ influx that produced the cortical Ca2+ influx [2+ influx was shown to be dependent on the integrity of the cortical actin cytoskeleton, as judged by the modulation of Ca2+ currents by actin drugs [2+ entry in starfish oocytes, as suggested for sea urchin eggs, was assessed pharmacologically using drugs known to disrupt acidic compartments. However, the treatment failed to inhibit the NAADP-induced plasma membrane depolarization [Patiria miniata showed that Ca2+ signals at fertilization are only slightly altered, raising doubts about the contribution of these channels to the initiation of the Ca2+ response at fertilization [In starfish, the global photo-activation of caged NAADP pre-injected into the cytoplasm of oocytes and eggs elicited a Caseawater . Electro+ influx . Interesin drugs . The posrization . Recentllization .2+ elevation following fertilization. A normal CF (shorter than that elicited in sea urchin eggs) and the Ca2+ wave occur when the sperm fertilizes the maturing starfish oocytes after GVBD and before the extrusion of the first polar body [3-dependent nature of the sperm-induced intracellular Ca2+ wave was demonstrated by experiments in which the microinjection of the SH2 domain of PLCγ delayed the initiation of the Ca2+ wave, leaving the CF unaffected [A. aranciacus) egg completely abolished CF, in addition to lowering the amplitude of the multiple, but abortive, intracellular Ca2+ waves that resulted from polyspermic egg [2+ releases triggered by uncaging of IP3 and cADPr [2+ [Starfish gametes have greatly contributed to the understanding of the signaling pathways leading to the intracellular Calar body , 177. Thaffected , 168. Hormic egg . It was rmic egg . The F-and cADPr , 104. ThADPr [2+  is suffiADPr [2+ , our resADPr [2+ .A. aranciacus (starfish) showed that a prolonged 1-MA-induced maturation  of freshly collected immature oocytes promoted pathological polyspermy at fertilization [2+ increases at fertilization. Indeed, the GV stage oocytes respond to insemination by eliciting multiple Ca2+ waves (arrowheads) that precede the occurrence of the CF  running from the sperm-egg interaction site to the opposite pole  as a result of polyspermic fertilization . Similarly, treatment of sea urchin eggs with agents that disrupt, relocate, or fuse cortical vesicles and CG also alter microvillar morphology and density, and these eggs exhibit altered Ca2+ kinetics at fertilization in its rise and fall [The roles played by the egg cortical actin cytoskeleton in regulating a normal fertilization reaction have also been demonstrated in sea urchin. Analysis of the effects of actin drugs promoting F-actin depolymerization or stabilization on the fertilization reaction of levation . The chalevation  were alsleavages . Seawateand fall . The cruand fall –186. Furand fall . Very reand fall .2+ elevation and the action of Ca2+-sensitive actin binding proteins [Within a few minutes after fertilization, the cortical actin cytoskeleton visibly reorganized at the egg surface from the point of sperm entry toward the entire egg surface , 189. Thproteins –196. Furproteins . Corticaproteins , 183.The interaction between the fertilizing sperm and sea urchin eggs occurs at the tip of microvilli containing bundles of actin filaments which may participate in sperm–egg binding and fusion . A largeAt variance with echinoderm eggs, the involvement of actin filaments for sperm incorporation in mammalian fertilization is controversial. This is probably due to the fact that different actin drugs have been used to depolymerize or stabilize the cortical actin in eggs before fertilization. These controversial results may be attributable to the specific and diverse mechanisms of action of actin drugs, which complicates comparison of their effects in perturbing the actin polymerization step .2+ response to fertilizing sperm, CG exocytosis, as well as monospermic penetration, spindle formation, and polar bodies extrusions. Results on overripe starfish eggs that are fertilized in a time window different from their optimal have shown that the deterioration of the cells induces dramatic morphological alterations of the surface and cortex that negatively impact the fertilization process. The same changes appear to happen in mammalian oocyte ageing. In view of the difficulties of gamete availability and accessibility as well as the ethical issues involved, extensive studies on the reproductive biology of human oocytes has met with obstacles. Thus, animal models of starfish and sea urchin have invaluable practical merits.Dynamic remodelling of the actin cytoskeleton is essential not only for the control of cell shape but also for the regulation of important cellular processes. An increasing body of evidence suggests that the structural organization of the actin cytoskeleton and its physical changes induced by external signals dynamically regulate a large number of events occurring during oocyte meiotic maturation and fertilization. During maturation, the acquisition of competence of the oocyte to be successfully fertilized is in major part controlled by the actin filaments of the egg cortex. Concerted reorganization of the actin cytoskeleton appears to regulate the membrane potential and the alignment of CG beneath the plasma membrane to facilitate the responses at egg activation: proper CaIn this review, we emphasize that one of the fundamental reactions of gametes at fertilization is concerted changes of the actin filaments in both gametes. For eggs, the cortical reaction is compromised when the egg is not healthy, thus a successful cortical reaction could be an indicator of the “quality” of the eggs, and the latter is mainly determined by the proper regulation of the actin cytoskeleton. Thus, technical aspects of ART to preserve functionality of the actin cytoskeleton in the human eggs would be also important. The usage of marine eggs as experimental systems to study oocyte maturation and fertilization is also advantageous in that perspective.


The autonomous pacemaker activity of SN DA neurons, in combination with low cytosolic Ca2+ buffering, leads to large somatodendritic fluctuations of intracellular Ca2+ levels that are linked to elevated mitochondrial oxidant stress. L-type voltage-gated Ca2+ channels (LTCCs) contribute to these Ca2+ oscillations in dendrites, and LTCC inhibition was beneficial in cellular and in vivo animal models of PD. However, in a recently completed phase 3 clinical trial, the dihydropyridine (DHP) LTCC inhibitor isradipine failed to slow disease progression in early PD patients, questioning the feasibility of DHPs for PD therapy. Novel evidence also suggests that R- and T-type Ca2+ channels  represent potential PD drug targets. This short review aims to (re)evaluate the therapeutic potential of LTCC, RTCC, and TTCC inhibition in light of novel preclinical and clinical data and the feasibility of available Ca2+ channel blockers to modify PD disease progression. I also summarize their cell-specific roles for SN DA neuron function and describe how their gating properties allow activity (and thus their contribution to stressful Ca2+ oscillations) during pacemaking.The loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SN) underlies the core motor symptoms of the progressive movement disorder Parkinson's disease (PD). To date, no treatment to prevent or slow SN DA neurodegeneration exists; thus, the identification of the underlying factors contributing to the high vulnerability of these neurons represents the basis for the development of novel therapies. Disrupted Ca However, a phase 3 clinical trial evaluating the efficacy of LTCC inhibition in early PD patients recently failed. This short review aims to give an overview of the (patho)physiological roles of Ca2+ channel activity in SN DA neurons and to (re)evaluate the therapeutic potential of Ca2+ channel inhibition  and the availability of clinically applicable selective drugs.The primary motor symptoms of the neurodegenerative disorder Parkinson's disease (PD) are caused by a progressive loss of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SN) and associated striatal DA depletion  . Inhibition of Na+ channels by tetrodotoxin abolished spike generation and revealed slow oscillatory membrane depolarizations  or A-type K+ channels] can also trigger the opposite—a functional coupling important for rhythmic activity   that allow to monitor the oxidation state of mitochondria established a link between Ca2+ influx and intracellular Ca2+ levels  were considered the main voltage-gated Ca2+ influx   was sufficient to lower mitochondrial oxidation   aggravated the observed effects on cell morphology and survival, which could be prevented by drugs targeting TTCCs as well as individual knockout of all TTCC isoforms. Two of the TTCC inhibitory compounds have complex mechanism of action besides inhibition of TTCCs, but one drug, ML218, specifically blocks TTCCs .Evidence for a role of TTCCs in PD pathology comes from a study employing DA neurons derived from PD patient-specific iPSCs as 2+ channels represent an attractive drug target for the therapy of PD. Their contribution to large somatodendritic Ca2+ oscillations, associated with increased mitochondrial oxidative stress, seems a likely mechanism by which their activity contributes to SN DA neuron degeneration. Pharmacological and genetic strategies to decrease the activity of LTCCs, RTCCs, and TTCCs showed promising neuroprotective effects in preclinical models of PD, but a phase 3 clinical trial found no slowing of disease progression in early PD patients upon treatment with the DHP LTCC inhibitor isradipine. Although DHPs are safe, brain permeable, and clinically available, their lack of selectivity for Cav1.3, the more likely LTCC target for neuroprotection, and adverse Cav1.2-mediated effects limit their potential for PD therapy. Selective TTCC blockers exist, show good tolerability and safety, and could be repurposed for the therapy of PD; however, evidence for neuroprotection in in vivo PD models is still missing. In contrast, no selective inhibitors for Cav1.3 and Cav2.3 exist. The convincing preclinical data described in this review and the discovery of activity-enhancing mutations in neurological diseases (Ortner et al., 2+ channel targeting blockers and, if once available, Cav1.3- and Cav2.3-selective inhibitors will help to uncover the full therapeutic potential of Ca2+ channel inhibition for neuroprotection in PD.There is accumulating evidence that voltage-gated CaThe author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Adipoq), peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), perilipin1 (Plin1), fatty acid-binding protein 4 (Fabp4), glucose transporter type 4 (Slc2a4), and CCAAT/enhancer-binding protein alpha (Cebpa). In OVX-induced obese mice, administration of scopolin promoted the reduction of body weight, total fat percentage, liver steatosis, and adipose cell size. In addition, the scopolin-treated OVX mice showed decreased serum levels of leptin and insulin. Taken together, these findings suggest that the use of scopolin prevented adipocyte differentiation and weight gain in vitro and in vivo, indicating that scopolin may be a potential bioactive compound for the treatment and prevention of obesity in humans.Obesity is prevalent in modern human societies. We examined the anti-obesity effects of scopolin on adipocyte differentiation in preadipocyte 3T3-L1 cells and weight loss in an ovariectomy (OVX)-induced obese mouse model. Scopolin inhibited adipocyte differentiation and lipid accumulation in the preadipocyte cells by suppressing the transcription of adipogenic-related factors, including adiponectin ( Obesity is a common metabolic health problem in modern societies . ObesityLycii radicis cortex (LRC) is used as a traditional medicine in East Asia owing to its beneficial effects on bone loss, rheumatoid arthritis, inflammation, and type 2 diabetes [The most common interventions for obesity are eating a healthy diet and exercising in order to lose weight. However, pharmacological drugs for decreasing food intake or reducing fat accumulation may be considered for obese patients . While vdiabetes ,16,17. Pdiabetes . Previoudiabetes ,20. DespThe aim of the present study was to evaluate the anti-obesity effects of scopolin under in vitro and in vivo conditions. We isolated and identified scopolin from the LRC as a primary bioactive compound. We investigated the effects of scopolin on adipogenesis in preadipocyte 3T3-L1 cells and weight loss in an ovariectomy (OVX)-induced obese mouse model.Adipoq), Pparg, (Lpl), Plin1, Fabp4, glucose transporter type 4 (Slc2a4), CCAAT/enhancer-binding protein alpha (Cebpa), and cells that stained positively for oil red O. However, scopolin treatment decreased the expression of adipogenic transcription factors  eight-week-old ddY female mice were divided into four groups, and two different concentrations of scopolin were administered for 12 weeks: (1) Sham, (2) OVX, (3) OVX administered with scopolin 20 mg/kg/day (Sco20), and (4) OVX administered with scopolin 40 mg/kg/day (Sco40). Food intake did not differ among the groups, and the body weight and body fat percentage were analyzed at 0, 6, and 12 weeks . As expeWe further examined the anti-obesity effects of scopolin in vivo by evaluating the serum concentrations of obesity-associated hormones such as leptin and insulin. Blood samples were collected from the mice after 12 weeks and the serum levels of leptin and insulin were assessed by enzyme-linked immunosorbent assay (ELISA). As a result, OVX mice showed increased serum levels of leptin and insulin compared to the Sham group, while the scopolin-administered OVX mice showed decreased serum levels of leptin and insulin compared to the OVX mice group . These rIn the present study, we demonstrated the anti-obesity effects of a bioactive compound, scopolin, under in vitro and in vivo conditions. We observed that scopolin prevented adipocyte differentiation by reducing the mRNA expression levels of adipogenic factors and lipid accumulation in preadipocyte 3T3-L1 cells. Oral administration of scopolin in an OVX-induced obesity mouse model revealed that scopolin prevented OVX-induced weight gain, an increase in body fat percentage, hepatic steatosis, and an increase in epididymal fat by downregulating serum leptin and insulin levels.Lpl, Pparg, Plin1, Fabp4, Slc2a4, and Cebpa. Pparg stimulates lipid uptake and increases insulin sensitivity by enhancing fatty acid storage in adipocytes [Plin1 is an essential factor required for lipid droplet accumulation, and Plin1 deficiency leads to lipid dysregulation in adipose tissue [Fabp4 regulates leptin secretion and mitochondrial fatty acid oxidation [Slc2a4 transports circulating glucose to adipocytes and skeletal muscle [Cebpa promotes new fat formation and accumulation of lipids in the adipocytes and the liver [Adipocytes play an important role in lipid storage and metabolism . Typicalipocytes . Plin1 ie tissue . Fabp4 rxidation . Slc2a4 l muscle . Cebpa phe liver . These ahe liver . In thisThe OVX animal model leads to obesity in both rats and mice and is a well-known feature of human obesity ,32. A nuThe clinical significance of serum leptin and insulin levels in obesity has also been reported in the literature because of the positive correlation with an increase in body weight and fat mass ,39. The 2.Mouse 3T3-L1 preadipocyte cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). For adipocyte differentiation, 3T3-L1 cells were cultured with 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 μg/mL insulin (MDI) for 3 days, then incubated a further 5 days with 1 μg/mL insulin for lipid accumulation. The medium was changed every 3 days. All cultured cells were maintained in a humidified atmosphere at 37 °C with 5% COThe 3T3-L1 cells were seeded in a 96-well plate and incubated with culture media containing different concentrations of scopolin (2 or 10 μM) for 48 h. Cell viability was determined by WST assay using an EZ-Cytox Cell Viability Assay Kit . WST solution  was added and incubated for 4 h. Absorbance was measured at 450 nm and 655 nm using a microplate reader .Adipoq, 5′-GGA AGA CCA CTC GCA TTC CTT-3′ and 5′-GTA ATC AGC AAC CAT TGG GTC A-3′ for mouse Pparg, 5′-ATG GAT GGA CGG TAA CGG GAA-3′ and 5′-CCC GAT ACA ACC AGT CTA CTA CA-3′ for mouse Lpl, 5′-CAA GCA CCT CTG ACA AGG TTC-3′ and 5′-GTT GGC GGC ATA TTC TGC TG-3′ for mouse Plin1, 5′-AAG GTG AAG AGC ATC ATA ACC CT-3′ and 5′-TCA CGC CTT TCA TAA CAC ATT CC-3′ for mouse Fabp4, 5’-ACA CTG GTC CTA GCT GTA TTC T-3’ and 5’-CCA GCC ACG TTG CAT TGT A-3’ for mouse Slc2a4, 5′-GCG GGA ACG CAA CAA CAT C-3′ and 5′-GTC ACT GGT CAA CTC CAG CAC-3′ for mouse Cebpa, and 5′-TGA CCA CAG TCC ATG CCA TC-3′ and 5′-GAC GGA CAC ATT GGG GGT AG-3′ for mouse Gapdh. Relative gene expression levels were normalized to that of mouse Gapdh. The values are expressed as fold changes compared to the control. Fold changes are presented as 2−ΔΔCt (ΔΔCt = ΔCtcontrol − ΔCttreatment).The primers targeting adipogenic markers were as follows: 5′-TGT TCC TCT TAA TCC TGC CCA-3′ and 5′-CCA ACC TGC ACA AGT TCC CTT-3′ for mouse Cells were fixed in 4% paraformaldehyde for 15 min, washed three times with phosphate buffered salin, and stained with oil red O dye for 2 h. Oil red O positive cells were visualized with an optical microscope , and representative images were taken using an attached camera.n = 24) or sham-operated  8-week-old female ddY mice were purchased from Shizuoka Laboratory Center Inc. . Mice were maintained on a diet of Formula-M07 (5.0 g/day)  and tap water (15 mL/day). All mice were housed individually in clear plastic cages under controlled temperature (23 ± 2 °C), humidity (55 ± 5%), and illumination (12 h light/dark cycle) conditions. Mice in the two treatment groups were administered different concentrations of scopolin (20 and 40 mg/kg/day) for 12 weeks. The animal research protocol was approved by the Animal Care and Use Committee of Ajou University School of Medicine , and all experiments were conducted in accordance with the institutional guidelines established by this committee.Either ovariectomized .Mice were euthanized before blood and tissue collection. Blood samples were collected from the left ventricle of the heart and the serum was separated by centrifugation at 1200× Fat and liver tissues were fixed in cold 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The samples were embedded in paraffin and cut into 3 μm coronal planes using a rotary microtome. Tissue sections were stained with hematoxylin and eosin (H&E) to evaluate histological changes in fat and liver tissues. The size of adipocytes in the fat tissue was evaluated using CaseViewer 2.1 version  and ImageJ 1.52r version  software.p < 0.05) was considered statistically significant.All experiments were performed in triplicate, and the data are presented as mean ± standard error of mean (SEM). All statistical analyses were performed using the software package SPSS v.11.0 for Windows . Comparisons of multiple groups were evaluated with a one-way analysis of variance (ANOVA), followed by Tukey’s HSD (honest significant difference) post-hoc test. A probability value less than 0.05 (We demonstrated that scopolin inhibited adipocyte differentiation and lipid accumulation in preadipocyte cells and prevented OVX-induced weight gain in an obese mouse model. Our results suggest that scopolin may be an attractive therapeutic natural medicine for the prevention and treatment of obesity.


Recent findings suggest TRPV4 channels produce localized Ca2+ transients at the endothelial cell plasma membrane that may allow targeted effector recruitment and promote large-scale Ca2+ events via release from internal stores (endoplasmic reticulum). However, the specific impact of TRPV4 channels on Ca2+ signaling in the intact arterial intima remains unknown. In the current study, we employ an endothelium-specific TRPV4 knockout mouse model (ecTRPV4−/−) to identify and characterize TRPV4-dependent endothelial Ca2+ dynamics. We find that carotid arteries from both ecTRPV4−/− and WT mice exhibit a range of basal and acetylcholine (ACh)-induced Ca2+ dynamics, similar in net frequency. Analysis of discrete Ca2+ event parameters  and event composite values reveals that while ecTRPV4−/− artery endothelium predominantly produces large Ca2+ events comparable to and in excess of those produced by WT endothelium, they are deficient in a particular population of small events, under both basal and ACh-stimulated conditions. These findings support the concept that TRPV4 channels are responsible for generating a distinct population of focal Ca2+ transients in the intact arterial endothelium, likely underlying their essential role in vascular homeostasis.Transient receptor potential vanilloid 4 channels (TRPV4) are pivotal regulators of vascular homeostasis. Altered TRPV4 signaling has recently been implicated in various cardiovascular diseases, including hypertension and atherosclerosis. These versatile nonselective cation channels increase endothelial Ca They control the degree and specificity of many responses from vasodilation to permeability and inflammation. In endothelial cells, discrete Ca2+ signals emit from internal Ca2+ stores  and along the plasma membrane  channels) in the form of transients and waves [2+ dynamics through automated detection and analysis approaches have revealed that spatial and temporal tuning of basally occurring Ca2+ dynamics drives graded stimulus responses  [2+ influx, type 4 vanilloid transient receptor potential (TRPV4) channels have emerged as crucial regulators of endothelial function and as pivotal players in the development of cardiovascular disease [2+-dependent pathways [2+-activated K+ channels (KCa) to elicit membrane potential hyperpolarization that can spread to smooth muscle and promote vasorelaxation [2+ influx, allowing widespread expansion of Ca2+ dynamics  [Dynamic Ca disease ,7,8. Thepathways ,12,13,14laxation . Endothem IP3Rs) ,16. Ca2.3 channel subunits [Dysregulation of TRPV4 signaling contributes to endothelial dysfunction and progressive disease. Diabetes and atherosclerosis are associated with loss of functional endothelial TRPV4 channels , and mulsubunits ,18. On tsubunits . These f2+ transients (or sparklets) along the endothelial cell plasma membrane [2+ stores were depletion [2+ dynamics and waves. This suggests that in intact vascular endothelium, fundamental TRPV4 Ca2+ events can be actively recruited, and they may act as triggers for much larger Ca2+ signals. In the current study, we employ a genetically altered mouse deficient in endothelial TRPV4 to determine the specific impact of TRPV4 channels on basal and stimulated Ca2+ signaling profiles in the arterial endothelium. Recent studies suggest influx through single or small clusters of TRPV4 channels give rise to highly localized Camembrane . Occurre−/− breeders by generating and crossing floxed TRPV4 mice with endothelial-specific CRE. Assessment of TRPV4 expression in carotid arteries isolated from ecTRPV4−/− mice and control wild-type (WT) mice  mice e reveals2+ signaling profile, we assessed the intima of opened carotid artery segments loaded with the Ca2+ indicator Fluo-4. −/− and WT mouse carotid artery endothelium before and after exposure to acetylcholine. The ecTRPV4−/− artery endothelium exhibited a similar number of Ca2+ signal origination sites and events as WT artery endothelium, both basally and after 10 nM ACh stimulation . Following ACh exposure, event amplitudes and durations remained higher in ecTRPV4−/− endothelium , whereas spatial spread was not significantly different than WT (62 vs. 64 µm2). Histogram analysis revealed a rightward displacement of the ecTRPV4−/− event parameter distributions relative to WT ). Notably, this index exposes events that are essentially larger or smaller than the mean basal Ca2+ signal, which would have an ADS value of 1. Because calculated ADS values were highly left skewed , they were plotted as log values for display and analysis . At the same time, the ecTRPV4−/− endothelium exhibits a broad distribution of larger events lacking in WT endothelium . In WT endothelium, stimulation with ACh caused a distinct bimodal distribution of Ca2+ signals, whereby a population of small events (ADS ~0.14) persisted and a new population of larger events (ADS 2-8) emerged. On the contrary, stimulation of the ecTRPV4−/− endothelium with ACh caused only expansion of the general basal distribution (ADS 2-8). Notably, although the differences between ecTRPV4−/− and WT event populations narrow with ACh treatment, the same population of small events are lacking in the ecTRPV4−/− endothelium.Assessment of individual event parameters suggested a specific type of dynamic Caanalysis ; a log AT events a, with Wstograms b reveals2+-permeable TRPV4 channels in cardiovascular disease has focused attention on their specific impact on vascular signaling and their potential as targets for therapeutic intervention. Studies pointing to TRPV4 channels as conduits of localized Ca2+ sparklets along plasma membranes of endothelial cells have suggested their role as key drivers of endothelial function [2+ signaling profile in intact endothelium has remained obscure. Here, we provide evidence that endothelium-specific ablation of TRPV4 channels substantially reduces a particular population of small Ca2+ events along the vascular intima and right-shifts the Ca2+ signal profile to a single broad population that is monolithically preserved under endothelial stimulation. These findings imply that TRPV4 channels are crucial for maintaining the normal dynamic range of endothelial Ca2+ signals and a progressive pattern of signal recruitment during endothelial stimulation.The TRP class of nonselective cation channels encompasses a diverse group of membrane proteins that play an essential role in cell signaling and homeostasis. Recent implication of Cafunction ,21, but −/− and WT arteries. This is particularly important since previous studies evaluating endothelial TRPV4 channels using agonists/antagonists or global TRPV4 knockout models may have included off-target effects [2+ signals along the arterial intima. We found that endothelial TRPV4 ablation had little overall influence on the frequency of events occurring along the intima although the time course of ACh-induced event firing was distinctly abbreviated . These findings may suggest an important role for TRPV4 in sustained endothelial functional responses. A key finding of the current study was that removal of endothelial TRPV4 channels considerably altered the specific attributes (event parameters) of the dynamic endothelial Ca2+ signals. Most importantly, the number of events with amplitudes <1.2F/F0 and durations < 3.5 s were greatly reduced. Surprisingly, this loss of small-amplitude/duration events was accompanied by a relative increase in larger/longer-lasting events. The deficiency of small Ca2+ events in ecTRPV4−/− endothelium is consistent with general loss of TRPV4 Ca2+ sparklets. In fact, the “missing” Ca2+ events in the ecTRPV4−/− endothelium describe a discrete a population of small transients  quite similar to previously described TRPV4 Ca2+ sparklets , spatial spread (5–11 µm2) [2+-induced Ca2+ release from internal stores. Indeed, the right-shifted mode we observed in the WT histogram  following ACh stimulation suggests conversion of some sparklet-type events to larger events, perhaps due to IP3-sensitzation of CICR. This is consistent with expansion of TRP-originating signals into Ca2+ waves as previously reported in intact arterial endothelium [−/− endothelium is unknown but may reflect a compensatory expansion of internal stores, perhaps through upregulation of IP3/IP3R signaling. Exploring this apparent adaptation is warranted given the known pathological implications of downregulated TRPV4 signaling. In the context of our current findings, it would appear that endothelial TRPV4 channels underlie a narrow range of physiological Ca2+ signals that may serve as important local regulators and key triggers for expanding Ca2+ signal profiles.Selective ablation of endothelial TRPV4 channels allowed us to specifically address the impact of TRPV4 channels within the intima while preserving TRPV4 expression and function in other cell types, including vascular smooth muscle and perivascular nerves . Indeed, effects ,12,22. W–11 µm2) . We mighothelium . The sou2+ events, each event is actually an amalgamation of all three parameters, and it cannot be assumed that any single event is actually represented by averaged individual parameters. Calculating the ADS product allowed us to gauge the relative “size” of each event (intensity in time and space) relative to the expected prototypical control event. Altogether, data suggest that in the absence of TRPV4 channels, the vascular endothelium largely loses the capacity to produce a specific event (ADS mode ~0.3) and may compensate by basally generating events that are an order of magnitude larger (primary ADS mode ~3). The later appears to be the same population of events recruited by normal endothelium upon stimulation . This finding predicts that chronic loss of TRPV4 channels may cause an amplification of basal signaling that mimics an endothelium under constant (agonist-mediated) stimulation. Whether this translates to amplified Ca2+ impact on basal endothelial function in ecTRPV4−/−  or causes desensitization of Ca2+-dependent signaling  is an intriguing question for future study. The findings may also indicate that the characteristic shift from one predominant mode of event to another is a crucial aspect of graded stimulus-dependent signaling and that TRPV4 channels play a key role in this tuning of endothelial response.While amplitude, duration and spatial spread provide valuable and definitive information about individual CaMice  were euthanized with pentobarbital sodium (50 mg/kg) and both common carotid arteries were harvested. All animal procedures were approved by the University of South Alabama Institutional Animal Care and Use Committee  and carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals.−/−) genotype. Primer pairs used were sense 5′-CATGAAATCTGACCTCTTGTCCC and antisense 5′-TTGTGTACTGTCTGCACACCAGGC, which yielded ~2.4 kB PCR products.Endothelium-specific TRPV4-deficient mice were produced by genetic targeting and cell-specific deletion as previously described . BrieflyCarotid artery segments were fixed in optimal cutting temperature compound (OCT) and then cut into 7 µm cross-sections. TRPV4 protein was labeled with rabbit TRPV4-specific primary antibody  and fluorescent anti-rabbit secondary antibody . The elastic lamina autofluorescence was assessed using the green channel (488 nm excitation/510 nm emission). Digital images were collected in 6 µm stacks (0.5 µm increments) using a Nikon A1R confocal microscope and NIS Elements .2+ indicator loading solution containing Fluo-4 AM  in HEPES-buffered PSS . After washing and allowing 15 min for equilibration, inserts were placed in glass-bottom chambers (separated from coverglass by 100 μm) containing HEPES. The chamber was mounted on an inverted microscope of an Andor Revolution spinning disk confocal system . Ca2+-dependent fluorescence  was measured at 8 frames/sec at 25 ˚C  using iQ software . The 16 bit raw data images were saved as 8 bit TIFF files and processed using the custom algorithm LC_Pro, implemented as a plug-in with ImageJ software . A detailed description of the algorithm is provided in [2+ change above statistical (p < 0.01) noise, 2) track full event spatial boarders, 3) define circular regions of interest  at active site centers, and 4) analyze average fluorescence intensities within ROIs. Fluorescence data are expressed as F/F0, where F0 is determined by a linear regression of base data at each ROI. The amplitude is taken as the peak F/F0 for each event. The duration of each event is measured at 50% of the maximal amplitude. This approach prevents the arbitrary extension of duration values for events that decay to values very near the baseline. Events that do not reach 50% decay are assigned duration values to the end of the recording. Spatial spread of each event is determined from the maximal-dimension ellipse fit by the algorithm resulting from continuous event tracking.Carotid artery segments were cut open longitudinally and mounted on sylgard inserts, intima side up, using tungsten micropins as previously described . Arterievided in . Briefly2+ event, we normalized the amplitude (A), duration (D) and spatial spread (S) of each event to the control base mean value for each parameter and calculated the ADS product for each event. Statistical analysis was performed with GraphPad Prism software  using a Mann–Whitney test (non-parametric) for single comparisons. p values < 0.05 were considered significant.Normally distributed data are presented as the mean ± standard error. Non-Gaussian data are presented as distributed histograms or presented as box-and-whisker plots. For composite ADS values that represent the combined parameter attributes of each Ca2+ transients in the intact vascular endothelium and alters the normal pattern of Ca2+ signal recruitment by endothelial stimulation. These findings suggest an important role of endothelial TRPV4 channels in the regulation of Ca2+-dependent vascular homeostasis.Genetic ablation of endothelial TRPV4 channels impairs the generation of small Ca


FGF5 is mutated in humans and other species. However, there has been no such report in rabbits. Thus, the first exon of rabbit FGF5 was disrupted by the CRISPR/Cas9 system, and the phenotype of -/-FGF5 rabbits was characterized while using hematoxylin and eosin (H&E) staining, immunohistochemistry, quantitative PCR, scanning electron microscopy, and western blotting. The results showed a significant and systemic long hair phenotype in the -/-FGF5 rabbits, which indicated that FGF5 is a negative regulator of hair growth. In addition, a decreased diameter of the fiber and a higher area proportion of hair follicle clusters were determined in FGF5-/- rabbits as compared with the WT rabbits. Further investigation verified that prolonging the anagen phase in rabbits, with decreased BMP2/4 pathway signaling and increased VERSICAN pathway signaling, caused the systemic long hair phenotype. Taken together, these results indicate a systemic long hair phenotype by prolonging anagen in -/-FGF5 rabbits, which could be widely used for Fur production and an ideal model for studying the mechanism of long hair in the future. Hair growth and morphology are generally regulated by the hair cycle in mammals. Fibroblast Growth Factor 5 (FGF5), which is a hair cycle regulator, has a role in regulating the hair cycle during the transition from the anagen phase to the catagen phase, and a hereditary long hair phenotype has been widely reported when  The rabbit is an important livestock species that provides a variety of products, including fiber, meat, and hides. Rabbit hair is one of the preferred natural fibers used in textile industries . In rabbFGF5 are associated with a long hair phenotype in humans FGF5 are n humans , alpaca n humans , Syrian n humans , and casn humans ,14. One n humans . Most ann humans ,16. Duri of DPCs ,17,18. Aand SHH) ,20,21,222, a higher follicular infundibulum volume, and a larger follicular infundibulum surface [FGF5 in rabbits by gene targeting. Based on this, the disruption of FGF5 in rabbits will benefit agricultural achievements and generate a more obvious hair phenotype than that in mice.Mouse models have been widely used to model abnormal hair development and even hair follicle morphology in a three-dimensional (3D) and stochastic way ,20. Howe surface . HoweverFGF5-/- rabbits via CRISPR/Cas9. Significant long hair fibers with rapid growth and decreased diameters were observed in different regions of fur of FGF5-/- rabbits. We also discovered a sex-dominant pattern of growth in FGF5-/- and WT rabbits. In addition, the results of this study show that disrupting FGF5 in rabbits improved the ratio of hair follicle clusters area, but produced no significant difference in the number of hair follicles. Altogether, this study suggests that rabbits with FGF5 disruption can produce excellent longer hair over their whole bodies, which will provide an ideal model to the Fur industry and study the molecular mechanism of hair cycle in the future.In this study, for the first time, we generated The rabbits that were involved in these experiments were New Zealand white. All of the animal studies were conducted according to experimental practices and standards that the Animal Welfare and Research Ethics Committee approved at Jilin University. New Zealand white rabbits were housed under the 12-hour light-dark cycles in individual cages, and fed twice daily with a basic commercial rabbit diet and water ad libitum.The Cas9 plasmid and constructed sgRNAs were transcribed in vitro, as previously described . BrieflyComplementary oligonucleotides, 20-nt guide sequences, were annealed at 95 °C for 5 min. and then ramped down to 25 °C to clone into the BbsI-digested pUC57-Simple vector (Addgene ID 51306) under the activity of the T7 promoter. PCR products, which were amplified by T7-F: (50-GAAATTAATACGACTCACTATA-3’) and T7-R: (50-AAAAAAAGCACCGA CTCGGTGCCAC-30), were used for in vitro sgRNA transcription (T7 RNA Synthesis Kit (Ambion)). The sequences were purified while using a TIANquick Midi Purification Kit (DP204) . The transcribed sgRNAs were then purified with a miRNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. The concentration and quality of RNAs were determined with a NanoDrop 2000  and by agarose gel electrophoresis, respectively.2, and 100% humidity conditions. Approximately 30–50 injected zygotes were transferred into the oviducts of recipient rabbits.Zygotes were collected from sexually mature New Zealand rabbits, as previously described . The oviFGF5 knockout and WT rabbits was isolated using a TIANamp Genomic DNA Kit , according to the manufacturer’s instructions. Genomic DNA from FGF5 knockout and WT rabbits were fixed with 4% paraformaldehyde for 48 h, embedded in paraffin wax, and then sectioned. The skin sections were stained with hematoxylin and eosin (H&E) and analyzed by microscopy . A primary antibody against FGF5  was used for immunohistochemistry. The slides were imaged with a microscope  and processed using Photoshop CS5 .Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed, as previously described . The ski-/-FGF5 and WT rabbits were attached to specimen stubs using double stick conductive tabs, and they were then sputter-coated with gold using a Polaron scanning electron microscope E-1010 . The samples were imaged using an S-3400N scanning electron microscope .Hair samples from the backs of -ΔΔCT formula was used to determine relative gene expression, which was normalized to GAPDH mRNA expression. All of the experiments were repeated three times for each gene. All the data are expressed as the mean ± SEM (Standard Error of Mean). The protocol for RNA extraction has been described previously . Briefly-/-FGF5 and WT rabbits were homogenized and lysed in RIPA buffer supplemented with a protease inhibitor cocktail , and they were incubated on ice for 30 min. The antibody against FGF5  was used as a primary antibody, while Tublin antibody  was used as the loading control.Western blot analyses were performed, as described previously . The samhttp://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). The WT forms of the FGF5 and FGF5s proteins are also shown.Two deletions were chosen for analysis to evaluate the effects of targeted deletion. The secondary structures of the truncated proteins were predicted by PHY2 software, and their 3D models constituted by the Phyre server  to analyze a photo of hair taken against a black background with a ruler. Pairwise data were analyzed with Student’s t-test while using GraphPad Prism 8.0 .p-value < 0.05 was considered to be statistically significant.The data were analyzed using GraphPad Prism 8.0 (T test) software (GraphPad Software Inc.). A http://tools.genomeengineering.org). The top five POTS were selected. Primers  of the sgRNAs were predicted using the CRISPR online design tool ( Primers  were useFGF5 gene was found to be highly conserved (91% identity) between rabbits and humans, as well as at the amino acid sequence level of FGF5 (FGF5 were designed to mimic mutations in human [FGF5 deletions in rabbits. One of these recipient mothers was pregnant to term and gave birth to seven live pups and one dead pup (No. 8)  and No. 8   . Genomic −58 bp) B,C.FGF5 could cause a significant long hair phenotype in rabbits, as shown in -/-FGF5 rabbits when compared to hair length in WT rabbits  for each sgRNA to test whether off-target effects could occur in these rabbits. We isolated the FGF5-/-) was mated with a WT rabbit to study the heritability of the long hair phenotype caused by disruption of FGF5. The F1 rabbits were all heterozygous, with a deletion of FGF5, though the typical symptom of long hair was not observed. A sexually mature F1 was mated within the F1 group to study the heritability of the long hair phenotype caused by disruption of FGF5  of hair [FGF5 could be observed on the body sites that usually have short hair with a high proportion of hair follicles in the telogen phase. Thus, hair was collected from eight different body sites of WT and -/-FGF5 rabbits  after birth, and every 10 days throughout the trial. The length of both guard and down hair had a sharp increase in FGF5-/- rabbits, when compared with those of the WT controls, as shown in As FGF5 in Syrian hamster caused a long hair phenotype with male dominant expression [FGF5-/- and WT rabbits were compared between male and female  staining  and a short form (FGF5s) through mRNA alternative splicing. FGF5s has been noted to suppress FGF5 activity by competitively binding to the FGF receptor 1, FGFR1 [FGF5 modification  [FGF5 protein of rabbit is necessary for the identification and investigation of genotype to the phenotype of -/-FGF5 rabbit in the further study.Studies of th cells , which i 29 kDa) . Thus, tFGF5 interacts with the receptor FGFR1, which is expressed in dermal papilla cells (DPCs); this signal is known to stimulate outer root sheath cell (ORSC) proliferation [FGF5-/- rabbits, we found increased VERSICAN levels and decreased BMP2/4 and TGF-β levels. The results from the FGF5-/- rabbits indicated that rabbits might share a similar mechanism with humans and rodents. This conversed hair development strategy may be very useful for studying abnormal hair growth, such as alopecia and polytrichosis. In the future, rabbits could be an excellent model for hair study due to their size and conversed hair cycle mechanism.The secretion of feration . Other mferation ,33. VERSferation . The expferation . In FGF5FGF5 could cause localized hypertrichosis in human legs, arms, and eyelashes, because these sites have a low anagen:telogen ratio [-/-FGF5 rabbit, a systemic long hair phenotype was determined, but the different body sites had different reactions to disruption of FGF5. Multi-scale modeling has shown that mouse skin behaves as a heterogeneous regenerative field, in which different regions of the body (fast cycling and slow cycling) have distinct cycling dynamics. Some areas, such as the ear, act as a hyper refractory domain with HFs in the extended rest phase [In humans, different body sites, such as the upper, eyelashes, and scalp, have variable percentages of hair follicles in anagen, from low to high, respectively ,39,40. Men ratio . In our st phase . TherefoFGF5 could induce occasional longitudinal hair fissures with broken or curled tips, while other reports suggested that the structure of secondary hair follicles (SHF) was not affected by the disruption of FGF5 [-/-FGF5 rabbit decreased. The cross section of skin from -/-FGF5 rabbits showed no obvious change in the number of hair follicles, but there was a change in the size of the hair follicle cluster area and the ratio of primary/secondary hair follicles. The higher ratio of hair follicle cluster that was observed in H&E staining could explain the bushy feeling when we touched the -/-FGF5 rabbits. The decreased primary/secondary follicle ratio demonstrated an increase in the number of secondary hair follicles, which can generate more down hair and would be useful in producing textiles.A previous study  showed t of FGF5 . The SEMFGF5 has no obvious effect on hair follicle development in mouse, but have an effect on early chick development [FGF5 during the development of the embryo, although additional work should be carried out. In conclusion, our results demonstrated that -/-FGF5 induced a long hair phenotype and had effects on the distribution of hair follicles among species.The BMP family and SHH balance the fate decision and generation of hair follicles during the embryonic period. In addition, previous work demonstrated that elopment ,42. BaseFGF5 can optimize features of hair fibers by producing long fibers that are thin and have a bushy distribution. These features are very important and useful in hair production. The spontaneous long hair that was caused by mutations of FGF5 in Syrian hamsters was affected by testosterone administration [FGF5. Moreover, the prolonged anagen phase could be sustained for a long time.The disruption of stration . Male do-/-FGF5 rabbits via CRISPR/Cas9 methodology. This result suggests that rabbits with FGF5 disruption can produce excellent long hair over their whole bodies, which can be very useful in agricultural areas and studies regarding the hair follicle cycle.In this study, for the first time, we generated -/-FGF5 rabbits could be a novel model to promote Fur production and ideal model to investigate mechanism of long hair.


While numerous studies implicate Ca2+ mishandling in AD, the cellular basis for loss of cognitive function remains under investigation. The process of synaptic degradation and degeneration in AD is slow, and constitutes a series of maladaptive processes each contributing to a further destabilization of the Ca2+ homeostatic machinery. Ca2+ homeostasis involves precise maintenance of cytosolic Ca2+ levels, despite extracellular influx via multiple synaptic Ca2+ channels, and intracellular release via organelles such as the endoplasmic reticulum (ER) via ryanodine receptor (RyRs) and IP3R, lysosomes via transient receptor potential mucolipin channel (TRPML) and two pore channel (TPC), and mitochondria via the permeability transition pore (PTP). Furthermore, functioning of these organelles relies upon regulated inter-organelle Ca2+ handling, with aberrant signaling resulting in synaptic dysfunction, protein mishandling, oxidative stress and defective bioenergetics, among other consequences consistent with AD. With few effective treatments currently available to mitigate AD, the past few years have seen a significant increase in the study of synaptic and cellular mechanisms as drivers of AD, including Ca2+ dyshomeostasis. Here, we detail some key findings and discuss implications for future AD treatments.Ca AD, as a progressive neurodegenerative disease, is characterized by Camopathy”  and synamopathy” ,4, and wmopathy” , and maye models ,7,8,9. Ceostasis ,11, botht models ,14,15,162+ entry points in the neuronal architecture, expressing both pre- and postsynaptic Ca2+ channels/receptors, including presynaptic RyRs, N/P/Q voltage gated Ca2+ channels (VGCCs), α7 nicotinic acetylcholine receptors (α7 nAChRs), and postsynaptic L-type VGCCs, RyRs and NMDA receptors (NMDARs). NMDARs in particular are one of the most well characterized postsynaptic glutamate receptors, with a high Ca2+ permeability and an established role in hippocampal synaptic plasticity [2+, but postsynaptic depolarization results in removal of Mg2+ block, and receptor disinhibition. Repeated NMDAR activation enhances postsynaptic Ca2+ entry, an effect which is facilitated by RyRs through Ca2+-induced-Ca2+ release (CICR), thus driving increased postsynaptic AMPA receptor (AMPAR) expression and subsequent synaptic long-term potentiation (LTP). This dual role of the NMDAR as coincidence detector and postsynaptic Ca2+ entry channel makes it uniquely positioned to mediate synaptic potentiation resulting from concurrent pre- and postsynaptic activation, thus forming a mechanistic basis for Hebbian plasticity and associative learning.Synapses are unique Caasticity . At hype2+ channel antagonists could serve as a useful pointer for a role for those Ca2+ channels in the pathophysiology of AD, with the failure of large scale clinical trials for L-type VGCC antagonists in particular, contrasting with the positive effects of memantine [2+ release within spines [Paradoxically, NMDARS, as well as playing a role in synaptic plasticity, may also play a role in synaptic loss  and cellemantine . AD is cemantine ,23, inclemantine , and simemantine ,27,28,29n spines ,31, and n spines , resultin spines ,34,35. In spines ,37,38. Sn spines .2+ hyperactivity in vivo, in presymptomatic AD mice, an effect which is blocked by AMPA receptor and NMDAR antagonists, suggesting that this hyperactivity is synaptically driven [2+ hyperactivity occurred mainly in the vicinity of insoluble dense core plaques, these plaques are also surrounded by soluble Aβ [2+ signaling in AD mice [2+ hyperactivity observed in an AD mouse model was inhibited by the sarcoplasmic endoplasmic Ca2+-ATPase (SERCA) pump inhibitor cyclopiazonic acid, indicating that this Ca2+ hyperactivity is driven by activation of presynaptic Ca2+ stores [2+ activated SK2 channels is increased in a 3xTg AD mouse model [+ current (IA) [2+ in hippocampal neurons [3 receptor activation [2+ hyperexcitability observed in vivo in AD mice.Hippocampal and cortical neurites in close proximity to amyloid plaques demonstrate Cay driven ,7. Altholuble Aβ ,41, whicluble Aβ . Further AD mice , raising+ stores . In cont+ stores ,44,45. Ise model , leadingent (IA) , thus fu neurons , possibltivation , effectstivation , isoflurWhile the last two decades have seen a large increase in the number of studies using AD mouse models, a more recent development has been in the use of human induced neurons (HiNs), which are neurons derived from tissue samples taken from patients, to study synaptic transmission ,52,53. I2+ and affect neurotransmitter release [2+ channels could serve as a therapeutic target for AD.More recently, proteomics has emerged as a method that allows for high throughput analysis of protein expression in small tissue samples , includi release ,67, and  release ,69, sugg2+ permeable homomeric α7nAChR isoform [2+ influx [nAChRs are essential for normal cognitive function ,71, and  isoform ,73. α7nA isoform , and als isoform ,76, and  isoform , and pos isoform ,79, with+ influx ,81,82,83+ influx ,81,84 an+ influx .2+ influx, both in oocytes and presynaptic terminals in hippocampus [2+ influx, an effect which was blocked by an α7nAChR antagonist [Aβ binds with high affinity to α7nAChRs ,87, resupocampus ,94. Low pocampus  and picopocampus . In addipocampus ,98 and cpocampus . Cells ttagonist . Galantatagonist .2+ effects in AD. In addition to α7nAChR mediated Ca2+ influx, activation of α7nAChRs triggers CICR via ryanodine sensitive Ca2+ stores [2+ overload via already increased RyR function [Although a role for α7nAChRs in the etiology of AD has not been established, many animal studies have demonstrated cognitive enhancing effects of compounds targeting α7nAChRs , includi+ stores , includi+ stores , which a+ stores . As RyR function , possiblAs of now, there are only two FDA-approved classes of drugs used in the symptomatic treatment of AD: the noncompetitive NMDA antagonist memantine, and the acetylcholinesterase inhibitors, donepezil, galantamine and rivastigmine, with both classes of drugs having a synaptic site of action. Although both memantine and donepezil have been shown to be moderately effective in the treatment of AD symptoms, there is an urgent need for disease-modifying approaches, which currently requires the identification of novel compounds and receptor targets at the pre- or postsynaptic level. While a number of studies have identified promising small molecules targeting NMDARs ,51, α7nA2+ levels are tightly regulated and maintained at low nM concentrations, despite a much higher extracellular Ca2+ concentration, and similarly elevated Ca2+ levels within intracellular organelles such as the ER. The ER is located throughout the cell, including pre- and postsynaptically, at synaptic terminals and dendritic spines respectively. RyRs, as well as having a role in gating ER Ca2+, are sensitive to changes in cytosolic Ca2+ through the process of CICR, with increases in postsynaptic Ca2+ resulting in RyR activation and release of ER Ca2+ into the cytosol. CICR has the effect of amplifying postsynaptic Ca2+ generated from influx via Ca2+ permeable receptors/ion channels such as NMDA receptors (NMDAR) and voltage gated Ca2+ channels (VGCCs), and RyR mediated CICR is upregulated in neurons in 3xTg AD mice [2+ allows for a large rapid increase in cytosolic Ca2+, this increased Ca2+ is usually rapidly removed from the cytosol, against a concentration gradient, by Ca2+ ATPases including the SERCA pump, which is also located on the ER membrane. The ER membrane also expresses IP3 receptors, although these are not thought to be synaptically expressed [Cytosolic Ca AD mice . Althougxpressed .2+ regulation. Although PS1 is a part of the γ-secretase complex which cleaves amyloid precursor protein (APP), it is also expressed on the ER membrane where it regulates RyR and IP3R channel properties [2+ leak channel [2+ regulation, and RyR dysfunction is implicated in the Ca2+ dysregulation observed in AD [2+ responses are increased in the soma and dendritic spines of hippocampal and cortical pyramidal neurons of AD mice expressing PS1 mutations (2+ dyshomeostasis observed in an AD mouse model [2+ release [2+ responses to high frequency stimulation (HFS) are increased in hippocampal and cortical neurons from several AD mouse models [2+ signaling is the restoration of reduced presynaptic vesicle stores observed in AD mice back to normal levels upon treatment with Ryanodex [Mutations in the presenilin 1 (PS1) gene are linked to familial AD, an early onset form of the disease, and these mutations have specific functional implications for Caoperties ,123,124, channel ,126. Mututations  [9,30,12se model . Human-i release , and RyR release ,130. Pose models ,9,131, e2+ signaling may also have implications for synaptic plasticity and LTP, which are impaired in AD mice [2+ and is accompanied by an increased release probability of glutamate. This form of short term plasticity is necessary for the synaptic tagging processes involved in LTP [2+ signaling in the impaired short and long-term synaptic plasticity observed in AD mice [2+ effects of RyR activation, due to decreased RyR expression [In addition to effects on basal synaptic transmission, changes in RyR-Ca AD mice ,45. Highd in LTP , howeverd in LTP . Chronic AD mice . Presenipression  and selepression . Thus, i2+ is maintained in part due to the actions of Ca2+-ATPases, which rapidly remove Ca2+ from the cell cytosol, against a concentration gradient. One of the major cellular Ca2+-ATPases is the SERCA pump, located on the ER membrane. SERCA function is facilitated by presenilin, and knockdown of the genes for presenilin 1 and presenilin 2 results in elevated cytosolic Ca2+, due to decreased SERCA mediated clearance of cytosolic Ca2+ [2+ channels on the ER membrane, results in increased Aβ [2+ response to SERCA inhibition by thapsigargin, indicating increased SERCA function [2+ hyperactivity observed in cortical neurons of AD mice in vivo [2+ sensors and Orai Ca2+ channels facilitate ER Ca2+ filling through the process of store-operated-Ca2+ entry (SOCE), a process which is deficient in PS1 mutant expressing neurons [2+ release is elevated in AD neurons , pyruvate dehydrogenase complex (PDHC), α-ketoglutarate dehydrogenase complex (KGDHC), malate dehydrogenase (MDH), and increasing ROS production while decreasing ATP production [2+ overload, contributed to mitochondrial dysfunction and impaired energy metabolism in AD.The main function of the mitochondria is the production of ATP. Unavoidably, the by-products of electron transport in aerobic respiration are reactive oxygen species (ROS), due to electron leaks at complex I and III. Caoduction . Redox poduction . Additiooduction . This su2+ transfer and cellular homeostasis. Mitochondrial Ca2+ uptake controls the rate of energy production, regulates intracellular Ca2+ signaling, and mediates cell death. Ca2+ transfer between these organelles is facilitated via the mitochondrial-associated membrane proteins (MAM). Numerous molecular proteins have been identified to support this physical interaction. Of interest, the glucose-regulated protein 75 (GRP75) is linked to IP3R and facilitates Ca2+ into the mitochondrial intermembrane space. From there, voltage-dependent anion-selective channel protein 1 (VDAC1) on the outer mitochondrial membrane, and the mitochondrial Ca2+ uniporter (MCU) on the inner mitochondrial membrane, transfer the Ca2+ to the mitochondrial matrix to stimulate the mitochondrial dehydrogenase and increase ETC activity and ATP synthesis. [2+ uptake. In AD, many genes involved in mitochondrial Ca2+ transport are altered [2+ influx, such as MCU, are downregulated whereas genes encoding mitochondrial Ca2+ efflux, such as NCLX, are upregulated, suggesting a compensatory mechanism to avoid excessive Ca2+ uptake. Additionally, studies report that, soluble Aβ aggregations increase cytosolic Ca2+, leading to mitochondrial Ca2+ overload via MCU. Excessive Ca2+ taken up by mitochondria leads to caspase activation and neuronal cell death [The dynamic function of the mitochondria requires crosstalk between other major organelles, such as the ER . Mitochonthesis. ,162,163. altered ,165,166.ll death .2+, VDAC1 supports transport of superoxide anions [3R and RyRs have been shown to be redox-sensitive, ROS and Ca2+ may play a regulatory role in ER-mitochondria communication. During AD- associated Ca2+ overload, mitochondrial Ca2+ influx elevates oxidative stress and increases ROS production. In AD, accumulated ROS has profound effects on cellular functions by oxidizing several proteins, such as the redox-sensitive RyR and IP3R channels. Thus, Ca2+ induced ROS increase and ROS-mediated Ca2+ increase creates a self-amplifying loop that furthers neurotoxicity and cellular dyshomeostasis, and neuronal death [PSEN1/2 may alter mitochondrial Ca2+ transport. Additionally, increased contact sites between ER and mitochondria, via MAM proteins in AD result in elevation in ER-mitochondrial Ca2+ signaling and increased mitochondrial superoxide production [In addition to Cae anions  and sincal death ,171,172.oduction ,177,178.2+ is released from the mitochondria through the Na+/Ca2+ exchanger (NCLX) and the permeability transition pore [2+ released; where the low conductance state amplifies Ca2+ waves and the high conductance state releases a surge of Ca2+ and apoptotic signals such as cytochrome C [2+ concentration, Ca2+ released from the mitochondria signals for apoptosis and increases AD pathology [Caion pore . The twochrome C ,181. Theathology . Howeverathology ,183. Addathology . Mitochoathology ,186,187.1 for fission and Mfn1, Mfn2, and OPA1 for fusion [2+ signaling led to increased DLP1 activation, resulting in excessive mitochondrial translocation and fission [2+ handling and increased oxidative stress disrupts mitochondrial function, advancing AD pathology. Disrupted DLP1 and Mfn2 function is also responsible for reduced mitochondrial distribution. In AD, mitochondria are less abundant in neuronal processes of susceptible pyramidal neurons [Mitochondrial morphology and distribution are also crucial for neuronal homeostasis and synaptic function. Mitochondria undergo fusion and fission in the cytoplasm, which is a process to maintain a healthy pool of mitochondria with proper distribution. These mechanisms are controlled by DLPr fusion . In AD,  fission ,190. Rec fission ,192. Exc neurons ,194. Inc neurons ,196.2+ signaling and colocalization of the two organelles. Intracellular Ca2+ signaling relieves the inhibitory mammalian target of rapamycin (mTOR) block, thus activating mitophagy and initiating autophagosome biogenesis  [2+ signaling, as seen in AD, may disrupt degradation of damaged organelle via inactivation of lysosomal proteolysis and increase accumulation of cellular debris [Damaged mitochondria are cleared through mitophagy, the selective degradation of mitochondria by autophagy following organelle damage or extreme cellular stress . Mitopha and 11) ,201,202.r debris ,204,205.2+ stores are responsible for regulating autophagy—a catabolic pathway utilizing the enzymatic activity of lysosomes to degrade and recycle large, bulky cellular debris, aggregated proteins, and damaged organelles. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy, however for simplicity, this review will focus on macroautophagy and be referred to as “autophagy”. More detailed descriptions of the aforementioned can be found in these reviews [2+ signaling hinders clearance of pathological protein deposits.Lysosomal Ca reviews ,206. Aut reviews ,208, theTranscriptomic profiles from the collective ongoing studies known as Rush Memory and Aging Project (ROSMAP), reveal clusters of genes that are associated with pathological protein handling. Specifically, higher expression of SORL1 and ABCA7 transcripts are associated with tau tangle pathology, while elevated BIN1 transcripts are associated with beta amyloid in AD brains . PLXNB1 + pump (vATPase) driving the influx of H+ into the lysosome [+ exchangers with AD suggest that proton leak pathways may regulate pathological Aβ generation and contribute to disease etiology [2+ signaling, as seen in AD, can hinder lysosomal acidification and impair proteolytic enzymes in lysosomes, further AD proteinopathy and impair lysososmal trafficking, resulting in neuritic dystrophy.The key, critical feature of lysosomes is the acidic lumen (pH ~4.5) necessary for protein degradation and autophagosome digestion. The acidic pH is maintained by an active vacuolar-ATPase Hlysosome ,212,213.etiology . In the etiology . Mutatioetiology ,216. Theetiology . Additioetiology ,219,220.+ channels (lysoNaV). The Na+ efflux potentiates the influx of protons by the vATPase to restore lysosomal acidity. However, lysoNaV are also Ca2+ permeable, therefore aberrant Ca2+ increase and changes in the Ca2+ concentration gradient, as seen in early pathology of AD, can hinder lysosomal acidity by reducing the driving force of H+ influx to restore lysosomal function [Additionally, an increase in the lysosomal pH disrupts the homeostatic mechanisms to maintain the lysosomal membrane potential. The alkaline lumen depolarizes the lysosomal membrane via activation of lysosomal voltage-activated Nafunction ,221,222.2+ efflux channels, lysosomal transient receptor potential Ca2+ channel mucolipin subfamily member (TRPML1) and two-pore channel (TPC) [2+ efflux through TRPML1, activates a calcineurin-dependent pathway that, via TFEB, enhances the transcription of genes involved in autophagy and lysosomal expression, such as LC3-II, ATG9B, UVRAG, WIPI, SQSTM1, MAPLC3B, GLA, GNS, HEXA, MCOLN1, TMEM55B, and ATP6V1H [This shift to a more alkaline lysosomal lumen causes hyperactivity of the lysosomal Cael (TPC) ,224,225 el (TPC) ,227,228) ATP6V1H ,230,231.2+ release is also responsible for fusion of autophagosomes to lysosomes. In a manner similar to neuronal vesicular fusion, lysosomal Ca2+ efflux channels such as, P/Q type VGCC, facilitate fusion between lysosomal tethering proteins, such as synaptogamin 7, SNAP29, and SNARE VAMP 7/8, to autophagosomal tethering proteins such as syntaxin 17 and possible SNARE proteins [2+ concentration, as in AD, can influence fusion of autophagosomes to malfunctioned lysosome, resulting in accumulation of cargo vesicles with maladaptive proteins, furthering neurotoxicity.This lysosomal-mediated Caproteins ,234,235.2+ stores are also involved in maintaining synaptic transmission. When mGluR1 is activated, NAADP-evoked lysosomal Ca2+ release from lysosomal Ca2+ channels, presumably through TPC, is amplified into Ca2+ waves via RyR activation [2+ entry through GluN receptors and facilitates the induction of LTP [2+ release facilitates fusion to the plasma membrane and release of cathepsin B. Cathepsin B then regulates structural plasticity and dendritic spine formation [Recent studies have shown that lysosomal functions go beyond their primary role as the degradative compartment within a neuron. Lysosomal Cativation . This sin of LTP . When VGormation ,239, howormation .2+ amplification suppresses autophagic flux [2+ signaling, as seen in neurodegenerative diseases, can alter this trigger zone and therefore disrupt autophagic clearance and synaptic plasticity.The systemic destruction of collective organelles leads to the neuron’s demise. The close proximity of the ER to the lysosome can enhance AD pathology  and PS mgic flux  and indugic flux ,238,239