Fibroblast growth factor receptors (FGFRs) in response to fibroblast growth factors (FGFs) transmit signals across the cell membrane, regulating important cellular processes, like differentiation, division, motility, and death. The aberrant activity of FGFRs is often observed in various diseases, especially in cancer. The uncontrolled FGFRs’ function may result from their overproduction, activating mutations, or generation of FGFRs’ fusion proteins. Besides their typical subcellular localization on the cell surface, FGFRs are often found inside the cells, in the nucleus and mitochondria. The intracellular pool of FGFRs utilizes different mechanisms to facilitate cancer cell survival and expansion. In this review, we summarize the current stage of knowledge about the role of FGFRs in oncogenic processes. We focused on the mechanisms of FGFRs’ cellular trafficking—internalization, nuclear translocation, and mitochondrial targeting, as well as their role in carcinogenesis. The subcellular sorting of FGFRs constitutes an attractive target for anti-cancer therapies. The blocking of FGFRs’ nuclear and mitochondrial translocation can lead to the inhibition of cancer invasion. Moreover, the endocytosis of FGFRs can serve as a tool for the efficient and highly selective delivery of drugs into cancer cells overproducing these receptors. Here, we provide up to date examples how the cellular sorting of FGFRs can be hijacked for selective cancer treatment.  Receptor tyrosine kinases (RTKs) constitute a large family of integral plasma membrane proteins that, by binding to appropriate ligands, transmit signals across the cell membrane. RTKs regulate a number of pivotal cellular processes, including cell division, motility, differentiation, metabolism, and survival. In humans, the RTK family constitutes of 58 members that share a similar structure and mode of action . RTKs arBecause of their critical function for cell homeostasis, the activity of RTKs is under tight control at various levels . AberranIn this review, we discuss the role of fibroblast growth factor receptors (FGFRs), a subfamily of RTKs, in cancer development. In particular, we focus on the subcellular trafficking of FGFRs and its implications in cancer treatment.FGFRs are integral membrane proteins with an overall structure typical for RTKs. Four major FGFR proteins have been identified, namely: FGFR1–FGFR4. FGFRs contain an extracellular region, a single transmembrane span, and an intracellular tyrosine kinase domain . The extFGFRs are activated by fibroblast growth factors (FGFs), which belong to the following two major groups: canonical FGFs  and endocrine FGFs . The members of the third group of FGFs, the intracellular FGFs (FGF11–FGF14), have no identified interaction with FGFRs, and thus are not able to stimulate FGFRs ,35,36,37The current model of FGFRs’ activation states that in an inactive state, FGFRs are present on the cell membrane as monomers. FGF binding induces conformational changes within the receptor, causing receptor dimerization and the activation of tyrosine kinase domains  37]. In. In37]. The ligand-induced FGFRs’ dimerization induces conformational changes within the intracellular regions of the receptor that trigger sequential tyrosine autophosphorylation events. The initial modification of tyrosine 653 largely increases the activity of the receptor kinase, which results in the phosphorylation of other tyrosine residues—Y583, Y463, Y766, Y585, and Y654 . This se2), generating the following secondary messengers: inositol triphosphate (IP3) and diacylglycerol (DAG). DAG activates protein kinase C (PKC), whereas IP3 alters the cellular calcium levels. The PLCγ signaling modulates a plethora of cellular processes, including cell division, calcium homeostasis, remodeling of cell membranes, and cytoskeleton . A. A179]. The function of nuclear FGFRs largely depends on the cell type. In the neurons, nuclear FGFR1 regulates the gene expression that governs cell proliferation and differentiation . NuclearMitochondria are organelles that perform numerous functions vital for cell homeostasis, like energy conversion, calcium ions regulation, biogenesis of iron-sulfur clusters, metabolism, and programmed cell death. This versatility of mitochondrial functions may be achieved by the dynamic properties of the mitochondrial proteome . MitochoImportantly, a fraction of FGFR1 were identified in the mitochondria of lung cancer cells . The bioAs altered FGFs/FGFRs signaling is frequently observed in various tumors, FGFRs became attractive molecular targets for the development of diverse anti-cancer therapeutics, excellently reviewed in the literature . TypicalTKIs are by far the most intensively explored anti-FGFRs agents, because of their simplicity and low costs of production . UnfortuThe elevated level of FGFRs on the surface of the cancer cells allows for the selective recognition of tumor entities by engineered molecules targeting these receptors. Interestingly, the naturally occurring cellular trafficking of FGFRs may serve as a highly efficient mechanism for the downregulation of receptor levels on the cell surface, and for the delivery of cytotoxic drugs into cancer cells. In this section, we focused on hijacking the cellular trafficking of FGFRs for the selective treatment of tumors with aberrant FGFRs. Antibody-drug conjugates (ADCs) constitute a novel group of anticancer therapeutics. ADC is composed of antibody and cytotoxic drugs connected by a specifically designed linker ,189. AntUp till now, several ADCs targeting FGFRs have been constructed, and their cytotoxic potential was evaluated . FGFR1-sN-methyl auristatin-W as a cytotoxic payload (BAY 1187982). BAY 1187982 demonstrates a high cytotoxicity against cancer cells producing FGFR2, and the cytotoxic activity correlates well with the level of FGFR2 on the cell surface, underscoring the relevance of receptor trafficking. FGFR2 is overproduced in gastric cancers and in triple negative breast cancers. BAY 1187982 displays a selective cytotoxic potential in gastric and triple negative breast cancer xenograft models, and in mouse cancer models [The FGFR2-specific antibody, BAY 1179470, was selected via the phage display approach using the n-CoDeR Fab library . BAY 117r models . Anotherr models . scFvF7-r models . LY30762r models . Not only antibodies or their engineered fragments have been used as targeting molecules for the selective delivery of cytotoxic drugs into cancer cells overproducing FGFRs. FGF1 and FGF2, natural FGFRs ligands, were successfully conjugated with MMAE in a site-specific manner, forming ligand drug conjugates (LDCs). The FGF-based conjugates retained the receptor binding and cellular trafficking properties of unconjugated FGFs, and demonstrated a selective cytotoxic potential against FGFR1-overproducing lung cancer cell lines ,198,199.Besides the cell surface, FGFRs are localized in the nucleus and in the mitochondria, where they promote oncogenesis by directly regulating the gene expression or by adjusting the cellular metabolism . ImportaBreast cancer cells contain FGFR1 truncation, which resides in the nucleus. The nuclear FGFR1 arises from the proteolytic processing of the full-length receptor by granzyme B. Importantly, the synthetic peptide inhibitor specific towards granzyme B blocked FGFR1 processing, nuclear translocation, and the migration of cancer cells . ComponeIn the mitochondria of cancer cells, FGFR1 promotes the Warburg effect by phosphorylating PDHK1 . BesidesFGFRs constitute a tightly regulated signaling system important for cell homeostasis. FGFRs are often overproduced or mutated in various tumors, and disrupted signaling facilitates carcinogenesis. Although recent years have brought about a large progress regarding understanding the FGFRs’ function, still little is known about the trafficking and intracellular role of these receptors. How do FGFRs reach the nucleus and mitochondria? Is mitochondrial trafficking specific only for FGFR1, or are other FGFRs are localized to this organelle as well? What are the other substrates of mitochondrial FGFRs? Which mitochondrial processes are affected by FGFRs? These and other questions remain to be answered. The internalization of RTKs occurs via numerous pathways. Up till now, FGFRs were reported to utilize three endocytic routes—CME, caveolae-mediated, and macropinocytosis. The involvement of other internalization pathways in FGFRs trafficking is unclear. As endocytic pathways regulate the duration and specificity of the propagated signals (and therefore cellular outcome), further studies are required for understanding the spatiotemporal control of FGFRs. FGFRs-mediated endocytosis is currently employed as a tool for the selective delivery of cytotoxic agents into cancer cells overproducing FGFRs in the ADC approach. In numerous cases, ADCs display side effects, which may be attributed to their partial mistargeting. An improvement of the selectivity and efficiency of drug delivery into the tumors may largely advance cancer treatment with ADCs. One of the possibilities is to improve the affinity of the targeting molecule within the ADCs. We have recently demonstrated that a high affinity promotes the internalization of engineered antibodies targeting FGFR1 in in vitro models ,192. TheOne of the major challenges in the targeted therapies against cancers with aberrant FGFRs is the development of resistance against applied therapeutics, including ADCs . A possiIn summary, the cellular trafficking of FGFRs can be employed for the selective treatment of cancers with aberrant FGFRs, either alone or in combination with other strategies. However, further detailed studies on the cellular trafficking of FGFRs are urgently required.


A new research journal named “Contact” has been recently established to facilitate the development of this research field. The current consensus is to define an organellar junction by the maximal distance between the participating organelles; and the gap of 30 nm or less is considered appropriate for classifying such structures as junctions or membrane contact sites. Ideally, the organellar junction should have a functional significance, i.e. facilitate transfer of calcium, sterols, phospholipids, iron and possibly other substances between the organelles (Carrasco and Meyer in Annu Rev Biochem 80:973–1000,  Tiewed in , 132) reiewed in , 142) atiewed in ). Other ns (e.g. ) further2+ store depletion and involves the activation of adenylyl cyclase 3 by STIM [2+ and cAMP signalling in the ER-PM junctions was extensively characterised in a number of elegant papers by D. Willoughby and colleagues from D. Cooper laboratory. This mechanism involves the direct interaction of adenylyl cyclase 8 and Orai1 [cAMP signalling is another signalling modality operating in the ER-PM junctions; studies from the A. Hofer laboratory recently defined a novel mechanism of cAMP signalling SOcAMPS  which is activated by ER Ca by STIM , 107. Annd Orai1 , 170. Innd Orai1 , 25, 152nd Orai1 , 146.The molecular mechanism of ER-PM junction formation was first discovered and characterised in yeasts where three groups of proteins Ist2, tricalbin proteins Tcb1–3 and Scs2/Sc22 contribute to the tethering of the organelles . Eganelles , 115. Reganelles .Membrane tethering between ER and Golgi is mediated by the oxysterol-binding protein (OSBP), which also serves as a conduit for the transfer of sterols and phospholipids between these two organelles , 115.Direct non-vesicular lipid transfer operates between the ER and peroxisomes . TethersInteraction between the ER and LDs is important for the lipid transfer to LDs; a complex consisting of fatty acid transport protein 1 (FATP1) and diacylglycerol O-acyltransferase 2 (DGAT2) have been identified as important for both ER-LD interaction and the lipid loading of LDs . Another2+ signals important for phagocytosis [2+ responses [ER contacts with phagosomes generate highly localised Caocytosis . Both juesponses , 124.Junctions between the ER and other cellular organelles are probably the most numerous inter-organellar junctions. However, junctions formed by other organelles have also been described and include contacts of LDs with peroxisomes and lysosomes (reviewed in ), and coMitochondria interact and form junctions with LD . PerilipThe components of the contact sites between mitochondria and peroxisomes have been characterised using a genome-wide screen in yeast. Pex11 and Mdm34 have been identified as interacting partners involved in the formation of junctions between these cellular organelles .2+ signalling in both organelles [Contacts between mitochondria and Golgi have been described in experimental papers utilising optical microscopy , 159. Inganelles .Mitochondria-lysosome contacts have also been described in mammalian cells , 171. InDirect contact between endosomes and mitochondria is utilised for the iron transfer from transferrin receptor-containing endosomes to the mitochondria , 149. InMitochondrial junctions with other cellular organelles are schematically illustrated on Fig. Early indications of connections between these two organelles have been published in the 1950s of the previous century . ConsideRecently, there was considerable progress in the characterisation of the molecular composition of the tethers linking ER with the outer mitochondrial membrane (OMM). In yeasts, a complex termed ERMES (ER-mitochondria encounter structure) has been identified by exceptionally elegant experiments combining expression of artificial tether linking ER with mitochondria and analysis of mutations in yeasts colonies. These experiments identified components of ERMES on the basis that mutations or deletions of these components results in growth deficiency that could be rescued by the expression of the artificial tether . The ide2+ transfer from the ER to mitochondria. Mitofusin 2 was suggested as the linker between the ER and mitochondria [ERMES complex/components are not retained in metazoans and other types of proteins are responsible for the formation of ER-mitochondria junctions in animal cells. Linkers formed by OMM protein VDAC1 (voltage-dependent anion channel 1), IP3R (located in the ER) and a molecular chaperone glucose-regulated protein 75 (grp75) was suggested by G. Szabadkai and colleagues from R. Rizzutto laboratory . The prechondria . This nochondria , 46) andchondria , 122. Rechondria ; oxysterchondria ; Bap31 tchondria  and ribochondria .Among the regulators of ER-mitochondrial junctions are ER-shaping proteins reticulons, which were identified as a result of ascorbate peroxidase proximity labelling . The abiMitochondrial fission requires the formation of specific contacts between mitochondrial membrane and the ER. Dynamin related protein 1 (Drp1) is essential for mitochondrial fission  and that it can be efficiently inhibited by Ruthenium Red (RuRed). The mitochondrial Ca2+ export system was characterised as Na+/Ca2+ exchanger, which can also transport Ca2+ when Na + is substituted by Li + and can be inhibited by CGP-37157. These crucially important early discoveries are reviewed in [2+ have also been conducted in the last three decades of the twentieth century. R. Denton’s group defined an important role of mitochondrial Ca2+ in the regulation of the Krebs cycle . At theiewed in ). Clear ed ..2+ signa2+ microdomains and the importance of the contacts between the ER and mitochondria for mitochondrial Ca2+ influx was emphasised by studies from the T. Pozzan laboratory [2+ [2+ entry and termed it mitochondrial calcium uniporter (MCU) [2+ import, MICU1 [2+-binding proteins [2+, which allows efficient Ca2+ entry into the mitochondria  cytosolic Ca2+ levels. This prevents futile Ca2+ cycle and the associated bioenergetics costs required to maintain acceptably low mitochondrial Ca2+ concentration. Such futile Ca2+ cycle and ATP expenditure were recently demonstrated in cells harbouring MICU1 mutation by G. Bhosale and colleagues from M. Duchen’s laboratory [2+ signalling and mitochondria. Importantly, it works in conjunction with Ca2+ signalling microdomains formed in the ER-mitochondrial junctions, which further increase the difference between bulk cytosolic Ca2+ rise and the Ca2+ rise in the proximity to the Ca2+-releasing channels and OMM region located in the junctional complex. Direct measurements of Ca2+ increases in the ER-Mitochondrial junctions have been conducted by G. Csordas and colleagues from the G. Hajnoczky laboratory by placing Ca2+ indicators into the junctions [2+ responses (> 9 μM) in the junctions  and the relative insensitivity of the junctional Ca2+ transients to slow Ca2+ buffering by EGTA [2+ signals in the junction and the rest of the cytosol enhances the signal-to-noise ratio for mitochondrial transfer of Ca2+ signals and facilitates this form of stimulus—metabolism coupling. The findings reported by G. Csordas and colleagues were consistent with results reported by M. Giacomello and colleagues who targeted Ca2+ indicator to the OMM and reported the appearance of Ca2+ hot spots where the Ca2+ concentration was found to be more than 5 times higher than that of the bulk cytosolic concentration [The role of Caboratory , 141. Thtory [2+ , 141. Thtory [2+ . In 2011er (MCU) . Approxit, MICU1 . This ret, MICU1  and EMREt, MICU1 . Both MIproteins , 134. Ania . There is now a sufficient body of evidence supporting the formation of SR-mitochondrial junctions and privileged local Ca2+ transfer from RyR into the mitochondria. Electron microscopy imaging revealed close contacts between mitochondrial and SR membranes (e.g. [2+ concentration hot-spots (> 20 μM) have been recorded on the OMM of cardiomyocytes [2+ increase following RyRs activation occurs in the presence of cytosolic calcium buffer in cardiac [2+ transfer by this mechanism is therefore important for stimulus-metabolism coupling in muscle cells ([RyRs form another group of intracellular Caes (e.g. ). High Cmyocytes . Mitocho cardiac , 155 and cardiac  muscle ce cells . MPTP is a high conductance mitochondrial channel permeable to molecules with molecular weight up to 1.5 kDa [2+ as permissive or initiating factor in physiological/pathophysiological settings involving MPTP is debated . Permisiewed in ) and neriewed in ), and th disease , whilst  disease . It is s2O2 nanodomains that were recently directly measured and reported by D. Booth and colleagues [2O2 sensor HyPer [2+-dependent redox nanodomains in the junctions between the organelles [2O2 transients potentiated ER Ca2+ release [ER-mitochondrial junctions are also sites of localised Hlleagues . In thisor HyPer  to the iganelles . Interes release . Redox r release  reviewed release ) and jun release ) and impThe mechanisms tethering mitochondria to the plasma membrane have been characterised in yeasts, where the Num1/Mdm36 anchors ER-mitochondria complex to the plasma membrane , 93, 1332+ indicators targeted to OMM and the cytosol, Giacomello and colleagues established that mitochondria adjacent to the plasma membrane did not show preferential Ca2+ uptake upon activation of store operated Ca2+ entry [2+ entry via SOCE was ineffective in producing Ca2+ hot spots on the OMM. Nevertheless, Ca2+ entry into the mitochondria was recorded in these experiments and the peak mitochondrial Ca2+ concentration was approximately one order of magnitude higher than in the cytosol [Using Ca2+ entry . Further cytosol . The abs cytosol .2+ entry and reduction in mitochondrial Ca2+ responses [2+ entry [2+ responses. This is achieved as a result of a local coordination of STIM/Orai channels, mitochondria and Ca2+ pumps of the plasma membrane. This study extends previous findings of the importance of mitochondria in the regulation of SOCE and its electrophysiological manifestation Ca2+ release-activated Ca2+ (CRAC) current (ICRAC)  . It is uffering . Notablyuffering . The aut2+ channels. This study reported that the subplasmalemmal mitochondria are exposed to higher Ca2+ concentrations and show stronger Ca2+ responses than mitochondria located in the deeper regions of the cytoplasm [2+ increases (hundreds of μM) in a subgroup of mitochondria upon activation of voltage-gated Ca2+ channels [2+ channels, RyR and mitochondria [Interestingly, the T.Pozzan group demonstrated privilege communication between mitochondria and voltage-gated Caytoplasm . Similarchannels . Intereschondria . A recenchondria . The str2+ channels but also Ca2+ pumps. This coordinated regulation is probably needed to ensure balance between Ca2+ signalling and Ca2+ homeostasis. The important role of subplasmalemmal mitochondrial group in the regulation of both SOCE- and PMCA-mediated Ca2+ fluxes was reported M. Frieden and colleagues [Subplasmalemmal mitochondria regulate not only Calleagues .2+ extrusion by PMCA and Ca2+ entry via STIM/Orai channels are redox sensitive processes . Bs . Development of techniques for selective labelling of junctional proteins and consequently identification of junctional proteomes should provide further impetus to this research area. It is likely that ER-mitochondrial junctions and mitochondria-PM junctions serve as important elements in stimulus-metabolism coupling and that this and other physiological functions of the junctional complexes will be actively investigated in the near future. Understanding the mechanisms involved in the formation and functioning of junctional complexes  will be beneficial for elucidating the pathophysiological implications of the disruption of these important transport/signalling platforms.


Toxoplasma gondii research have facilitated a broader understanding as well as a more integrated view of this complex cascade of events and have unraveled the importance of phosphatidic acid (PA) as a lipid mediator at multiple steps in this process.To elicit effective invasion and egress from infected cells, obligate intracellular parasites of the phylum Apicomplexa rely on the timely and spatially controlled exocytosis of specialized secretory organelles termed the micronemes. The effector molecules and signaling events underpinning this process are intricate; however, recent advances within the field of  T. gondii tachyzoite egress from infected cells via activation of microneme exocytosis and the actomyosin system. Extrinsic and intrinsic signals are likely to govern parasite egress from infected cells; however, the studies performed to date have implicated only specific extrinsic stimuli including low potassium (K+), low pH P2) to generate diacylglycerol (DAG) and inositol triphosphate (IP3) to produce PA and to mobilize calcium, respectively, to ultimately trigger microneme exocytosis [At any point during intracellular replication, deleterious environmental changes resulting in a loss of host-cell integrity can trigger , low pH , 2, and , low pH . During tection [,5P2) to Cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are important second messengers with diverse roles in eukaryotic cells. Within the Apicomplexa, cGMP is a vital upstream mediator of signaling events leading presumably to PI-PLC activation and subsequent microneme secretion. Protein kinase G (PKG) senses fluctuations in cGMP levels, and its association with the parasite plasma membrane by acylation is a necessary modification to function in controlling motility and invasion . PKG criT. gondii [Cyclic nucleotides are degraded by phosphodiesterases (PDEs), of which there are 18 putative genes in . gondii , 6 or th. gondii , 6. BIPPT. gondii cAMP-dependent protein kinase A catalytic subunit 1 (PKAc1), which is targeted to the parasite pellicle via its association with the dually acylated PKA regulatory subunit (PKAr) [2+ signaling upon host-cell invasion [Also involved in egress and invasion is t (PKAr) . Remarkat (PKAr) . The hosinvasion  and switinvasion  , a process previously reported to be sensitive to IP3 receptor inhibitors [3 receptors cannot be identified. Apicomplexan parasites possess several putative Ca2+ stores, including the acidocalcisomes, the mitochondrion, the inner membrane complex (IMC), and most relevantly, the endoplasmic reticulum (ER). Treatment of parasites with the sarcoplasmic/ER Ca2+-ATPase (SERCA) inhibitor thapsigargin has been shown to prevent reentry of Ca2+ in the ER and to stimulate microneme exocytosis, presumably by blocking the influx of leaked Ca2+ from the ER [2+ indicators has offered a powerful tool to visualize Ca2+ during egress and was exploited to identify compounds modulating Ca2+ signaling via a cell-based phenotypic screen for compounds that modulate Ca2+ signaling [2+ is proposed to be mobilized in response to abscisic acid (ABA) via the production of cyclic ADP (cADP) ribose [T. gondii possesses ADP ribosyl cyclase and hydrolase [Downstream of PKG, PKA, and PI-PLC activity is IPhibitors . Despitem the ER . The recignaling –15. Addi) ribose . Althougydrolase  and a pu2+ effector kinases  and instead utilize phylum-specific calcium-dependent protein kinases (CDPKs) that are activated by the direct binding of Ca2+ to their EF-hands. Among the 14 genes coding for T. gondii CDPKs, CDPK1 was shown to be critical for microneme secretion. CDPK3, the only other member implicated to date in egress is dispensable and plays a more specific role in parasite egress [The Apicomplexa lack typical Cae egress , 18, 19.e egress –22. Of re egress  , and PA phosphatases (PAPs) [T. gondii [T. gondii with the PAP inhibitor propranolol, implying that the ensuing buildup of PA facilitates exocytosis [APH gene resulted in a selective block in microneme secretion, leading to impairments in parasite motility, invasion, and egress [2+ and membrane curvature. In both T. gondii and Plasmodium falciparum, a conserved C2 domain–containing protein termed DOC2.1 has been shown to play a role in microneme secretion, as well as the associated events of egress, motility, and invasion [2+-dependent manner to prompt microneme–plasma membrane fusion [The production of IPs (PAPs) . In mamm. gondii . Moreoveocytosis . Althougocytosis . Conditid egress . APH is d egress , which id egress . SNARES invasion . Althouge fusion   and is also implicated in PA signaling events leading to egress [T. gondii [In addition to DGK1, o egress . PA prod. gondii . Remarka. gondii  Fig 1)..T. gondiMotility and host-cell entry and egress involve a parasite-derived molecular motor termed the glideosome, components of which are located between the parasite plasma membrane and IMC, as well as specific parasite surface adhesins. An armadillo repeat–containing protein identified as glideosome-associated connector (GAC) was found to be crucial for motility, invasion, and egress without impacting microneme secretion . GAC notMicroneme exocytosis is the culmination of a complex series of events that are slowly being unraveled. Further investigations into the roles of CDPKs, PI-PLC regulation, cyclic nucleotides, and integration of potassium, pH, and PA sensing will be required to give a global overview of how tachyzoites govern natural and induced egress as well as invasion to perpetuate their pathogenicity.


Acute myeloid leukemia (AML) is a myeloid malignancy characterized by the proliferation of abnormal and immature myeloid blasts in the bone marrow. Circular RNA (circRNA) is a novel class of long non-coding RNA with a stable circular conformation that regulates various biological processes. The aberrant expression of circRNA and its impact on AML progression has been reported by a number of studies. Despite recent advances in circRNA research, our understanding of the leukemogenic mechanism of circRNA remains very limited, and translating the current circRNA-related research into clinical practice is challenging. This review provides an update on the functional roles of and research progress on circRNAs in AML with an emphasis on mechanistic insights. The challenges and opportunities associated with circRNA-based diagonostic and therapeutic development in AML are also outlined. Acute myeloid leukemia (AML) is the most common leukemia in adults, presenting great biological and clinical heterogeneity . In 2018Investigations on the genetic and molecular mechanisms of cancer have recently shifted from protein-coding genes to non-coding transcripts . About 9This review compiles the roles of recently reported circRNAs that show altered expression in AML. Moreover, we also present the potential molecular mechanisms of certain circRNAs. Finally, we discuss the main challenges related to research on circRNA.The unique characteristic of circRNAs is associated with their production, which mostly occurs via the canonical and non-canonical splicing  of exonscis- and trans-acting factors (Based on their origin of production and their determination by RNA-sequencing (RNA-seq), the circRNAs are classified into four types: exonic circRNA (ecircRNA) , 7, circ factors , with th factors  10). So. Socis-  factors  8). Uni. Unicis- factors  ..cis- andThe most established function of circRNAs is through sponge formation with miRNAs and proteins . In the Although circRNAs contain an open reading frame (ORF), they generally lack the essential components necessary for translation, such as a poly(A) tail and a 7-methylguanosine cap. Nevertheless, mounting evidence suggests the translation ability of circRNAs  13). Fo. Fo13). Various molecular-based biomarkers  have been reported in AML and have been comprehensively reviewed . circRNAten miRNAs, miR-153-5p, miR-183-5p, miR-338-3p, miR-346, miR-545-3p, miR-574-5p, miR-599, miR-653-5p, miR-766-3p, and miR-767-3p. Functional analysis via the KEGG and GO databases predicted that the target genes of these miRNAs are associated with various cellular processes and signaling pathways involved in cancer  has also been associated with resistance to vincristine in AML , and knoin vitro proliferation of leukemic cells, blocked apoptosis, and promoted the formation of AML tumor in vivo. On the other hand, Morens et al. reported the deletion of the DLEU2 locus and embedded miRNA cluster miR-15a/16-1 in adult leukemia but its transcriptional repression due to hypermethylation in young AML patients  was highly expressed in AML samples . circ-DLpatients . The DLEpatients . These fRecently, Fan et al. reported that hsa_circ_100290, upregulated in AML, possesses oncogenic potential by enhancing cellular proliferation, inhibiting cell-cycle arrest, and suppressing cell apoptosis . They foin vitro and in vivo  encodes a multifunctional, highly conserved nuclear chaperone protein that is involved in ribosomal biogenesis, apoptosis, and cell proliferation . An insecircBase, developed by Glazar et al., collects and integrates data related to circRNAs from public references and hosts information from various species such as model organisms. The database not only provides free access to the user to browse and download gene annotations but also allows the user to obtain evidence supporting the expression details of a particular circRNA  functional networks  and its potential functional aspects . These Rzartinib  73). Ci. Ciin vizartinib   are also present in the nucleus. These circular isoforms interact with chromatin modifiers, thereby causing repression or activation of the gene , 78. EIcSome circRNAs, for instance, circ-ZEB1.19, circZEB-1.17, circZEB1.5, and circZEB1.33, by trapping miRNA-200, are involved in the suppression of lung cancer progression . The dowcircRNAs were initially considered non-coding RNA due to their disability to recruit ribosome , 86. HowOf note, the circRNA-miRNA-mRNA axis is a widely accepted mechanism in the establishment of leukemia. Circularization-based approaches can be used as a treatment strategy. Generally, a circRNA contains multiple miRNA binding sites (MRE). Therefore, targeting the inhibition of circRNA expression rather than a single miRNA/gene offers treatment advantages. Inhibiting circRNA expression will promote the protective effect of the corresponding miRNA in suppressing oncogenes, for instance, XIAP , Rab10 , PRACKB Advancements in Next-Generation Sequencing (NGS) technologies and the availability of user-friendly bioinformatics tools have facilitated circRNA biology-related research in cancer. These advancements have helped in the transformation of circRNA research from considering it a redundant splicing byproduct to exploring its possible potential targeted therapeutic application in AML. However, the field is in its infancy and faces enormous challenges  and raisIn conclusion, the role of circRNA in various cancers, including AML, is a major focus in cancer research. Although the detailed mechanisms of its biogenesis, transportation, function, and metabolism have not been fully studied, yet the glimpses provided by modern research suggest that circRNA as a potential candidate for AML diagnosis and therapy.MJ, QZ, and LS designed and conceived the study. TS, BC, ZH, and TX helped with figure design and literature selection and drafted the manuscript. MF, LS, YW, and SP revised the language. QY helped in manuscript formatting and language revision. All of the authors read and approved the final manuscript.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.


Unfortunately, the limited confining potential in semiconductor QWs causes formidable challenges in scaling such a scheme to the visible/near-infrared (NIR) frequencies for more vital nonlinear-optic applications. Here, we introduce a metal/dielectric heterostructured platform, i.e., TiN/Al2O3 epitaxial multilayers, to overcome that limitation. This platform has an extremely high χ(2) of approximately 1500 pm/V at NIR frequencies. By combining the aforementioned heterostructure with the large electric field enhancement afforded by a nanostructured metasurface, the power efficiency of second harmonic generation (SHG) achieved 10−4 at an incident pulse intensity of 10 GW/cm2, which is an improvement of several orders of magnitude compared to that of previous demonstrations from nonlinear surfaces at similar frequencies. The proposed quantum-engineered heterostructures enable efficient wave mixing at visible/NIR frequencies into ultracompact nonlinear optical devices.New materials that exhibit strong second-order optical nonlinearities at a desired operational frequency are of paramount importance for nonlinear optics. Giant second-order susceptibility  However, it is problematic to extend this mechanism into the visible/near-infrared (NIR) frequencies, where the majority of optical nonlinearities play crucial roles in optoelectronics and photonics8. Recent advances in ultrathin gold films introduce a possible solution for the creation of giant nonlinearities in a higher energy range of the spectrum due to the large depth of these QWs9. However, restricted by the accessible growth technologies, these ultrathin gold films cannot be used for sophisticated coupled QW heterostructures as epitaxial semiconductor wells.Large second-order optical nonlinearity in the infrared frequency range has been achieved by leveraging electronic intersubband transitions in semiconductor quantum well (QW) heterostructures11. These plasmonic materials have optical responses similar to those of gold while showing vastly enhanced chemical and thermal stabilities13, rendering them suitable for the study of optical nonlinearity. Among these materials, titanium nitride (TiN) can exist in a range of nonstoichiometric compositions and can be grown epitaxially on various transparent substrates such as sapphire15. Therefore, epitaxial heterostructures of TiN/Al2O3 have become excellent candidates for obtaining ultra-large χ(2) in the frequency range from visible to NIR spectra.In a different context, transition metal nitrides have recently received increased attention for their use in plasmonicsχ(2) of approximately 1500 pm/V in the NIR frequency range for a visible-frequency second harmonic generation (SHG) due to the electronic intersubband transition of asymmetric coupled metallic QWs (cMQWs) made of TiN/Al2O3 epitaxial multilayers. This value is more than one order of magnitude higher than that of traditional nonlinear crystals, and it is a few orders of magnitude higher than that of classic plasmonic metal structures16. The cMQWs were carefully designed to support three electronic subbands with equal energy spacing , such that χ(2) near the double transition frequency Ω is17Here, we report an extremely high ni, ε0, and e represent the electron density in the ith subband, the vacuum permittivity, and the electron charge, respectively. The dipole moment ezi,j and the decay rate Γi,j are associated with optically active subbands. Only the lowest of the three electronic subbands Ei−1 was below the Fermi level EF in order to increase the electron density of (ni−1 − ni) and thus χ(2). The product of the dipole moments e3zi−1,izi,i+1zi+1,i−1 is large in asymmetric QWs17, which also contributes to a large χ(2). In addition, a significantly increased χ(2) is exhibited in the cMQWs when the incident frequency ω is resonant with the double transition frequency Ω. Moreover, the peak χ(2) can be tuned to the visible/NIR spectrum due to the extreme depth of the metallic QW. Therefore, due to the large dipole moment and the high carrier concentration within a single QW, and the double resonant transition between equally spaced electronic bound states formed by the coupled QWs, we expect to observe a giant χ(2) in the visible/NIR range of the spectrum in these proposed cMQWs.where 2O3 dielectric barrier, as shown in Fig. 9, in order to produce an SHG output at the visible frequency of 460 nm (2Ω), the widths of these wells (1.0 and 2.2 nm) and the barrier (0.5 nm) were chosen to have a double transition frequency Ω at 920 nm. The TiN/Al2O3 cMQWs were fabricated via an epitaxial growth method using the reactive magnetron sputtering technique  . Therefore, an in-plane polarized pump light  is obliquely incident on the sample surface with a fixed incident angle of θ = 30° , can be converted to the pulse intensity. We clearly observe that the spectrum features a sharp peak centered at the SHG wavelength of 460 nm (2Ω). It is worth noting that SHG signals from the substrate (sapphire) and the thick TiN film (30 nm) are below the noise floor under the same experimental conditions. The measured dependence of the total (transmitted and reflected) SHG emission intensity on the incident power is shown in the right inset of Fig. 6.The equipment and apparatus used in the SHG measurement are schematically depicted in Fig. φ = 0°) to the in-plane (φ = 90ο), with a fixed incident angle of θ = 30°. The SHG signals exhibit a sharp peak localized at the collection angle of θ = 30° in the back-aperture plane. This demonstrates that the SHG signals are directly aligned with the reflection of the incident light (not a photoluminescence process), as illustrated in Fig. 2 peak intensity, SHG emission has a sin4(φ) dependence arising from the intersubband transition polarization selection in QW structures18, which states that SHG from an intersubband transition is proportional to the square of the out-of-plane polarization intensity Iz which scales as E2sin2(φ), where E is the incident electric field.In general, phase matching is a critical requirement for an SHG nonlinear optical process. However, because the propagation distance (less than 10 nm for the single cMQW unit) is significantly shorter than the SHG coherence length, the phase-matching condition is automatically satisfied in this cMQW system. Consequently, distinct visible-frequency SHG signals in both the reflection and transmission directions were clearly observed Fig. . To deteχ(2) for visible-frequency SHG enabled by cMQWs relies on the double resonant transition. Figure χ(2) (Supplementary Section 19. This χ(2) spectrum has a resonant peak centered at the double transition frequency Ω (920 nm) as expected in Fig. χ(2) is as high as 1500 pm/V, which is more than 20 times higher than that of traditional nonlinear crystals (75 pm/V for LiNbO3) and is several orders of magnitude higher than that of typical metal structures (~1 pm/V)16. Such a large χ(2) at NIR frequency shows that the cMQWs are excellent candidates for ultracompact nonlinear components.The giant 20, leading to ultracompact nonlinear optoelectronic devices21. Plasmonic enhancement could be combined with the ultra-large χ(2) for surface SHG at visible frequency by constructing a metasurface embedded with cMQWs. Here, the local electric fields are enhanced due to two plasmonic resonances at both the double transition frequency Ω (920 nm) and the SHG frequency 2Ω (460 nm). The absorption of photons at the fundamental frequency Ω in cMQWs will be enhanced by the first plasmonic resonance, while the radiative decay of the cMQWs at the SHG frequency 2Ω will be boosted by the other plasmonic resonance, leading to an efficient SHG. Such metasurfaces also give rise to efficient conversion of the impinging transverse electric field polarization into the desired z-direction17, enabling normal excitation of the incident beam to expand the usability of the extremely high nonlinear cMQWs.Plasmonically enabled electric field enhancements have recently been used in conjunction with micro- and nanostructures whose characteristic lengths are far below the light wavelength 2O3 (380 nm thick), and a 50-nm TiN layer. The array of monocrystalline silver nanocubes was self-assembled and then transferred on top of the epitaxial TiN/Al2O3 multilayer , and the results are summarized in Fig. Figure PSHG/PFF, where PSHG (PFF) is the average power at the SHG  wavelength; it reaches 10−4, which is more than four orders of magnitude higher than that of the previous demonstrations of plasmonically enhanced SHG at a visible/NIR wavelength28. Such a giant visible-frequency SHG power efficiency enables on-chip integration of these optical metasurfaces in ultracompact nonlinear devices.Figure χ(2) at the NIR frequency and demonstrated a giant visible-frequency SHG power efficiency in such a cMQW-embedded metasurface. Our material system can be readily integrated into more advanced configurations. For example, by tailoring the quantum states of the cMQWs, high χ(2) could be realized in a desired wavelength range; therefore, a broadband efficient SHG is possible by stacking cMQW ultrathin films with different double transition frequencies. Likewise, combinations of optical nonlinearities, e.g., both a high χ(2) and a large χ(3)9, would be conceivable in a single material system in a concise manner. Furthermore, a nearly isotropic high χ(2) may be achievable in material systems by combining cMQWs with high out-of-plane χ(2) and the emerging two-dimensional materials with large in-plane χ(2)29. Nanostructured materials with such giant SHG power efficiencies are expected to enable new on-chip surface nonlinear optical applications.We have shown a new cMQW material platform that exhibits an extremely high Supplementary information


We adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DN using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF. While MSC-derived DNs have been characterized for neuronal markers and electrophysiological properties, we investigated store-operated calcium entry (SOCE) mechanisms of these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP+). Overall, we show that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP+ exposure dysregulates calcium signaling, making them vulnerable to neurodegeneration. Since in-vitro differentiation of MSCs into DNs is an important vehicle for PD disease modeling and regenerative medicine, the results of this study may help with understanding of the pathological mechanisms underlying PD.Parkinson’s disease is a neurodegenerative disorder involving the progressive loss of dopaminergic neurons (DNs), with currently available therapeutics, such as L-Dopa, only able to relieve some symptoms. Stem cell replacement is an attractive therapeutic option for PD patients, and DNs derived by differentiating patient specific stem cells under defined  The prevalence rates are likely to increase as the population ages3, and by 2040, the annual cost to US society is expected to exceed $50 billion4. PD drastically affects quality of life as patients develop motor symptoms, such as bradykinesia, resting tremor, shuffling gait, rigidity, flexed posture, freezing, loss of postural reflexes, and non-motor symptoms, such as depression, lack of motivation, passivity and dementia.Parkinson’s disease (PD) involves a progressive loss of DNs, and is the second most common chronic neurodegenerative disease after Alzheimer’s disease. PD prevalence is age associated, increasing significantly in incidence above the age of 655, only a few percent of PD cases involve mutations in single genes, and 90% of PD patients have no family history of PD6. Epidemiologic studies have implicated numerous environmental risk factors in PD pathogenesis8. One such environmental risk factor was identified by Langston and colleagues as 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)9. MPTP is formed by the dehydration of an intermediate compound required for the synthesis of an active narcotic compound, 1-methyl-4-propionoxypyridine (MPPP). Admixed with MPPP, MPTP was sold as “synthetic heroin”, which resulted in PD symptoms and ER visits by intravenous drug users of San Jose, California in 198210. Now, MPTP is commonly used to recapitulate some element of PD pathology in cell-culture based and animal models of PD.The cause of majority of PD cases remains unknown. Although numerous genes and genetic loci have been implicated12. One of the most important signaling molecules, and a common denominator in these cellular dysfunctions, is intracellular calcium [Ca2+]i14. Ca2+ levels are tightly controlled throughout neurons, including within major organelles such as mitochondria and ER. Ca2+ regulates several cellular functions such as enzyme phosphorylation and dephsophorylation, cytoskeletal mediated motility, neurotransmitter release, transcription of numerous genes, and the process of programmed cell death. Ca2+ signaling depends on increased levels of intracellular Ca2+ derived either from outside the cell or from stores within the ER. Alterations in Ca2+ levels may occur due to excessive Ca2+ influx in the cytosol, overloading of mitochondria with Ca2+or depletion of ER Ca2+ stores.Pathologically, the hallmarks of PD are the death of DNs in the substantia nigra pars compacta, and the presence of cytoplasmic protein aggregates, known as Lewy bodies. The cellular dysfunctions underlying these pathological hallmarks include abnormal mitochondrial metabolism, oxidative stress, neuroinflammation and upregulation of unfolded protein response (UPR) in the endoplasmic reticulum (ER)2+ homeostasis. Several proteins regulate Ca2+ movement across the ER membrane, and includes IP3 receptors which serve to release Ca2+ from the ER in response to cell surface stimulation16. A plant toxin, called Thapsigargin, also releases the same pools of Ca2+, as does IP317. Upon ER Ca2+ store depletion, store-operated Ca2+ entry (SOCE) mechanisms in the plasma membrane are believed to be activated to replenish intracellular stores in ER. Influx through this pathway may provide direct Ca2+ signals to localized, spatially restricted target areas close to the sites of Ca2+ entry, thus initiating specific signaling pathways18 important for neurosecretion, sensation, long-term potentiation or depression of synaptic transmission, synaptic plasticity, gene regulation, neuronal growth and differentiation19. While the molecular identity of SOCE has not been conclusively identified, especially in neuronal cells, the transient receptor potential canonical (TRPC) channels, even if not direct components, interact with them21. These channels are important in maintaining ER Ca2+ levels, and movement of the ER away from its Ca2+ set point can compromise proteostasis, thereby triggering UPR, and ultimately neuronal death24.ER plays an important role in Ca26. In this regard, cell-based treatments to restore nigrostriatal neuronal function by transplanting cells with DA-producing capacity into the striatum holds great potential for PD. Several cellular sources have been investigated for the potential to act as renewable supply of transplantable dopaminergic neurons for PD, including fetal brain tissue, embryonic stem cells, induced pluripotent stem cells, neural stem cell precursors, and mesenchymal stem cells (MSCs)27.The current treatment strategies for PD rely mostly on pharmacologic replacement of dopamine (DA) with L-dopa, which provides some symptomatic relief, but does little to reverse or slow down the progression of the diseasein vitro and in vivo27 including DNs41. These reports of differentiation, along with those of MSC-mediated disease improvement in experimental animal models of PD42, have garnered much interest in the use of MSC-derived DNs as therapeutics to replace dying neurons in specific brain regions of PD patients. Several differentiation protocols have thus far been adapted, which have characterized the MSC-derived DNs to varying extents; however, a complete characterization of these neurons is still missing. Before the proposed stem-cell based therapy can effectively translate into the clinic, it is important to fully understand the function of MSC-derived DNs under physiological and pathological conditions. Towards this end, we investigated Ca2+ signaling, specifically SOCE mechanisms, TRPC1 expression, and the effect of MPP+, an environmental neurotoxin and a proposed risk factor for PD on SOCE and TRPC1 expression in these neurons.MSCs are multi-potent stem cells, with the ability to proliferate, symmetrically divide and produce multi-lineage mesodermal and non-mesodermal derivatives et al.’s protocol was adopted43, and is depicted in Fig. 2 incubator. Neuronal differentiation was initiated by replacing the MSC-culture media with neurobasal media supplemented with 0.5% B27 , 250 ng/ml SHH, 100 ng/ml FGF-8, and 50 ng/ml bFGF . The cells were maintained in the same media throughout the 12-day differentiation protocol. On day 9 of differentiation, 50 ng/ml BDNF was spiked into the differentiation media. Undifferentiated MSCs were maintained as negative controls, and SH-SY5Y cells  differentiated into neurons with the help of retinoic acid (10 µM for 7 days), were used as positive controls in the functional characterization experiments.Bone-marrow derived MSCs were obtained from Lonza Group Ltd. , and maintained in culture, as per manufacturer’s instructions. For dopaminergic neuronal differentiation of MSCs, Trzaska K. +  or corresponding dilution of dimethylsulfoxide (vehicle control) for times indicated in the results or figures. For calcium measurements, IP3 (10 µM) or Thapsigargin (1 or 2 µM) was added just before gathering the data. For non-specifically blocking the Transient Receptor Potential Cation (TRPC) channel, cells were pre-treated with 100 µM SKF96365 for 30 mins.At the end of 12-day differentiation protocol, cells were treated with 500 µM MPPTM, Life Technologies, Carlsbad, CA, USA), transferred to polyvinylidene difluoride (PVDF) membranes, and probed with respective antibodies. A 1:500 dilution of the primary antibody  was used to probe for Tyrosine Hydroxylase (TH) and Dopamine active transporter (DAT). The remaining antibodies were used at a 1:1000 dilution. Corresponding peroxidase-conjugated secondary antibodies , ECL reagent  and Quantity One Analysis software  were used to detect the protein bands.At the end of 12-day differentiation protocol, cells were harvested and stored at −80 °C, until use. Whole cell lysates were prepared in RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Total protein concentrations were determined using Bradford reagent , resolved on NuPAGE 4–12% Bis-Tris gels . For dopamine release, cells (differentiated or undifferentiated MSCs or those treated with SKF96365) were depolarized with 50 mM of K24. Differentiated MSCs, after treatment with MPP+ for 2, 4, 6 12, and 24 hours, were incubated with 2 µM Fura-2 , a calcium indicator, for 45 mins. at 37 °C, in a 5% CO2 incubator. After washing the cells with Ca2+ free buffer , the fluorescence intensity of Fura-2 loaded control cells was monitored with a CCD camera-based imaging system  mounted on an Olympus XL70 inverted microscope, equipped with an Olympus 40x (1.3 NA) objective lens. A monochrometer dual wavelength enabled alternative excitations at 340 and 380 nm, whereas the emission fluorescence was monitored at 510 nm with an Okra Imaging camera . The images of multiple cells, collected at each excitation wavelength, were processed using the C imaging, PCI software  to provide F340/F380 nm fluorescence ratios. The analog plots represent the intracellular ([Ca2+]i) values averaged from 30–40 cells, and are representative results from 3–4 separate experiments.The intracellular calcium concentrations were measured as previously described44. Coverslips with cells were transferred to the recording chamber and perfused with external Ringer’s solution . Whole cell currents were recorded in a tight-seal whole cell configuration at RT, using Axopatch 200B amplifier . The patch pipette had resistances between 3 and 5 MΏ after filling with the standard intracellular solution . Voltage ramps ranging from −100 mV to +100 mV, at a holding potential of 0 mV were delivered to the cells, for 100 ms, at 2 s intervals. Currents were recorded at 2 kHz and digitized at 5–8 kHz. pClamp 10.1 software  was used for data acquisition and analysis. Basal leak was subtracted from the final current measurements, and average currents are shown.Cell-attached patch clamp measurements were performed, as previously describedCells were grown on coverslips, washed twice with phosphate-buffered saline (PBS), and fixed for 30 min using 3% paraformaldehyde. The cells were permeabilized using cold methanol and blocked for 20 mins using 5% donkey serum. Cells were probed with the primary antibodies  at a 1:100 dilution for 1 h. After washing thrice with PBS + 0.5% bovine serum albumin, the cells were labeled with rhodamine or FITC conjugated anti-rabbit secondary antibodies  at a 1:100 dilution. Confocal images were collected using a MRC 1024 krypton/argon laser scanning confocal equipped with a Zeiss LSM 510 Meta photomicroscope .6 cells/well. The cultures were pretreated with 100 µM SKF96365 for 30 mins. Cell viability was determined using Vybrant® MTT cell proliferation assay kit , as previously described44. 30 µl of MTT reagent (0.5 mg/ml MTT in PBS containing 10 µM HEPES) was added to each well, and cells were incubated in a CO2 incubator for 2 h. The medium was aspirated from each well, and the culture plate was dried at 37 °C for 1 h. The resulting formazan dye was extracted with 100 µl of 0.04 N HCl in isopropanol, and the absorbance was measured in a micro plate reader  at 570 and 630 nm. Cell viability was expressed as a percentage of the control (untreated MSC-derived dopaminergic neurons).Cells were seeded in 96-well plates at a density of 0.5 × 10All results are expressed as mean ± S.E. Origin 7.0  was used to calculate statistics; differences between groups were tested with two-tailed, paired student’s t-test. In all cases, P ˂ 0.05 was considered statistically significant.Materials, data and associated protocols will be promptly made available to the editorial board and readers without undue qualifications in material transfer agreements as outlined in the Scientific Reports Editing and Publishing Policies.Ih currents which was induced by a hyperpolarizing pulse of 60 mV for 1.5 s duration (in voltage clamp)  using several published protocols, and found that the one reported in this paper Fig. , worked mp) Fig.  that wermp) Fig. , we coul2+ concentration is tightly regulated, and disturbances in Ca2+ homeostasis have been implicated in several neuropathological conditions20. Alterations in Ca2+ homeostasis may be caused by exposure to MPP+, which in turn contributes to loss of dopaminergic neurons via apoptosis21. We initially investigated the expression of various calcium entry channels in both control MSCs and 12 day post differentiation of MSCs. As shown in Fig. The neuronal Ca+ exposure can alter store-operated Ca2+ entry (SOCE) mechanisms in MSC-derived DNs. Addition of 2 µM Thapsigargin (Tg) in the absence of extracellular Ca2+ resulted in a slight increase in intracellular Ca2+ ([Ca2+]i) . Together these results suggest that extended treatment with MPP+ results in diminished SOCE, which in turn decreases ER derived cytosolic Ca2+ that would lead to ER stress followed by the loss of dopaminergic neurons).We next evaluated if MPPeak Fig.  in these3), for depleting the ER stores and initiating SOCE. Addition of IP3 in the patch pipet induced an inward current which was non-selective in nature, and reversed between 0 and −5 mV . Importantly, the current properties were not changed upon differentiation Fig. ; however mV Fig. . The inwtry Fig. , as comptry Fig. .While tr+-treated (500 µM MPP+ for 24 hours) MSC-derived dopaminergic neurons  (data not shown).The data presented above strongly implicated that the endogenous calcium channel properties were similar to that of TRPC1. This prompted us to investigate the expression of TRPC1 protein in MPPlls Fig. . Again, 2+ entry in dopamine release, and cell viability. For this purpose, we treated MSC-derived DNs with SKF 96365 to block the TRPC channel. As compared with untreated cells, SKF 96365 treated cells showed a significant decrease in dopamine release  as compared with untreated cells . Taken together, these findings suggest that TRPC1 mediates SOCE in MSC-derived dopaminergic neurons and decreased TRPC1 function, either by the addition of neurotoxin (MPP+) or TRPC channel blocker (SKF 96365) leads to decrease in dopamine release and survival of DNs.Next, we evaluated the role of TRPC mediated Caase Fig. . Since wlls Fig. . Similarin-vitro conditions are an attractive therapeutic option for PD patients. In this study, we adopted a previously published approach to differentiate Mesenchymal Stem Cells (MSCs) into DNs using a 12-day protocol involving FGF-2, bFGF, SHH ligand and BDNF43. While MSC-derived DNs have been previously characterized for neuronal markers and electrophysiological properties46, we investigated the store-operated calcium entry (SOCE) mechanisms in these DNs under normal conditions, and upon exposure to environmental neurotoxin, 1-methyl, 4-phenyl pyridinium ion (MPP+). Our data implicated that like native neurons, MSC-derived DNs utilized SOCE mechanisms, which potentially involved TRPC1. Moreover, similar to native neurons, addition of neurotoxin (MPP+) decreased SOCE and TRPC1 protein expression in MSC-derived DNs. Blocking of TRPC channels in these MSC-derived DNs resulted in decreased dopamine release and cell viability. Overall, our report shows that MSC-derived DNs are functional with regard to SOCE mechanisms, and MPP+ exposure dysregulates calcium signaling in these neurons, making them vulnerable to neurodegeneration.Dopaminergic Neurons (DNs) derived by differentiating stem cells under defined 48, embryonic stem cells50, neural precursor stem cells53, induced pluripotent stem cells54 and MSCs41. The use of MSCs for cell-based therapy is viewed upon as an attractive approach, since these cells can be (a) easily isolated from small aspirates of bone marrow, (b) easily expanded in culture with low tumorigenicity and teratoma formation, (c) display immunosuppressive properties that are advantageous for transplantation56, and (d) may allow autolgous transplantation under ideal settings27. Additionally, several studies demonstrated expression of dopaminergic neuronal markers by un-differentiated hMSCs59, thereby suggesting their neural pre-disposition, and provide support for their differentiation capacity to functional DNs, and for their use in cell-based therapy for PD. Lastly, MSCs injected into the brains of PD patients are likely to function not only by differentiating into DNs, but also by secreting cytokines and growth factors that may function in a paracrine manner to promote endogenous neuronal growth, decrease apoptosis, reduce levels of free radicals, encourage synaptic connection from damaged neurons and regulate inflammation61.Several cellular sources have been investigated for the potential to act as renewable supply of transplantable DNs for PD, including fetal brain tissuein-vitro setting has been attempted by several researchers64. The majority of these studies employed the addition of extrinsic differentiation inducing factors, such as cytokines, small molecules, gene transfection, conditioned medium, and co-culturing with cells such as astrocytes27. These studies have exhibited varying degrees of success involving generation of TH-positive neurons, physiological properties, dopamine secretion, and symptomatic improvement in rodent models of PD. Several earlier differentiation studies were limited in their potential for clinical translation, as they utilized gene transfection29, conditioned medium or co-culture systems with other cell types such as astrocytes28. Earlier differentiation protocols utilizing a combination of cytokines or small molecules weren’t efficient in terms of the % of cells expressing dopaminergic neuronal markers or lacked demonstration of electrophysiological properties which characterized neurons. However, the approach that we utilized for our study has been shown to result in ~70% cells expressing tyrosine hydroxylase (enzyme required for dopamine synthesis), and >90% of cells expressed neuronal markers, NeuN and beta-III-tubulin46. Additionally, these cells showed the presence of voltage-gated Ca2+ channels, exhibited inward spontaneous post-synaptic currents, and secreted dopamine in response to Ca2+ depolarization46. These findings indicated the presence of functional neuronal circuits in these neurons, similar to native neurons.Dopaminergic neuronal differentiation of rodent and human MSCs in an 65. Mitochondria are important cellular organelles as ATP producers and Ca2+ signal regulators. The environmental neurotoxin, MPTP, functions as an inhibitor of mitochondrial respiratory chain Complex 1, causing Parkinson-like symptoms. MPTP is converted to MPP+ by glial cells and taken up by DNs selectively through dopamine transporter (DAT), which is exclusively expressed in DNs12. Our data shows that unlike un-differentiated hMSCs, the differentiated hMSCs (DNs) express DAT. The fact that MSC-derived DNs are sensitive to the actions of MPP+, as demonstrated by reduced SOCE, TRPC1 and TH expression suggests that MSC-derived DNs not only express DAT, but are also capable of taking up MPP+. Thus, we propose that this MSC-derived dopaminergic neuronal differentiation model can be used to study mitochondrial homeostasis in the presence of MPP+.Mitochondrial dysfunction in DNs has been implicated as an important factor in the pathogenesis of sporadic and familial PD, and leads to apoptotic death of these neurons. This represents a potentially valuable therapeutic target in these patients+ was found to dysregulate Ca2+ homeostasis in our previous studies utilizing neuroblastoma cell line (SH-SY5Y cells)21. We demonstrated that endogenous SOCE, which is critical for maintaining ER Ca2+ levels , is dependent on TRPC1 activity. While the molecular identity of SOCE channels remains ambiguous, our study provided evidence that TRPC1, even if not direct component of SOCE channel, did regulate SOCE21 in these cells. TRPC1 mediated Ca2+ entry was found to be essential for maintaining ER Ca2+ stores, and consequent prevention of ER stress and unfolded protein response (UPR) is protective against neurodegeneration. Most importantly, this study demonstrated that in SH-SY5Y neuroblastoma cells, MPP+ treatment decreased TRPC1 expression, TRPC1 interaction with the SOCE modulator stromal interaction molecule 1 (STIM1), and Ca2+ entry into the cells. These results were instrumental in showing the mechanism involving SOCE and TRPC1 channel, by which a widely used model compound (MPP+) causes neurodegeneration by altering Ca2+ homeostasis and inducing ER stress in PD21. While the mechanism of MPP+ induced neurodegeneration was exciting, the study was limited in the fact that it utilized a neuroblastoma cell line originally derived from a malignant tumor66, which is far from mimicking native neurons.MPP+ induced Ca2+ dyshomeostasis in the physiologically relevant model MSC-derived DNs. Like in SH-SY5Y cells, MPP+ treatment decreased TRPC1 expression and Ca2+ entry into the cells in a time-dependent manner. The endogenous SOC showed I-V relationships that were similar to those observed for TRPC1-dependent currents45. TRPC blockage with an inhibitor compound decreased dopamine release and MSC-derived DN cell viability. These results reinforce our previous findings that Ca2+ homeostasis and ER Ca2+ levels, maintained with the help of SOCE, and potentially involving TRPC1, are neuroprotective. Exposure to MPP+ may cause neurodegeneration by altering SOCE and TRPC1 mediated Ca2+ homeostasis, which may induce ER stress, UPR and ultimately lead to neuronal death. These results underscore the fact that MSC-derived DNs characterized in the current report are similar to native neurons in expressing TRPC1 channels, SOCE mechanisms, and are sensitive to PD model compound MPP+.In the current report, we tested the MPP+, on SOCE mechanisms in these neurons. We have shown that these neurons express neuronal markers, electrophysiological properties, SOCE mechanisms, TRPC1 protein expression and are sensitive to MPP+, like native neurons. This broadens the prospect of utilizing this differentiation protocol for regenerative therapy in PD patients. By investigating the effect of MPP+ on MSC-derived DNs, we propose a mechanism by which MPP+ is possibly neurodegenerative, by dysregulating SOCE mediated Ca2+ homeostasis, which potentially involves TRPC1. This serves as an example of how MSC-differentiation to DN described here, may serve as a model for neuronal development and disease modeling for PD. Besides generating DNs similar to native neurons, the length of this model (12 days) is apt for analyzing genetic and epigenetic changes associated with dopaminergic neuronal differentiation, under physiological and PD relevant pathological conditions.In summary, our study provides the first comprehensive analysis of SOCE mechanisms in MSC-derived DNs, and the effect of an environmental neurotoxin, MPPDataset 1


Our data reveal that Ca2+ influx manipulation leaves the distributions of sEPSC amplitude and charge largely unchanged. Regardless the type of manipulation, the rate of sEPSC decreased with the reduction in Ca2+ influx. The fraction of compact sEPSCs was increased in the presence of BayK, an effect that was abolished when combined with decreased [Ca2+]e. In conclusion, we propose that UVR is the prevailing mode of exocytosis at cochlear IHCs of hearing rats, whereby the rate of exocytosis and the kinetics of SV fusion are regulated by Ca2+ influx.The early auditory pathway processes information at high rates and with utmost temporal fidelity. Consequently, the synapses in the auditory pathway are highly specialized to meet the extraordinary requirements on signal transmission. The calyceal synapses in the auditory brainstem feature more than a hundred active zones (AZs) with thousands of releasable synaptic vesicles (SVs). In contrast, the first auditory synapse, the afferent synapse of inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs), typically exhibits a single ribbon-type AZ tethering only tens of SVs resulting in a highly stochastic pattern of transmitter release. Spontaneous excitatory postsynaptic currents (sEPSCs), besides more conventional EPSCs with a single peak, fast rise and decay (compact), also include EPSCs with multiple peaks, variable rise and decay times (non-compact). The strong heterogeneity in size and shape of spontaneous EPSCs has led to the hypothesis of multivesicular release (MVR) that is more (compact) or less (non-compact) synchronized by coordination of release sites. Alternatively, univesicular release (UVR), potentially involving glutamate release through a flickering fusion pore for non-compact EPSCs, has been suggested to underlie IHC exocytosis. Here, we further investigated the mode of release by recording sEPSCs from SGNs of hearing rats while manipulating presynaptic IHC Ca Established by Figure 1). Sound encoding at this synapse is thought to rely on a single AZ to drive all spiking in a bipolar postsynaptic neuron -provides an impressive example . Testing potential mechanisms of coordinated MVR, this study did not find larger vesicles at the base of stimulated synapses as would be expected for compound exocytosis and still observed large EPSCs in the absence of extracellular Ca2+ (0 mM [Ca2+]e with 2 mM EGTA), arguing against the common Ca2+ nanodomain hypothesis of MVR. Interestingly, application of the dihydropyridine agonist, BayK, that promotes the bursting opening mode of L-type Ca2+ channels, increased the fraction of compact sEPSCs : 141.7 NaCl, 5.36 KCl, 10 HEPES, 0.5 MgSO2GTP, 3.5 MgCl2, 0.1 CaCl2, 5 EGTA, 5 HEPES, and 2.5 Na2ATP . The extracellular solution for recording contained (in mM): 5.8 KCl, 144 NaCl, 0.9 MgCl2, 1.3 CaCl2, 0.7 NaH2PO4, 10 D-glucose, and 10 HEPES . In the “0 Ca2+” experiment, no CaCl2 but instead 2 mM of the Ca2+ chelator EGTA was added to the extracellular solution (3 mM MgCl2 were included to compensate for the omission of Ca2+). All chemicals were purchased from Sigma-Aldrich , unless stated otherwise. In all recordings except for the measurement of the resting membrane potential, tetrodotoxin  was added to block voltage-gated Na+ channels. Five micromolars BayK (Tocris) and 1 μM isradipine (Tocris) were mixed with the extracellular solution prior to perfusion in the experiments of manipulating Ca2+ channels. In those experiments, after the control condition, extracellular solution with BayK or isradipine was perfused into the recording chamber through a separate perfusion line. The volume of perfusion line and recording chamber (2 ml) was 5–7 ml. Only those sEPSCs recorded after 10 ml solution flowed in were taken for analysis to ensure the chemical has been applied to the cells. An EPC-10 amplifier controlled by Patchmaster software  was used to sample and filter currents at 20–50 kHz and 5–10 kHz, respectively. sEPSCs were recorded at a holding potential of −90 mV  at room temperature (21–24°C).The postsynaptic boutons of type I SGNs innervating the IHCs were visually identified on a monitor using a 40×/63× water immersion objective lens attached to an upright microscope with differential interference contrast optics , and a 4×/2.5× magnification camera (TILL Photonics). Whole-cell voltage-clamp recordings from postsynaptic boutons of rat type I SGNs of the apical cochlear coil were performed largely as described in previous studies . The recs) was calculated from the height of capacitive transient in response to a 10 mV voltage step. The actual series resistance (Rs) was post hoc calculated because the aRs is affected by the filtering of amplifier. To obtain the actual amplitude of capacitive transient, we fitted the decay phase of capacitive transient with a double exponential, and extrapolated the fitting curve to time 0. The amplitude of fitting curve at 10 μs was taken for Rs calculation. The Rs is roughly 50% smaller than aRs. Average Rs values are summarized in Table 1. A recording was discarded if Rs > 100 MΩ or the aRs fluctuates more than 50 MΩ during recording.The apparent series resistance  and prepared for presentation using Adobe Illustrator. Averages were expressed as mean ± standard error of the mean (SEM). In order to compare two samples, data sets were tested for normal distribution (Jarque–Bera test) and equality of variances (F-test) followed by two-tailed paired Student’s t-test, or, when data were not normally distributed and/or variance was unequal between samples, the paired two-tailed Mann–Whitney-Wilcoxon test was used.For detection and analysis of EPSCs, Mini Analysis software (Synaptosoft) was used with a detection threshold set at 3–5 times greater than the root mean square (rms) of the baseline noise. To classify EPSCs into compact or non-compact EPSCs, the method described by  was emplFigure 1) and its regulation by Ca2+ at the rat IHC synapse after hearing onset by performing whole-cell patch-clamp recordings of sEPSCs from the postsynaptic SGNs at postnatal days (P) P17–P19, while manipulating Ca2+ influx in the unclamped IHCs. In some recordings, where the release frequency was too low to collect enough events for sufficient statistics, 10–15 mM [K+]e was applied to depolarize the IHC by 10–20 mV . Under the basal conditions  the membrane potential of IHCs measured in current-clamp was stable and more hyperpolarized (∼−74 mV) than that thought to occur in vivo  diminishing [Ca2+]e to 0 mM or (ii) reducing the Ca2+ channel opening probability with the dihydropyridine Ca2+ channel antagonist, isradipine.To evaluate if the observed sEPSCs could be further broken down to smaller units , we reduced the presynaptic Ca2+] was slowly replaced by nominally Ca2+-free extracellular solution with 2 mM EGTA added. Instead of starting the recording after the extracellular Ca2+ was completely removed for at least 5 min as done in 2+ washout. Due to the near complete absence of release upon total Ca2+ deprivation, analysis was performed on sEPSCs recorded during the perfusion of the Ca2+-free solution after the frequency had substantially dropped and before the sEPSCs completely stopped .In the first set of experiments, the extracellular solution containing 1.3 mM e . As reported previously  when lowering [Ca2+]e to 0.3 mM in the continued presence of BayK . At 0.3 mM [Ca2+]e, Ca2+ influx of IHCs at −50 mV was close to the one observed during the control before the perfusion with BayK . Therefore, this manipulation removed the effect of higher Ca2+ influx and allowed us to isolate the impact of the increased open probability. The amplitude and charge of compact sEPSCs were not affected regardless of the [Ca2+]e . This suggests that sEPSCs in the presence of BayK and without depolarization of IHCs result from UVR. Importantly, the proportion of compact sEPSCs was increased with BayK, but this increase was reversed when lowering [Ca2+]e . The results suggest that the shift from non-compact to compact sEPSCs requires an increase of the total Ca2+ entry (incoming Ca2+ charge) at the release sites.If sEPSCs recorded in the postsynaptic bouton of SGNs indeed reflect UVR at IHC AZs, compact and non-compact EPSCs likely represent two kinetic variants of SV fusion, for example, full-collapse fusion and fusion preceded by fusion pore flickering. To decipher the mechanism regulating the kinetics of SV fusion, we recorded sEPSCs under three subsequent conditions: (i) control, (ii) with 5 μM BayK, and (iii) with 5 μM BayK + 0.3 mM  at the release site favors rapid transition to irreversible fusion pore opening.The mode of SV release is a key property of synaptic transmission. Which release mode – coordinated MVR or UVR – prevails at the rat inner hair cell (IHC) ribbon synapse has remained an open question. Here we studied the effects of manipulating CaRecordings of postsynaptic currents from the IHC synapse reveal a striking heterogeneity in amplitude and shape of sEPSCs, which seemed at odds with the quantal hypothesis of neurotransmission by 2+ influx reduction, did not change the amplitude and charge distributions of compact sEPSCs , indicating that compact sEPSCs most likely reflect release from single SVs and argues that UVR prevails at the IHC synapse of hearing rats. Since the charge of compact and non-compact sEPSCs were not significantly different (Table 1), we suggest that non-compact sEPSCs, also reflect UVR likely involving release through a dynamic fusion pore.The rationale of the current study followed the general principle to characterize the minimal quantal size of a synapse by reducing release probability to elicit the minimum output causing the mEPSC. Assuming that the amount of neurotransmitter per SV follows a normal distribution and the postsynaptic receptors are not saturated (the relationship of the amount of neurotransmitter and EPSC amplitude is linear), the mEPSC amplitude distribution should be Gaussian with a small coefficient of variation  sEPSCs was influential for phrasing the concept of MVR at the IHC synapse and therein they were interpreted as less synchronized fusion of SVs . The abuThe canonical scheme of full-collapse fusion of SVs onto the cell membrane and the neurotransmitter being released completely at once has also been favored for retinal ribbon synapses . We favo2+ influx: abolishing Ca2+ influx at IHC synapses by omitting extracellular Ca2+ in mice and increasing Ca2+ influx by application of BayK in rats . However, this effect was reversed upon subsequently decreasing [Ca2+]e to 0.3 mM in the continued presence of BayK, which in separate experiments was shown to closely match the IHC Ca2+ influx to the pre-BayK level. A potential explanation within the framework of the UVR hypothesis is that the increased CaV1.3 channel open probability with BayK favors rapid transition to irreversible fusion pore opening due to higher time-averaged [Ca2+] at the release site. This interpretation, however, contrasts previous findings in chromaffin cells, where higher Ca2+ supported longer fusion pore openings  might determines the mode of fusion process. A longer duration of Ca2+ elevation at the release site would promote full-collapse fusion. Further experiments, such as paired recordings of IHC and post-synaptic bouton and cell-attached membrane capacitance measurements from IHCs, and computational modeling are required to further study the mechanism of exocytosis at the IHC synapse.The alternative interpretation that the gating mode of Caeraction  seems leThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.C-HH and TM designed the study. C-HH performed the patch-clamp measurements and analysis. C-HH and TM prepared the manuscript.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.