Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by a progressive paralysis due to the loss of particular neurons in our nervous system called motor neurons, that exert voluntary control of all our skeletal muscles. It is not entirely understood why motor neurons are particularly vulnerable in ALS, neither is it completely clear why certain groups of motor neurons, including those that regulate eye movement, are rather resilient to this disease. However, both vulnerability and resilience to ALS likely reflect cell intrinsic properties of different motor neuron subpopulations as well as non-cell autonomous events regulated by surrounding cell types. In this review we dissect the particular properties of different motor neuron types and their responses to disease that may underlie their respective vulnerabilities and resilience. Disease progression in ALS involves multiple cell types that are closely connected to motor neurons and we here also discuss their contributions to the differential vulnerability of motor neurons.Amyotrophic lateral sclerosis (ALS) is defined by the loss of upper motor neurons (MNs) that project from the cerebral cortex to the brain stem and spinal cord and of lower MNs in the brain stem and spinal cord which innervate skeletal muscles, leading to spasticity, muscle atrophy, and paralysis. ALS involves several disease stages, and multiple cell types show dysfunction and play important roles during distinct phases of disease initiation and progression, subsequently leading to selective MN loss. Why MNs are particularly vulnerable in this lethal disease is still not entirely clear. Neither is it fully understood why certain MNs are more resilient to degeneration in ALS than others. Brain stem MNs of cranial nerves III, IV, and VI, which innervate our eye muscles, are highly resistant and persist until the end-stage of the disease, enabling paralyzed patients to communicate through ocular tracking devices. MNs of the Onuf’s nucleus in the sacral spinal cord, that innervate sphincter muscles and control urogenital functions, are also spared throughout the disease. There is also a differential vulnerability among MNs that are intermingled throughout the spinal cord, that directly relate to their physiological properties. Here, fast-twitch fatigable (FF) MNs, which innervate type IIb muscle fibers, are affected early, before onset of clinical symptoms, while slow-twitch (S) MNs, that innervate type I muscle fibers, remain longer throughout the disease progression. The resilience of particular MN subpopulations has been attributed to intrinsic determinants and multiple studies have demonstrated their unique gene regulation and protein content in health and in response to disease. Identified factors within resilient MNs have been utilized to protect more vulnerable cells. Selective vulnerability may also, in part, be driven by non-cell autonomous processes and the unique surroundings and constantly changing environment close to particular MN groups. In this article, we review in detail the cell intrinsic properties of resilient and vulnerable MN groups, as well as multiple additional cell types involved in disease initiation and progression and explain how these may contribute to the selective MN resilience and vulnerability in ALS. The primary targets of amyotrophic lateral sclerosis (ALS) are corticospinal motor neurons in the motor cortex , and somatic MNs in the brainstem and spinal cord (termed “lower MNs”). The lower MNs control voluntary contraction of striated skeletal muscles and thus their progressive breakdown and the loss of synapses between MNs and muscle  in ALS is responsible for the irreversible paralysis of the body . Lethal (1)Alpha MNs (αMNs), which innervate extrafusal muscle fibers, are vulnerable in contrast to gamma MNs (γMNs), which innervate the muscle spindles in the same muscle group .(2)Specific lower MN groups are more resistant than others. In particular, oculomotor, trochlear, and abducens MNs are spared in ALS until the end-stage of the disease ,9 as wel(3)There are gradients of vulnerability among somatic αMNs that innervate one particular muscle, correlating with MN and muscle activity and innervated fiber types. Specifically, fast-twitch fatigable (FF) alpha MNs are more sensitive to degeneration than slow (S) MNs, as shown in ALS patients  and ALS In order to understand these differences in vulnerability and resilience, we explored the motor system and its particular dysfunction in the course of the disease and discussed the possible contributions of several non-neuronal cell types, including interneurons, astrocytes, oligodendrocytes, microglia, Schwann cells, muscle, and peripheral macrophages.The human motor system encompasses central nervous system structures in the cerebral cortex, brainstem, spinal cord, and cerebellum as well as nerves, skeletal muscles, and the specialized synapses between somatic MNs and muscles, the NMJs. Voluntary movement emerges from a cascade of neuronal activity across the components of the primary motor circuit; the planning and initiation of movement originates in the motor cortex by activation of the upper MNs, which are large pyramidal cells that are called Betz cells in primates. They project their axons through pyramidal tracts to lower MNs that execute motor functions. Here, the corticobulbar tracts connect to cranial nerve nuclei in the brainstem while the corticospinal tracts connect to MNs that are located in the ventral horn of the spinal cord . The lowmotor units. Their functional specialization has greatly diversified during vertebrate evolution. In the human body, there are an estimated 100,000–450,000 spinal and brain stem MNs that innervate the approximately 100–200 million muscle fibers organized in more than 300 paired muscles [Lower MNs and their target muscle fibers form functional entities called  muscles . Distinc muscles . The mot muscles ,25.Phox2b, similarly to cranial MNs [The ancestral medial motor column (MMC) is found in all vertebrates, spans the full spinal cord lengthwise, and innervates dorsal epaxial musculature (vertebrate extensor muscles). The ventral hypaxial musculature is innervated by the hypaxial motor column (HMC). At thoracic levels, the intercostal and thoraco-abdominal muscles belong to hypaxial musculature, and at postcrural level, prevertebral and the pelvic floor muscles. The diaphragm also arises from hypaxial muscles at the cervical level in the embryo but migrates caudally during development . The phrnial MNs ,30.hox gene activation [Hox genes regulate two aspects of the formation of the motor system. The colinear activation of sequential hox genes informs the position within the rostro-caudal body axis and columnar specification. Within each motor column, the hox gene expression further segregates it into individual motor pools [hox genes orchestrate both the motor neuron pool specification and the connectivity of these motor neurons through regulating expression levels of Ret (encoding a receptor tyrosine kinase for members of the GDNF family) and the Gfrα co-receptor (which GDNF binds to) [On the molecular level, the motor columns are distinguished by the expression of specific transcription factors during embryonic development ,31,32 antivation ,34. Hox or pools . In factinds to) .hox gene expression ensures faithful and proper connectivity of the motor units.Motor axons from several MNs intermingle in the spinal nerves as they grow and consolidate into nervous plexi that are exposed to the same guidance signals. At the base of the limb, motor axons group into muscle-specific fascicles, a step that is regulated by PSA-NCAM and required for correct targeting of muscles during development ,38,39 anLhx3 is initially expressed in all MN progenitors and adjacent V2 interneuron progenitors [Lhx3 is not required for the generation of MN progenitors but for their diversification into motor columns, and it becomes restricted to the MMC in mice at later stages [Foxp1 [Foxp1 is clearly pivotal to LMC identity as removal of this transcription factor causes loss of this motor column and the reversal to a more ancestral state where HMG MNs are generated, but not LMC or PGC MNs, followed by the expected alterations in axonal projection [The columnar identities are further determined by interaction with downstream transcription factors. genitors ,42. Lhx3r stages . LMC ands [Foxp1 . Foxp1 iojection .Somatic MNs are classified into alpha (α), gamma (γ), and beta (β) based on the innervation pattern in skeletal muscle fibers ,44,45 F. There aGfra1 than the other MN subtypes [+,-K+-ATPase) is a selective marker of small axon fibers that innervate skeletal muscle stretch receptors, which presumably are γMN axons [The differences between these three MN classes emerge late in development  ,52,53,54subtypes ,55,57. γsubtypes . ImmunohMN axons ,59.Tns1 as a novel and specific marker of somatic MNs in the spinal cord [+ and PRPH+ neurons [Zeb2 and Fbn2 are visceral MNs [Zeb2 demarks a visceral MN population [Sv2b and Stk32a were colocalized by in situ hybridization with Rbfox3 in large cholinergic ventral horn neurons that had C-boutons on their somas and VGLUT1+ synapses, strongly supporting that these are new markers for αMNs [Esrrg and Gfra1 [Transcriptomic characterization using single cell or single nuclei sequencing has corroborated the diversity of neurons in the developing and adult spinal cord ,61,62. Anal cord ,64. Its  neurons ,66 in theral MNs ,64, instpulation . Both stpulation ,64. Howepulation  as therepulation . Sv2b annd Gfra1 ,57. Howend Gfra1 ,64 that G93A, Prp::hTARDBPA315T, tON::hFUSP525L mouse models, which showed that γMN numbers remained unchanged at the end-stage of the disease when more than half of αMNs had been lost [A sensory fibers while γMNs lack this, similar to oculomotor MNs, which are also resilient in ALS [G93A mice with Egr3 knock out mice, in which muscle spindles degenerate and muscle spindle afferents are eliminated, was protective to αMNs. Likewise, the dampening of afferent sensory signal by selective elimination of half the γMNs in the SOD1G93A mouse, due to knock out of muscle spindle-derived GDNF on which γMNs depend, increased the survival of the mice [A proprioceptive inputs onto γMNs protects them from ALS [A proprioceptors is not generally required to elicit MN degeneration because some vulnerable αMN populations also lack this. For example, there is an early disruption of NMJs of MNs that innervate the cutaneous maximus muscle (a type II muscle)  in the SOD1G93A mouse [+ synapses [A sensory input could contribute to the difference between αMN and γMN sensitivity in ALS as it may exacerbate hyperexcitability of αMNs, but it does not appear to underlie the vulnerability of αMNs fundamentally.αMNs are vulnerable in ALS, while βMNs and γMNs appear resilient to this devastating disease. This has been demonstrated by careful quantifications in SOD1een lost . So why een lost . αMNs hat in ALS ,68. MNs t in ALS ,69,70, a3A mouse , althoug3A mouse . Likewissynapses , althougThe identity of αMNs in the motor unit is even further informed by the contractile properties of the skeletal muscle fibers they innervate; αMNs controlling either fast-twitch fatigable (FF) muscle fibers, or fast-twitch fatigue-resistant (FR) muscle fibers, or slow-twitch (S) muscle fibers form separate subtypes . TransduAmong spinal αMNs, there is also a gradient of vulnerability. The NMJs between FF MNs and muscle are denervated first before NMJs of FR and S MNs are affected. S MNs show the highest resistance among these spinal MN groups ,83,84 F. FF MNs Epha4 signaling successfully prolonged the life span of ALS mice [Eph signaling and stimulating growth factor signaling may be the way forward.S MNs can partially compensate for the denervation of NMJs on FF muscles by axon sprouting and reinnervation of vacated endplates before they themselves also succumb to the disease and retract their axons ,20,86. AALS mice . Axon spALS mice . CombiniNot all somatic lower MNs in the brain stem and spinal cord are vulnerable to degeneration in ALS. MNs belonging to three cranial nerves , oculomotor, trochlear, and abducens, are particularly resilient to degeneration in ALS ,9,11,12,Akt signaling and promote survival [G93A mice using adeno-associated viral (AAV) delivery protected MNs and their synapses with muscle and caused a regenerative response in motor axons and prolonged the life span of the mice by 10% [G93A mice by 14%. Remarkably, SYT13 also improved the survival of SMA mice by 40%. The specific function of SYT13 in MN protection across MN diseases is still under investigation, but it is evident that SYT13 protects both MN somas and axons from degeneration across ALS causations [Part of the signature of CNIII/IV MNs that we believe renders them resilient in ALS are their relatively high intrinsic levels of IGF1 and IGF2 ,94. CNIIsurvival . Delivere by 10% . Oculomoe by 10% . SYT13 pusations .Several studies have indicated that OMNs may have a reduced susceptibility to excitotoxicity which may be mediated partly through enhanced GABAergic transmission and high calcium buffering capacity ,90,92,94G93A mouse, while suppression of MMP9 delayed denervation. However, overexpression of MMP9 in resistant OMNs or slow MNs innervating the soleus muscle in the SOD1G93A mouse was insufficient to induce degeneration of these neurons [OMNs may not only be resistant by expressing a number of neuroprotective factors, but perhaps also by lacking a combination of degeneration-inducing molecules. Kaplan and colleagues showed that vulnerable FF MNs have higher levels of the metalloprotease MMP9 than OMNs and Onuf’s neurons . In this neurons . Thus, iMIF (macrophage migration inhibitory factor) [Are resilient neurons intrinsically similar in some way that explains their resilience in ALS or do they respond to disease in a way that clearly counteracts induced toxicity or both? To investigate this matter, we conducted RNA sequencing of MNs isolated from the oculomotor nucleus and the Onuf’s nucleus in human postmortem tissues. We then compared the genes that were differentially expressed in these resistant neurons compared to vulnerable spinal MNs. We found that the vast majority of differentially expressed genes were unique to each resistant motor nucleus, which indicates a resilience-signature that is unique to each nucleus. Nonetheless, a number of transcripts were common to both resilient MN groups and among these were  factor)  which ha factor) .It remains to be studied how oculomotor MNs respond dynamically to mutations that cause ALS and if such a response is unique and further explains their resilient nature. From neuropathological studies of TDP43 mislocalization and aggregation in ALS, we do know that oculomotor MNs show signs of pathology, while still remaining apparently intact.In fact, phosphorylated-TDP43 (pTDP43) inclusions can be seen in OMNs in ALS (50% of cases in one study) but also in normal aging . This coSYT13 levels remained high at the end-stage of the disease in both OMNs and in remaining relatively resilient spinal MNs [SYT13 was initially high in the spinal MNs that remained until end-stage, and presumably were of an S MN type, or if SYT13 was upregulated in a protective response to disease. Future single-cell analysis in tissues and in iPSC-derived MNs could resolve this matter. Secondly, we also identified relatively high levels of NDNF, (neuron-derived neurotrophic factor), NRSN1 (Neurensin), and PLPPR4 (phospholipid phosphatase-related protein type 4) across relatively resilient neurons at the end-stage of the disease compared to control, which may aid in neuroprotective responses [Gdf15, Lif, Chl1, Cplx2, and Aldh4a1, as well as regulation of motor proteins Kif5a and Kif3a, which could aid in axon transport [KIF5A is associated with ALS [GDF15 and LIF are trophic factors that appear to have important roles in maintaining MN survival and connectivity with muscle in vivo [We have so far analyzed the transcriptomic profiles of human OMNs in control and ALS at disease end point, which gave some important clues. Firstly, we found that inal MNs . It remaesponses . Towardsransport . Intereswith ALS ,101 and with ALS , highlig in vivo ,105,106. in vivo . This fu in vivo ,90,93,94A receptors [G93A mouse model [Interneurons form essential regulatory circuits in the central nervous system and, as such, modulate both upper and lower MN activity. ALS patients display cortical hyperexcitability that is thought to arise from an imbalance between excitatory cortical MNs and inhibitory cortical interneurons. The underlying mechanisms are not resolved, but ALS patients appear to have abnormal expression patterns of neurotransmitter receptors in the motor cortex, e.g., reduced level of GABAeceptors  and a geeceptors . Furthereceptors ,110. Evise model ,112,113.The selective vulnerability of different lower MN subtypes in ALS correlates with their function and intrinsic properties. However, they also integrate specific regulatory inputs from interneurons which, therefore, also correlate with the patterns of vulnerability in MN diseases . Some loA receptors that modulate their excitability and firing rates [D and V0S interneurons, V1 interneurons including Ia interneurons and Renshaw cells, as well as V2b interneurons [G93A mouse models starting as early as from day 60 before MNs are lost [Lower MNs in the brainstem and spinal cord express high levels of glycine and GABAng rates . Glycinerneurons ,121. Therneurons , likely rneurons . Moreoveare lost ,126,127.G93A mice [Chrna2 in Renshaw cells and Slc18a3 (Vacht) on the MN side [G93A mice that indicate pruning, possibly because of reinnervation attempts to remaining MN somas [V1 Renshaw cells are recurrently inhibitory interneurons on MNs that receive their input from motor axon collaterals in the spinal cord. They inhibit the same MN that activates them alongside proximal MNs and reduce their firing, allowing for higher pulse rates . Notably93A mice ,130. How MN side . At the MN somas . It is nMN somas . TherefoG93A mice [G93A disease progression. The loss of spinal glycinergic inputs on αMNs resulted in specific locomotor defects, including slowed down locomotion and reduced stride length. This locomotor phenotype agrees with the selective loss of V1 interneurons (marked by EN1) in ALS mice and is phenocopied in wild-type mice with reversible silencing of spinal EN1+ neurons, specifically [Allodi and colleagues uncovered MN subtype-specific alterations in the interneuron MN transmission in the spinal cord of SOD193A mice . They foifically . Similarifically .V1 interneurons form the majority of glycinergic interneurons in the spinal cord (ca. 80%) and they contribute substantial input to αMNs with 50% of near soma MN synapses. While the strong reduction of the V1 glycinergic inhibitory inputs to MNs could improve excitability at early stages , the lowAstrocytes are non-myelinating glial cells in the central nervous system that have a multitude of functions and an astounding number of processes that envelop neuronal synapses. In humans, a single astrocyte can interact with up to 2 million synapses at a time . Many asAstrocytes promote neuronal survival, induce functional synapse formation (irrespective of the age of the host the astrocytes originate from), and engulf synaptosomes. Astrocytes are heavily involved in spatially regulating extracellular potassium concentrations through the expression of a number of potassium ion channels, KIRs (potassium inward rectifying channels). Repolarization of neurons tend to raise potassium concentrations in the extracellular fluid . If the rise is significant, it can interfere with neuronal signaling by depolarizing neurons. Astrocytes remove potassium ions from the extracellular space and distribute the ions throughout their cytoplasm and further to their neighbors through gap junctions, keeping potassium at levels extracellularly that do not interfere with normal propagation of an action potential . AstrocyAstrocytes are clearly affected by ALS in multiple ways and take an active part in the disease. In transgenic SOD1 mice, astrocytes show early Lewy-body-like inclusions, immunoreactive for SOD1, prior to glial activation and prior to the accumulation of inclusions in neurons .Mislocalization of TDP43 is a common neuropathological finding across ALS causations, with implications for both toxic gain of function as well as loss of function. In addition to the mislocalization of TDP43 in neurons, astrocytes in ALS patient tissues show cytoplasmic TDP43 inclusions .G37R ALS mouse (using a GFAP-Cre mouse), astrocytes appear to drive late disease progression. Here, disease onset was unaffected but microglial activation was reduced (fewer MAC2+ iNOS+ microglia) and concomitantly with that, later disease progression was slowed down [As evidenced from animal studies where mutant SOD1 was removed specifically from astrocytes in the SOD1wed down . While awed down .G85R ALS mice phenocopy this feature of the human disease and show a clear downregulation of EAAT2 in spinal cord [G93A rats show a downregulation of EEAT2 in the ventral horn at symptomatic stages and a complete absence at end-stage [Astrocytes also play a critical role in regulating extracellular neurotransmitter levels, particularly important for excess glutamate which is sequestered through excitatory amino acid transporter 2 (EAAT2 or GLT1) on astrocytes. Loss of rat GLAST (EAAT1) or EAAT2 after injection of antisense oligos into lateral ventricles resulted in elevated extracellular glutamate levels and induced signs of neuropathology, including cytoplasmic vacuolization in neurons in the striatum and hippocampus . ALS patnal cord . Similarnd-stage . Thus, tnd-stage , but as Regarding regional specificity, it was early on shown that the shape and branching of midbrain dopamine (mDA) neurons can be influenced by coculturing these with astrocytes originating from different regions of the brain. When mDA neurons were grown on a monolayer of astrocytes from the striatum, they showed minimal extension of processes. However, when the mDA neurons were instead cultured on astrocytes derived from the mesencephalon, they extended a large number of highly branched processes ,149.+ channel, compared to astrocytes in outer CNS regions, including the dorsal horn of the spinal cord. Astrocytes abutting MMP9+ MNs, marking FF MNs, showed a higher level of KIR4.1 than those surrounding MMP9-negative MNs , and termed these AbA cells (aberrant astrocytes). These AbA cells had relatively high levels of S100B and connexin 43 (CX43) while lacking GLT1, compared to control embryonic astrocytes . Only 10% of primary MNs survived 48 h coculture with adult AbA cells from the symptomatic SOD1G93A rat, while 60% of MNs survived coculture with neonatal astrocytes from the SOD1G93A rat, as compared to 100% of MNs cultured on control neonatal astrocytes. Conditioned media from AbA cells gave the same result in MN death, and dilution of the media resulted in greater MN survival, indicating that AbA cells secreted factors that are directly toxic to MNs rather than being less supportive to MNs. The toxic effect of the conditioned media appeared selective to MNs as hippocampal neurons were not affected [G93A astrocytes in an effort to better understand MN toxicity, selectivity, and resilience and vulnerability.Researchers have identified astrocytes with an aberrant phenotype in SOD1affected . This stMicroglia are the resident macrophages of the central nervous system (CNS) and the most abundant glial cell of the CNS, making up 10–15% of all cells found in the brain. Microglia originate from yolk sac primitive macrophages and populate the CNS during early embryonic development and persist throughout adulthood. This early colonization of the embryonic CNS by microglia is conserved across vertebrate species, which implies that it is essential for early brain development. It has indeed been shown that microglia are pertinent for the wiring of circuitry in the forebrain , and in E. coli showed that cerebellar microglia were able to better control the net replication of bacteria than cortical microglia [Microglia are rather uniformly distributed in the brain and spinal cord but show increased densities in neuronal nuclei, including, for example, the substantia nigra pars compacta. It was recently shown that there is significant heterogeneity in microglia across brain regions. Specifically, transcriptional profiling of microglia isolated from the cerebellum, cortex, hippocampus, and striatum of the adult brain revealed their heterogeneity and networks that set them apart were energy metabolism, immune response and regulation, oxidative stress, cell death/apoptosis, and endocytosis/vesicle transport . The proicroglia .G93A ALS mouse microglia have a defined profile that is distinct from that of other types of activated microglia, for example, after LPS stimulation, and show upregulation of both toxic factors and potentially neurotrophic factors, e.g., IGF1 [Microglia become activated early on in ALS, at or before disease onset in mutant SOD1 mice and with the number of cells increasing with progression ,167 Fig. ActivatG37R was removed from microglia and macrophages in transgenic ALS mice, through crossing with a Cd11b-Cre mouse line, ALS disease progression was delayed significantly, while onset was unchanged, thus demonstrating that these cells are important for disease progression [G93A mouse model with wild-type myeloid progenitors delayed disease progression [When mutant SOD1gression . Similargression .G93A mouse [G93A microglia  isolated from either brains or spinal cords of transgenic mice induced degeneration of 50% of MNs within three days. Reduction of mSOD1G93A in adult microglia with 75% through shRNA left MNs unharmed, showing that the toxicity of the microglia was related to the prolonged overexpression of mutant SOD1. Suppression of NF-κB signaling in the SOD1G93A microglia normalized the production of TNFα and nitric oxide (NO) and prevented microglia-induced MN death and axonal damage in vitro [It has been suggested that microglia induce MN death via the NF-κB pathway, which is highly activated in microglia already from disease onset in the SOD13A mouse . Cocultuin vitro .+ MNs in vitro to degenerate within 72 h of coculture, thus indicating that mutant SOD1G93A microglia become toxic due to NF-κB activation. Knockdown of NF-κB signaling in microglia in vivo by Cre-mediated heterozygous removal of IKKβ, using the CSF1R promoter  did not affect onset of disease but extended the disease progression phase by 47%. When animals were analyzed at the end-stage, there was a similar level of gliosis in the CNS of all mice, but if animals were age-matched and CSF1R-Cre mice were thus analyzed at the time of the Cre– littermate end point, astrogliosis and microgliosis was decreased by 30% and 25%, respectively. This indicates that NF-κB signaling drives part of the toxicity of microglia and is one of the drivers of the disease progression phase.Activation of NF-κB in wild-type microglia caused 50% of the Hb9::GFPIt would have been interesting to conduct single-cell profiling of all MNs prior to initiating the coculture to investigate if there were particular MN subpopulations present with potential selective resilience to degeneration (S versus FR and FF MNs) as 50% of the cells remained after 72 h of coculture with microglia.It would also have been enticing to conduct longitudinal analysis of the transcriptional dynamics of individual MNs during their coculture with microglia to reveal if particular MNs can withstand the toxicity from microglia due to modulation of cell intrinsic signaling. TNFα can be both toxic and neuroprotective depending on the status of the neuron and NF-κB transcriptional activation can stimulate expression of prosurvival genes that will block apoptosis such as BCL2 and cellular inhibitors of apoptosis proteins. Perhaps it is a lack of TNF receptors 1 and 2 on particular MNs which underlies resilience, or it is mainly a maturity issue and lack of downstream signaling of NF-κB, but this remains to be investigated.G93A mutation and slowly progressing mice harboring the SOD1G37R mutation [SOD1 or C9ORF72 mutations [Immune activation occurs early on in ALS in the peripheral nervous system, with marked infiltration of peripheral macrophages along motor nerves, as shown in mutant SOD1 ALS mice, both fast progressing mice harboring the SOD1mutation ,173. Thiutations . MacrophG93A mouse model that replacing peripheral macrophages with wild-type macrophages had no effect on disease severity. However, replacing them with macrophages in which either wild-type SOD1 was overexpressed or where Nox2 had been knocked down, which in both cases would result in reduced neurotoxic reactive oxygen species, attenuated both peripheral and central pathology in the mice, without any major recruitment of the peripheral macrophages into the CNS, and improved the survival of the mice [Chiot and colleagues elegantly demonstrated in the SOD1the mice . The desOligodendrocytes are the myelinating glial cells of the CNS. Oligodendrocytes also provide energy in the form of lactate to axons through monocarboxylate transporters (MCTs). It has been shown that MCT1 (SLC16A1) is highly expressed in oligodendrocytes .+/− mice developed axonopathy without disturbing the myelination of axons [G93A mice as a function of disease and in ALS patients [G93A mice using an AAV9 vector did not improve MN loss or the survival of the mice [MCT1 is also present on MNs and other neuron types in the CNS, as well as on sensory neurons in the dorsal root ganglia, as evidenced by in situ hybridization experiments in the Allen Brain Atlas . General knock down of MCT1 in vitro and in vivo reduced MN survival. It would have been interesting to perform transcriptional analysis of individual MNs prior to and after MCT1 reduction to see the individual responses as only a portion of MNs were lost. Moreover, it is not clear if these were FF or S or both. Furthermore, MCT1of axons . Thus, fpatients ,175. It C9ORF72 mutant, sporadic ALS, as well as SOD1 and TARDBP mutant oligodendrocytes all resulted in reduced MN survival and so did conditioned media from all lines, indicating that it was secreted factors that mediated the effect. All oligo lines, except C9ORF72 mutants, showed reduced lactate levels in the media and conditioned-media-induced MN death was associated with decreased lactate production. Thus, there appears to be mutation-specific modulations in oligodendrocytes. Notably, toxicity in coculture with oligodendrocytes appeared to be lactate-independent and is likely mainly mediated by cell–cell contact [Ferraiuolo and colleagues showed that mouse MNs cocultured with ALS-oligodendrocytes derived from either mSOD1 mice or ALS patients had lower survival than MNs cocultured onto control oligodendrocytes . C9ORF72 contact .G93A mice in combination with an enhanced generation of NG2+ cells and oligodendrocytes. The newly generated oligodendrocytes failed to remyelinate axons, reminiscent of multiple sclerosis [G37R from NG2+ cells prolonged the life span of the mice by delaying the onset and early progression of the disease.Not only neurons are targets in ALS. In fact, there appears to be an increased degeneration of oligodendrocytes in the spinal cord of SOD1clerosis . Oligodeclerosis . ExcisioInterestingly, MNs are also degenerating in multiple sclerosis , linkingThere is yet no data linking oligodendrocyte heterogeneity and selective vulnerability in ALS, but there is a diversity among mature oligodendrocytes . It was + GAP43+ precursors (at E14 in mouse) into immature Schwann cells marked by expression of SOX10, p75, S100, EGR2 (KROX20), and OCT6 (at E17 in mouse). They then separate into the myelin generating lineage that ensheath somatic motor nerves or the non-myelinating lineage of the somatosensory and autonomic nerves, see review [Schwann cells are the primary glial cells of the peripheral nerves and encompass both myelin generating and non-myelinating cells. They share a developmental origin in neural crest and progress from SOX10e review . The myeG93A protein in MPZ+ Schwann cells, specifically, yielded mice identical to controls with no changes in locomotion, neuronal loss, or axonal degeneration [The biology of Schwann cells is tied to MNs and vice versa. Not only is their interaction crucial for their mutual development and survival, neuron-derived factors guide the differentiation of Schwann cells along the axon  while Scneration . This miG37R protein, that retains its dismutase activity, within Schwann cells (myelinating and non-myelinating) did not alter disease onset, but surprisingly exacerbated disease progression. This would indicate that a high level of dismutase activity in Schwann cells is protective in ALS, as has been shown for wild-type SOD1 in injury [G85R, which is lacking dismutase activity, from Schwann cells delayed disease onset and slowed progression slightly [G37R was depleted from Schwann cells, there was a concomitant 50% decrease in the neurotrophic factor IGF1 (insulin-like growth factor I) in sciatic nerve Schwann cells [Igf signaling in the SOD1G85R-depleted nerve.However, Schwann cells may play a role in the disease that is more subtle. The selective reduction (with 70%) of mutant SOD1n injury ,190,191,n injury . If thisslightly . So, whann cells . The autnn cells . It remaInterestingly, sensory neurons signal peripheral nerve injury through induced nitric oxide and upregulate erythropoietin receptors. Schwann cells sense the nitric oxide through the hypoxia inducible factor (HIF1) and secrete neurotrophic erythropoietin (EPO) that activates axon-protective pathways in the neurons . Future Sema3a is an axonal guidance factor which is specifically increased in the perisynaptic Schwann cells of fast-fatigable muscle fibers in SODG93A mice [Sema3a could bias plasticity during regenerative innervation of vacated NMJs [G93A mice and, thus, might be involved in the initial retraction of the motor axons at these terminals. The implication of such a repellant axon guidance factor in the disease also supports the notion that miscommunication between vulnerable neurons and perisynaptic Schwann cells in muscle might contribute to the denervation of the NMJ and selective vulnerability.Recently, a distinct non-myelinating perisynaptic Schwann cell was described to participate in the NMJ, reviewed in . Perisyn93A mice . This inted NMJs . FurtherSomatic MNs that are vulnerable to ALS control the skeletal muscles in the body. These are striated muscles that connect to bones via tendons and move the skeleton by their contraction. The striated muscles can be voluntarily controlled in contrast to the smooth and cardiac muscles that are innervated by visceral MNs instead of somatic ones. The selective vulnerability of the motor units occurs at different levels in ALS and is reflected on the side of the muscle as well the MN . For exaG93A mice. Already by P30, approximately 40% of NMJs in fast-twitch muscles are denervated at a presymptomatic timepoint, whereas MN somas show only a 20% reduction one month later, by P60 [G93A mice occurs even in MNs that offset apoptosis by a deletion of BAX [MN death is strongly correlated with the decline in motor function in SOD1 mouse models ,200. Neu, by P60 ,201, and, by P60 ,202. Then of BAX ,203. We n of BAX ,204, we Both sporadic and familial ALS cases present skeletal muscle pathology that includes signs of mitochondrial dysfunction ,207,208.G93A mice is preceded by an increase of proinflammatory cytokines and autophagy markers. Autophagy contributes to decreased myofiber size initially, and caspase activity contributes to atrophy only at late stages following denervation [G93A mice [G93A mice, but it does not extend life span [G93A expression in skeletal muscle specifically to understand the role of muscle in ALS. The selective expression of SOD1G93A in skeletal muscle, under the myosin light chain promoter, elicited progressive muscular atrophy, muscular weakness, and mitochondrial dysfunction, but did not trigger degeneration of spinal MNs [G37R mice. Here, the suppression of mutant SOD1 was estimated to be about 50% [G93A mice clearly shows that pathology starts presynaptically and is subsequently followed by pathology on the postsynaptic muscle side [G93A mice [The particular molecular changes leading up to muscular atrophy have been described and include an increase in reactive oxygen species , but alservation ,217. Fur93A mice . PGC1α i93A mice ,220. Thuife span . There iife span  and a hiife span . A numbeinal MNs . Furtherbout 50% . Such a bout 50% , and it cle side . These r93A mice ,225,226.93A mice . This de93A mice . This sh93A mice . HoweverPrimary myoblasts isolated from ALS patients have a reduced ability to form myofibers and display fusion defects . These pG93A mice showed that such markers were upregulated already at 4 weeks of age and even stronger at 8 weeks, a time point when NMJ disruption has started to initiate in these mice and prior to when astrocyte and microglia activation occur. Perivascular markers were also increased in TARDBPQ331K mice at relatively early disease stages. This would indicate that the vasculature is an early pathological target in ALS, preceding inflammation. Analysis of a large number of plasma samples from sporadic ALS patients with recent ALS diagnosis showed a correlation between elevated levels of the protein marker SPP1 for perivascular fibroblasts and a more aggressive disease progression and thus shorter survival from onset of disease, which indicates a connection between the vasculature and sporadic ALS [Blood–brain barrier disruption has been associated with multiple neurodegenerative diseases including ALS, reviewed in . Severaladic ALS .However, a recent study that tried to connect blood–brain barrier disruption and disease severity using the ratio of CSF to serum albumin levels as a marker failed to associate disease aggressiveness with disruption of the blood–brain barrier except for in patients with limb-onset . FurtherThe progressive dysfunction and loss of motor neurons in ALS results in the decay of motor function. This degeneration involves both motor neuron intrinsic changes and several other cell types across the motor system, from the motor cortex to the neuromuscular junctions . The difALS, like many human neurodegenerative diseases, is difficult to interrogate mechanistically and we are limited by insight into the human nervous system. Our conclusions rest, therefore, on studies in suitable model systems that are validated or corroborated in humans wherever possible. Several mouse models based on other ALS-causative genes have been created in the last decades reviewed in ,241,242 Given the multiple sites of pathology in the body during the course of ALS and the many cell types involved, it is an important challenge to establish timelines of the cellular and molecular changes and causal links between alterations where possible.


Mitochondria take part in a network of intracellular processes that regulate homeostasis. Defects in mitochondrial function are key pathophysiological changes during AKI. Although Wnt/β-catenin signaling mediates mitochondrial dysfunction in chronic kidney fibrosis, little is known of the influence of β-catenin on mitochondrial function in AKI. To decipher this interaction, we generated an inducible mouse model of tubule-specific β-catenin overexpression (TubCat), and a model of tubule-specific β-catenin depletion (TubcatKO), and induced septic AKI in these mice with lipopolysaccharide (LPS) and aseptic AKI with bilateral ischemia-reperfusion. In both AKI models, tubular β-catenin stabilization in TubCat animals significantly reduced BUN/serum creatinine, tubular damage (NGAL-positive tubules), apoptosis (TUNEL-positive cells) and necroptosis (phosphorylation of MLKL and RIP3) through activating AKT phosphorylation and p53 suppression; enhanced mitochondrial biogenesis (increased PGC-1α and NRF1) and restored mitochondrial mass (increased TIM23) to re-establish mitochondrial homeostasis  through the FOXO3/PGC-1α signaling cascade. Conversely, kidney function loss and histological damage, tubular cell death, and mitochondrial dysfunction were all aggravated in TubCatKO mice. Mechanistically, β-catenin transfection maintained mitochondrial mass and activated PGC-1α via FOXO3 in LPS-exposed HK-2 cells. Collectively, these findings provide evidence that tubular β-catenin mitigates cell death and restores mitochondrial homeostasis in AKI through the common mechanisms associated with activation of AKT/p53 and FOXO3/PGC-1α signaling pathways. Kidney tubular dysfunction and cell death contribute heavily to the pathophysiology of acute kidney injury (AKI). Catenin beta-1, also known as β-catenin, is a crucial signal transducer that regulates cell adhesion and maintains kidney cell homeostasis. Its activity remains largely quiescent in the adult kidney where in the absence of Wnt stimulation, β-catenin is degraded by ubiquitination-dependent proteolysis in the proteosome after its phosphorylation through the assembly of the destruction complex consisting of tumor suppressor proteins adenomatous polyposis coli (APC), dishevelled (DVL) protein, and two serine-threonine kinases, casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β). β-catenin becomes reactivated in pathological states such as glomerulonephritis, diabetic kidney disease and lupus nephritis . In the Mitochondrial dysfunction orchestrates key pathophysiological changes during AKI in which mitochondrial biogenesis, mitochondrial dynamics and mitophagy were altered –20. MitoIn this study, we adopt a strategy of loss- and gain-of-β-catenin function by generating two strains of transgenic mice with tubule-specific β-catenin deletion or stabilization (through exon 3 deletion) to delineate the role of tubular β-catenin and the mechanistic pathways associated with mitochondrial biogenesis and cell death in murine septic and aseptic AKI.KspCreERT2; Catnblox(ex3)/wt)) was previously generated in our laboratory [KspCreERT2 allele served as control /wt). The homozygous β-catenin-floxed (exon 2-exon 6) mouse was generated as previously described [KspCreERT2 mice, renal tubular epithelial cell-specific conditional knockout mice  were obtained. Their littermates without the KspCreERT2 allele served as the corresponding control . Mouse ear punch samples were used for genotyping by KAPA2G Fast HS Genotyping Mix kit  according to the manufacturer’s instructions. The genotyping primer sequences were listed in Supplemental Table Cre recombination.All animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research  and followed strictly the National Institute of Health Guide for the Care and Use of Laboratory Animals. A tubular cell-specific β-catenin stabilization mouse .(1) Ischemia-reperfusion injury (IRI)-induced AKI was established as previously described with modifications . After aTM Urea Assay Kit  according to the manufacturer’s instructions.Serum creatinine (sCr) and blood urea nitrogen (BUN) were determined by Stanbio Direct Creatinine LiquiColor Procedure Kit  and QuantiChromParaffin-embedded mouse kidney sections (4-μm thickness) were prepared for Periodic acid Schiff (PAS) staining . Percentages of intact, moderately, and severely damaged renal tubules were quantified on ten non-overlapping high-power cortical fields of PAS-stained sections by three blinded examiners as previously described  using th3 blocks and fixed in 2.5% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate-HCl buffer pH 7.4) at 4 °C overnight and cut into 100 nm ultra-thin sections before staining with 2% aqueous uranyl acetate . Digital images were obtained with a Philips CM 100 transmission electron microscope  at an operating voltage of 100 kV.Mouse kidney samples were freshly collected in 1 mmΤerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay  was used to detect apoptosis following the manufacturer’s procedure.Paraffin-embedded mouse kidney sections (4-μm thickness) and paraformaldehyde-fixed HK-2 cells were used for immunofluorescence staining, and reagents were obtained from Abcam. Briefly, kidney paraffin-sections were dewaxed and rehydrated. Citrate buffer (pH 6.0) was used for antigen retrieval. After treating with hydrogen peroxide blocking reagent for 10 min, slides were blocked with mouse-on-mouse blocking solution followed by 5% goat serum for 30 min. Sections were incubated with monoclonal anti-mouse β-catenin  plus rabbit anti-mouse PGC-1α  overnight, followed by incubation with AlexaFluor 488 goat anti-rabbit plus AlexaFluor 594 goat anti-mouse secondary antibodies . For immunofluorescent staining on tubular epithelial cells, HK-2 cells were cultured on LabTek chamberslides (Thermo Fisher Scientific). After pretreatment with or without LPS treatment for 30 minutes, cells were fixed by 4% paraformaldehyde for 15 min, following by 2% BSA blocking for 30 min. Subsequently, cells were incubated with anti-mouse β-catenin monoclonal antibody  plus rabbit anti-mouse FOXO3 antibody  overnight, followed by incubation with AlexaFluor 488 goat anti-mouse plus AlexaFluor 594 goat anti-rabbit secondary antibodies (Thermo Fisher Scientific) for 1 h. Both kidney sections and fixed cells were co-stained with DAPI for 10 min, then mounted with antifade mounting medium . Fluorescence images were visualized and captured under a fluorescence microscope .Nuclear fractions were isolated from kidney cortical tissue and HK-2 cells by NE-PER kit . Protein concentrations of nuclear fractions were determined by BCA Protein Assay Kit (Pierce).TM supplement medium  containing 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) in an incubator at 37 °C with 5% CO2. HK-2 cells were treated with 100 ng/µl LPS for various time points. RNA and protein were collected for subsequent experiments. To stabilize β-catenin, an empty vector pcDNA3-2AB Flag and β-catenin-4A mutant (S33/S37/T41/S45) plasmid  were transiently transfected into HK-2 cells with Lipofectamine 2000  according to manufacturer’s instructions. In all experiments, transfected cells were treated with 100 ng/µl LPS.Human proximal tubular epithelial cell line, HK-2 cells, was purchased from American Type Culture Collection , cultured in DMEM/F12, GlutaMAX4 cells per well. Cells were transfected with empty vector or β-catenin-4A mutant plasmid followed by LPS treatment. MitoTracker® Red CMXRos  was added into culture media and incubated at 37 °C for 30 min. Cells were then fixed by cold methanol and co-stained with DAPI. MitoTracker staining was visualized by confocal microscopy . Intensity of MitoTracker was quantified by Image J software on 10 random fields at 400 × magnification.HK-2 cells were seeded onto Chamber Slide (Thermo Fisher Scientific) at 2 × 10The interaction of β-catenin and FOXO3 was determined by co-immunoprecipitation assay  as previously described . HK-2 ceChromatin immunoprecipitation (ChIP) assay was performed using a Magnetic ChIP kit . Briefly, HK-2 cells were transfected with vector plasmid or β-catenin-4A mutant plasmid. 24 h after transfection, protein-DNA complex was cross-linked with 1% formaldehyde for 10 min at RT. Chromatin fragmentation was achieved by micrococcal nuclease digestion and ultrasound sonication. 10% sonicated samples were saved as 10% input. The remaining samples were immunoprecipitated with either FOXO3 ChIP-grade antibody  or normal rabbit IgG  at 4 °C overnight. ChIP-grade protein A/G magnetic beads were added and incubated for 2 h at 4 °C. After DNA recovery and purification, FOXO3-PGC-1α promoter binding within precipitated chromatin was analyzed by qPCR. The PGC-1α promoter region primers were designed by Primer-BLAST and listed in Supplementary Table Kidney cortical RNA was extracted by using NucleoSpin® kit . High-capacity cDNA Reverse Transcription Kit  was used for RNA reverse transcription to cDNA. Real-time PCR was performed using specific primers . Total protein concentration was quantified using BCA Protein Assay Kit (Pierce). Western blot was used to determine protein expression . Primaryp < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad Prism 9 . All data were expressed as mean ± SEM. Comparisons among groups were made by one-way ANOVA, followed by the Turkey test. TubCat (tubule-specific β-catenin stabilized) and TubCatKO (tubule-specific β-catenin deleted) mice and their respective control animals (CTL and KO CTL) were subjected to bilateral renal ischemia followed by 24 h reperfusion Fig. . AKI wasIn the septic AKI model Fig. , increasIRI-induced tubule cell apoptosis was reduced in TubCat mice but increased in TubCatKO animals Fig. . IRI alsThe protective role of β-catenin against tubular apoptosis and necroptosis was confirmed in septic AKI induced by LPS in which apoptosis and overexpression of p-MLKL and p-RIP3 were ameliorated in TubCat mice but exacerbated in TubCatKO mice Fig. A–D. ChanDouble-immunofluorescence staining showed that PGC-1α, the master regulator of mitochondrial biogenesis, was upregulated in TubCat-IRI kidneys and co-stained with β-catenin-positive tubules Fig. , whereasTransmission EM Fig. E, F showForkhead box O3 (FOXO3), an upstream regulator of PGC-1α, was significantly downregulated in cortical kidney nuclear fraction after IRI Fig. . It was Similar rescue of mitochondrial function was reproduced in the LPS-induced septic AKI model Fig. , althougFusion-fission dynamics are critical in maintaining mitochondrial homeostasis. Οptic atrophy 1 (OPA1) and mitofusin-2 (MFN2) are two molecules regulating mitochondrial fusion, and dynamin-related protein 1 (DRP1) is a GTPase that controls mitochondrial fission. Mitochondrial dynamics shifts towards fission during IRI with downregulation of fusion regulators OPA1 and MFN2, but upregulation of fission regulator DRP1 Fig. . In TubCIn HK-2 cells exposed to LPS, the mitochondrial biogenesis proteins PGC-1α and NRF1 were reduced in a dose-dependent manner Fig. . In HK-2To ascertain the mechanism through which tubular β-catenin maintains mitochondrial biogenesis, we studied FOXO3 expression in nuclear lysates of LPS-treated HK-2 cells . β-catenin is mainly expressed in the cytoplasm and cell-cell junctions under the unstimulated condition, whereas FOXO3 is predominantly expressed in the nucleus Fig. . After LTo investigate whether such binding affected the downstream FOXO3/PGC-1α signaling, which regulates mitochondrial biogenesis, FOXO3 ChIP antibody or IgG antibody was used for immunoprecipitation of the fragmented chromatin of HK-2 cells under vector or β-catenin plasmid transfection. ChIP output DNA and input DNA were used as the template in ChIP-qPCR with three predicted FOXO3-binding PGC-1α promoter primer sets  , 47, 48.The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) has been proposed as a positive regulator of PGC-1α transcription in neural cells , 53. In C. elegans, the FOXOs transcription factor DAF-16 requires the β-catenin gene, bar-1, for longevity [FOXO3 belongs to the FOXO family, which regulates vital cellular processes including metabolism, proliferation, stress resistance, and apoptosis –59. In Congevity , suggestongevity . Shiftinongevity , 62. Theongevity . In endoongevity . Deacetyongevity . These oAlthough the protective effect of β-catenin accumulation was ascertained in TubCat-IRI and TubCat-LPS models, the detrimental effect of β-catenin deficiency in renal tubules remained debatable in AKI models. In TubCatKO mice, IRI only marginally increased sCr and kidney function loss was not different between KO CTL-LPS and TubCatKO-LPS mice. Histologically, TubCatKO mice had slightly higher proportion of severely damaged tubules than KO CTL mice in both septic and aseptic AKI models. Nevertheless, the acute tubular injury marker (NGAL), cell death molecules, and mitochondrial-related proteins showed a significant difference between TubCatKO and KO CTL mice. One explanation for this discrepancy could be that the reduction in β-catenin levels in TubCatKO-IRI/LPS vs. KO CTL-IRI/LPS mice was only sufficient to affect mitochondrial function and cell death, but not kidney function. The discrepancy in kidney function between the IRI and LPS models could arise from the fact that IRI is a direct ischemia injury to the kidney, whereas LPS is a systemic inflammatory syndrome that induces a more subtle in situ injury to the kidney. It is therefore not surprising that IRI induced more kidney function loss and histologic damage than LPS. Another explanation might be the relatively short timepoint (24 h). A longer timepoint, up to 48 h or even 7 days, might cast a more global view of the effect of tubular β-catenin deficiency in AKI. Finally, the discrepancy in TUNEL-positive cells between TubCat and TubCatKO mice in the IRI model could arise from their heterogeneous genetic background that affects their propensity to apoptosis induced by IRI.In conclusion, tubular β-catenin activation alleviates septic and aseptic AKI by reducing kidney cell death and restoring mitochondrial biogenesis via β-catenin/AKT/p53 and β-catenin/FOXO3/PGC-1α signaling pathways. A schema for the protective mechanism is shown in Fig. Supplemental MaterialsOriginal Western blots


Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3β S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-β oligomers (AβO) in mice. Finally, quercitrin rescues AβO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/β-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.The Wnt/β-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer’s disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with  The Wnt/β-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis . DeregulThe absence of efficient treatment for AD inspires the quest for novel therapeutic candidates . In thisO-α-l-rhamnoside), a quercetin derivative, through a natural compounds cell-based screening as a Wnt/β-catenin signaling positive modulator. Quercitrin can be commonly found in food, beverages, and in traditional Chinese medicine  Gaertn.) [We have encountered quercitrin (quercetin-3-medicine . TraditiGaertn.) . QuercitGaertn.) , antileiGaertn.) , osteogeGaertn.)  and antiGaertn.) . Our groGaertn.) ,37 and iGaertn.) ,39 as WnXenopus laevis. We used Xenopus early development, a gold standard canonical Wnt signaling model, to describe quercitrin as a Wnt/β-catenin signaling potentiator that hinders Glycogen Synthase Kinase-3 (GSK3) kinase activity. Moreover, quercitrin potentiates the canonical Wnt synaptogenic effect in hippocampal neuronal cultures and rescues AβO-induced memory impairment in mice. Overall, we characterize quercitrin as a Wnt/β-catenin potentiator and a promising therapeutic candidate for treating AD.In the present work, we combine cell-based assays with in vivo approaches using different vertebrate animal models, including mice and Through a cell-based screening of natural compounds, we noticed that quercitrin increases the Wnt/β-catenin signaling activity. Considering that Wnt signaling is a well-conserved signaling pathway across the various eukhariotic cells , we studCanonical Wnt signaling functions through β-catenin degradation regulation. GSK3 and CK1, two kinases present in the destruction complex, also composed of Axin and APC, phosphorylate β-catenin, marking it for proteasomal degradation. The presence of Wnt ligands triggers the destruction complex recruitment to the membrane in a dynamic manner leading to β-catenin stabilization and target genes transcription ,43. RKO Xenopus laevis early development, a gold standard model to assay Wnt signaling modulation because of its critical relevance to axial patterning [Xenopus early development leads to a secondary axis formation. To investigate if quercitrin potentiates Xwnt8  induced Xenopus secondary axis formation , a Wnt/β-catenin signaling-specific reporter gene [Xenopus embryos , β-catenin WT, or LRP6 a–c and mThe LRP6 overexpression inhibits GSK3 (through the PPPSPxS motifs)  and disaTo test the quercitrin impact on GSK3 activity, we transfected HEK293T cells with a GSK3 biosensor  consistip < 0.001, Welch t-test statistical analysis). The addition of 10 μM quercitrin simultaneously to hWnt3a enhanced the hWnt3a effect on synapse number by 2-fold (4.5-fold increase when compared to the untreated condition)  a–e. The ndition) e. Treatmndition) . These dSynapse loss is an essential hallmark of several memory dysfunctions . We thusOur finding that quercitrin presents synaptogenic potential in vitro and prevents AβO-induced synapse loss prompted us to examine its impact on memory dysfunction triggered by the oligomers m. As expIn summary, quercitrin rescues synaptic damage and memory impairment induced by AβO in mice. Furthermore, the following evidence correlates the quercitrin effect with Wnt/β-catenin signaling modulation: (i) the β-catenin levels are simultaneously modulated with the memory impairment rescue, and (ii) the downstream inhibition by XAV939 abrogates quercitrin beneficial effect.The Wnt signaling origin coincided with metazoa and was crucial for patterning axes during embryonic development . Among vXenopus laevis development, in which quercitrin increased the occurrence of the Xwnt8-induced secondary axis ; RRID: Addgene_29689) [HEK293T was purchased from ATCC, and RKO pBAR/Renilla (B/R), and SW480 B/R cell lines were kindly donated by Professor Xi He . Every cell line was cultured with DMEM-F12  supplemented with 10% FBS (Gibco). The flavonoid quercitrin  was solubilized into DMSO  at 50 mM and stored at −20 °C. XAV939  was solubilized into DMSO at 10 mM and stored at −20 °C. BIO was purchased from Sigma (#B1686—CAS Number 667463–62-9) and solubilized into DMSO  at 10 mM. L-cell and L-Wnt3a cell lines were obtained from ATCC, and the conditioned medium was obtained according to ATCC protocol. pCS2 GFP-GSK3-MAPK was a gift from Professor Edward De Robertis, University of California, Los Angeles  . For tesXenopus embryos were obtained by in vitro fertilization and staged according to Nieuwkoop and Farber [Xwnt8 mRNA was synthesized by linearizing Xwnt8-containing plasmid with NotI and transcribing the mRNA using SP6 RNA polymerase . For canonical Wnt signaling reporter gene assay, two 4-cell stage embryos transverse blastomeres were injected with 4 nL containing 280 pg of S01234-luciferase plasmid, 50 pg of Tk-Renilla plasmid, 10 pg of Xwnt8 mRNA, and 0.8 pmol of quercitrin or DMSO each, for a total of 1.6 pmol of quercitrin per embryo. For inducing a secondary axis, two 4-cell stage ventral blastomeres were ventrally injected around the equatorial region with 1, 3, or 10 pg of Xwnt8 mRNA per embryo together with 1.6 pmol quercitrin or DMSO. We determined the quercitrin injection concentration based on other compounds [3; 0.08 mM MgSO4·7H2O; 1 mM Hepes; 0.03 mM Ca(NO3)2.4H2O; 0.04 mM CaCl2·2H2O; pH 7.7) until control embryos reached stage 10. Triplicates of four embryos were lysed using PLB  and assayed for Firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay. All experiments were performed at 22 °C and in triplicate. Xwnt8 mRNA-injected embryos were considered a positive control group, while uninjected embryos were considered the negative control group.Adult frogs  were stimulated with 1000 IU human chorionic gonadotropin  according to the Animal Care and Use Ethics Committee from the Federal University of Rio de Janeiro and were approved by this committee under permission number 152/13. d Farber . Xwnt8 mompounds ,87. Foll2, 95% air atmosphere for 12 days in vitro. After, cultures were cotreated for 24 h with 100 ng/mL recombinant hWnt3a plus DMSO or 10 μM quercitrin. To inhibit the Tankyrase (TNKS) activity and therefore suppress Wnt signaling, neuronal cultures were cotreated with 100 ng/mL recombinant hWnt3a plus DMSO or 10 μM quercitrin in the presence of 5 μM XAV939 for 24 h. The experiment was performed in triplicate.Primary hippocampal neuronal cultures were performed according to  using E1Sixteen embryonic days (E16) Swiss mice were used for neuronal cultures. For rodent in vivo experiments, three-month-old male Swiss mice from our animal facilities, housed in groups of five per cage with free access to food and water, under a 12 h light/dark cycle, controlled temperature, and humidity were used. All procedures followed the “Principles of Laboratory Animal Care”  and were approved by our local Animal Care and Use Committee . Experiments were performed according to Brazilian Guidelines on Care and Use of Animals for Scientific and Teaching Purposes (DBCA).For infusion into the lateral ventricle (i.c.v.), animals were anesthetized with 2.5% isoflurane  using a vaporizer system  and were gently restrained briefly during the injection procedure. A 2.5 mm-long 30 G needle was unilaterally inserted approximately 1 mm to the midline point equidistant from each eye and parallel to a line drawn through the anterior base of the eye as previously described. Using a Hamilton syringe, solutions (3 µL) containing vehicle , recombinant Wnt3a (100 ng), quercitrin (300 pmol), XAV939 (100 pmol) or combined quercitrin/XAV939  were slowly infused. After one hour, mice were again anesthetized and received a second injection (3 µL) with vehicle (PBS) or AβOs (10 pmol) at the same place. Mice showing any signs of misplaced injections or brain hemorrhage  were excluded from further analysis.t-test comparing the mean exploration percentage time for each object with the chance value of 50%. Animals that recognize the familiar object as such  explore the novel object > 50% of the total time. For the NOR test, the AβO + DMSO group was considered the positive control group. The DMSO-only injected group was considered the negative control group.The behavioral evaluation was performed on the following day of pharmacological treatment. Mice were placed in the experimental room under controlled light (indirect fluorescent light) and temperature (23 °C) conditions for at least one hour before analysis. First, mice were submitted to a five min-long open-field arena test (habituation phase), in which they were placed at the center of an arena (30 cm × 30 cm × 45 cm) divided into nine equal quadrants by imaginary lines on the floor and allowed to explore it for 5 min freely. Total locomotor activity and time spent at the center or the periphery of the arena were recorded using ANY-maze software . The arena was thoroughly cleaned with 70% ethanol in between trials to eliminate olfactory cues. Immediately after habituation, mice were submitted to the novel object recognition test. During a 5-min long training session, animals were placed at the center of the same arena, now in the presence of two identical objects. During sessions, objects were fixed to the arena’s floor using tape to prevent displacement caused by the exploratory activity of the animals. A trained researcher recorded the amount of time mice spent exploring each object. Sniffing and touching the object were considered exploratory behavior. The arena and objects were cleaned thoroughly with 70% ethanol in between trials to eliminate olfactory cues. Two hours after training, animals were again placed in the arena for the test session, with one of the objects used in the training session having been replaced by a new one. Again, the amount of time spent exploring familiar and novel objects was measured. Test objects were made of plastic and had different shapes and colors. Preliminary tests showed that none of the objects used evoked innate preference. Results were expressed as a percentage of time exploring each object during the training or test sessions or as total exploration during each session. Data were analyzed using a one-sample Student’s 4) cells were seeded into 96-well plates and transfected using Lipofectamine 3000  with TOPFLASH (100 ng/well) and Tk-Renilla (50 ng/well) according to manufacturer protocol. RKO B/R (1.5 × 104) cells were seeded into 96-well plates. Cells were treated with control media, L-cell conditioned medium (CM), Wnt3a CM, recombinant hWnt3a , or other specified conditions. After treatment, cells were lysed with Passive Lysis Buffer  and assayed for Firefly and Renilla luciferase activity using the Dual-Luciferase Reporter Assay . Luminescence was measured using the Modulus IITM Microplate Multimode Reader . Every assay was repeated at least three times and in triplicate.HEK293T  or Triton  lysis buffer and phosphatase/protease inhibitors . Cell lysates were mixed with Laemmli buffer  and heated at 95 °C for 5 min. Protein samples were separated on the SDS-PAGE gel and transferred to the Immobilon-E membrane . Membranes were blocked for 1 h or overnight with TBS-T 2% PVP . Indirect immunochemistry using horseradish peroxidase-conjugated secondary antibodies  was detected using SuperSignal West Pico chemiluminescent substrate  or SuperSignal Femto Maximum Sensitivity Substrate  and exposed in Kodak X-OMAT film. The immunoblot of the GSK3 was transferred to an Immobilon-FL membrane , blocked with the Blocking Buffer , and incubated with the IRDye 680 , IRDye 800  secondary antibodies and exposed in the Odyssey Imaging System .The following primary antibodies were used according to manufactures concentration: α-tubulin , β-actin , β-catenin , phosphorylated β-catenin S33, S37 , Cyclophilin B , Flag-M2 , GAPDH , GSK3β , phosphorylated GSK3β S9 , LRP6 , phosphorylated LRP6 S1490 , synaptophysin  and PSD-95 . Densitometric analyses of the immunoblots were performed using NIH ImageJ software, Bethesda, MD, USA (FIJI version 1.53c).Immunocytochemistry and synaptic puncta analysis were performed according to . BrieflyHippocampal neurons were randomly selected based on DAPI staining, and 10–20 fields were imaged per experimental condition. The green and red channels were merged and quantified using the Puncta Analyzer plug-in in NIH ImageJ. The experiments were performed in duplicate, and each result represents the mean value of three independent hippocampal neuronal cultures.t-test or one-way analysis of the variance (ANOVA) and Dunnett post hoc comparison test unless declared otherwise. Statistical significance was defined as * p < 0.05, ** p < 0.01 or *** p < 0.001, unless specified otherwise, and ns stands for not significant. DMSO-treated conditions were considered as the control group for the statistical analyses unless stated otherwise. Every experiment was repeated at least three times unless specified otherwise. Behavioral experiments were performed with ten mice for each condition. Every graph displays the mean ± SD or mean ± SEM, depicted in every figure legend. The statistical analyses were performed using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla, CA, USA, www.graphpad.com.Comparison among treatments was performed employing the Student


To this end, solution-processed a-IZO films were prepared with respect to the Zn molar ratio, and their structural characteristics were analyzed. For the structural characteristic analysis of the a-IZO film, the cross-section, morphology, crystallinity, and atomic composition characteristics were used as the measurement results. Furthermore, the chemical reaction pathway of the nitrate precursor-based IZO solution was evaluated for the growth process of the a-IZO film structure. These interpretations of the growth process and chemical reaction pathway of the a-IZO film were assumed to be due to the thermal decomposition of the IZO solution and the structural rearrangement after annealing. Finally, based on the structural/chemical results, the electrical performance of the fabricated a-IZO TFT depending on the Zn concentration was evaluated, and the electrical behavior was discussed in relation to the structural characteristics.Understanding the chemical reaction pathway of the metal–salt precursor is essential for modifying the properties of solution-processed metal-oxide thin films and further improving their electrical performance. In this study, we focused on the structural growth of solution-processed amorphous indium-zinc-oxide ( Recently, as the demand for flexible displays has increased and direct printing of display products has been attempted, interest in solution processes has drastically expanded ,2,3,4. IAmong the many metal–salt reactions, the metal–nitrate precursor has many advantages over other conventionally used precursor reactions such as metal hydroxide, metal chloride, and metal acetate ,16,17. TEventually, the electrical properties of AOS TFTs are significantly affected by the bonding structure and composition ratio of the atoms constituting the semiconductor material ,25. AOS a-IZO TFTs were analyzed based on the structural/stoichiometric characteristics of solution-processed a-IZO semiconductor films. For this, a-IZO films were prepared with respect to the Zn molarity, and structural characteristics such as the bonding structure, crystallinity, and composition were analyzed. Furthermore, based on the thickness-dependent properties and thermal decomposition results of the a-IZO film, it is speculated that the chemical reaction pathway and growth process of the film are determined. Finally, an a-IZO TFT with respect to Zn concentration was fabricated, and its electrical performance was evaluated. The electrical behavior of a-IZO TFT is discussed in relation to the structural/chemical characteristics of the a-IZO film. In this study, the electrical characteristics of amorphous 3(NO3)3∙xH2O)  and zinc nitrate hydrate (Zn2(NO3)2∙xH2O)  in 2-methoxyethanol (CH3O(CH2)2OH) . For proper dissolution, the solution was mixed using a magnetic bar at 60 °C for 12 h or longer. The molarity ratios, as listed in As shown in a-IZO TFT, a p-type (p-doped) silicon  wafer sputtered with a 100 nm thick SiNx dielectric was used. Before spin coating the solution-processed semiconductor layer, the substrate was cleaned using an ultrasonicator  in the order of acetone, isopropyl alcohol, and deionized water.To fabricate the solution-processed Thereafter, to lower the surface energy of the substrate, it was exposed to oxygen plasma  at a chamber pressure of 3 mTorr for 1 min. Spin coating was carried out at 2000 rpm for 1 min to prepare the IZO solution, and soft banking was started for 5 min at 110 °C, immediately after coating. To anneal the semiconductor film, the spin-coated substrate was thermally annealed at 600 °C for approximately 1 h under a nitrogen atmosphere. Substrate heating and cooling were continued slowly for 1 and 6 h, respectively, and additionally cooled at room temperature for 6 h or more.a-IZO TFT was a finger-type, as shown in −6 Torr, and the thickness was approximately 50 nm. The width and length of the finger-type channel were 2000 and 80 μm, respectively, and W/L = 40.The fabricated source/drain of a-IZO films. The surface and cross-sectional characteristics of the a-IZO films were measured using field-emission scanning electron microscopy (FE-SEM) (JS-6701F) . Furthermore, the crystal structure and atomic composition of the prepared a-IZO films were analyzed using high-resolution X-ray diffraction (HR-XRD)  and X-ray photoelectron spectroscopy (XPS)  , respectively. Auger electron spectroscopy (AES) (PHI 700)  was used to analyze the constituent atoms according to the depth of the a-IZO film, and the thermal decomposition process of the solution was analyzed using thermogravimetric analysis (TGA) (TGA N-1000) . Finally, all the electrical properties of the solution-processed a-IZO TFT were measured using a semiconductor analyzer (ELECS-420)  at room temperature.The following equipment was used to analyze the fabricated a-IZO film, the properties of the fabricated film with respect to the Zn molarity were evaluated. a-IZO films with various Zn molar ratios. For this analysis, 0.2, 0.25, 0.3, 0.4, 0.5, and 0.6 M of the Zn molarity were used, and the In molarity ratio was fixed at 0.05 M. As shown in a-IZO film increased from 20 to 70 nm as the Zn molar ratio increased from 0.2 to 0.6 M. The surface properties also exhibited noticeable differences depending on the Zn concentration, as shown in a-IZO layer formed uniformly in the 20 nm thin film. This was confirmed by comparing the enlarged surface SEM images of the SiNx and a-IZO. Even at a low Zn molarity ratio, the ZnO aggregates began to appear as bright seeds, and as the Zn molarity increased, the seeds expanded over the entire surface. In the a-IZO film produced by the solution process, 20 nm of the thin a-IZO layer was first formed from the bottom of the interface, regardless of the Zn molarity ratio. It is presumed that with an increase in the Zn concentration, an excess ZnO-aggregated layer sequentially emerges on the surface.To discuss the structural characteristics of the a-IZO films were measured. a-IZO/SiNx/Si bulk film, and the measurement result in the GI mode shows the surface crystallinity of the film. Here, the peak characteristic in the gray box of the left-side graph in x/Si bulk substrate; thus, it does not appear in the surface characteristic of the GI mode result of the right-side graph. a-IZO thin film with respect to the Zn molar ratio. In addition to the Zn molarity characteristics of the a-IZO film, IZO and ZnO reference peaks are included at the top of each graph. It should be noted that unlike the results in a-IZO film produced by the solution process, a 20 nm of thin a-IZO layer is first formed at the bottom of the interface regardless of the Zn molarity ratio. Then, with an increase in the Zn concentration, excess ZnO aggregations are formed as multi-layers on the surface.To determine the crystallinity characteristics, the XRD patterns of the fabricated a-IZO films, the XPS results were analytically measured. a-IZO layer, whereas the 0.3, 0.4, and 0.5 M samples show the characteristics of the ZnO aggregation layer. Particularly, a noticeable difference in the XPS analysis appeared in To determine the stoichiometric properties of the solution-processed a-IZO films with respect to the Zn molar ratio can be summarized as follows: To fabricate the IZO film by a solution process, it was assumed that a relatively uniform a-IZO film was formed on the SiNx dielectric, with a small In concentration; Zn molarity of 0.2 M is considered an a-IZO film. As the Zn molarity increased, the excess Zn accumulated as an aggregation layer on the a-IZO film.The structural characteristics of the solution-processed a-IZO film, a chemical understanding of the reaction pathway of the solution process can help improve the physical properties  of the film. a-IZO sample with a Zn molarity of 0.25 M. The dashed line in the graph represents the measurement result after spin coating , and the solid line represents the measurement result after annealing at 600 °C for more than one hour. The grey box in a-IZO film were clearly observed in the solid line. As shown by the solid line in a-IZO sol–gel state can be found in the characteristics of the rearrangement of atoms. In the dashed line, the atoms are relatively uniform in the gel state (after spin coating); however, the distribution of the Zn and In atoms is abruptly rearranged toward the top and bottom after annealing. This atomic rearrangement can occur because of the various reactions in the solution process, and in this study, a chemical reaction pathway of the nitrate precursor was applied for a more valid evaluation.In addition to the structural characteristics of the a-IZO film can be determined based on the decomposition process of the TGA graph. a-IZO film. The analysis of the nitrate-based thermal decomposition was referenced to the theory in this study [The stoichiometric analysis of the growth pathways of the solution-processed is study . As showMoreover, in solution, nitrate forms a nitric acid–water azetrope via a hydrolysis/condensation reaction and can be expressed as Equation (2):3-OH) structure was generated in the solution by the olation reaction, as shown in Equation (3) below, and the nitric acid–water mixture was removed by evaporation with a solvent at a temperature of ~120 °C, as indicated in After spin coating, as the annealing temperature increased, a metal–nitrate-hydroxide  and (5), and the remaining nitrate is exhausted as gas.2, and N2O gases. Depending on the final by-product of nitrate , the thermal decomposition of nitrate can be completed at a relatively high temperature of ~335 °C (iv). Finally, an M-O-M oxo link structure (oxo linkage) is formed by the oxolation reaction in Equation (7):In the thermal decomposition of Equations (4) and (5), the decomposition temperature of the metal ion was affected by the magnitude of the charge density (CD). This charge density is proportional to the charge of ions and inversely proportional to the radius of atoms and is expressed as Equation (6) :(6)CD~αTa-IZO/ZnO film, as discussed in the structural characteristics, is closely related to the subsequent occurrence of the thermal decomposition of the In-nitrate and Zn-nitrate at different temperatures.After the completion of reactions (ii) to (iv) in a-IZO films can be summarized as follows. The sol film formed by spin-coating was sintered into a gel film through solvent and azetrope evaporation. In gel-type a-IZO thin films, nitrate is terminated as a gas through thermal decomposition, and a multi-layer film is formed in the order of the a-IZO semiconductor and ZnO aggregation layers. Subsequently, the remaining -OH is removed in the form of H2O and O2 through oxidation, and the thin film is finally condensed in the form of an M-O-M oxo-link structure.The growth characteristics of solution-processed a-IZO TFT was evaluated with respect to the Zn molar ratio. a-IZO TFT with respect to the Zn molarity ratio. As shown in The electrical performance of the a-IZO thin film. a-IZO film and the atomic bonding model of the by-product after annealing. In InO), and oxygen vacancies (VO) [+–DInO− bond can be formed, which can act as a donor via DInO–→DInO0 + e−.Eventually, the decrease in the electrical performance of the TFT due to the increase in Zn molarity can be interpreted as an increase in the total series resistance owing to the ZnO aggregation layer, as shown in ies (VO) ,34. Parta-IZO film with respect to the Zn molarity ratio, the cause of the multi-layer thin-film structure in d_In ~183 °C) by thermal decomposition. Simultaneously, ~1011 cm−3 of N+ fixed charges in the dielectric and DInO− charges are attracted to each other by Coulombic interaction, and an In-N covalent bond is formed through the annealing process. The extra electrons increased the free electron density in the a-IZO film. Through this process, it can be speculated that ~20 nm of the a-IZO film was formed at the semiconductor–dielectric interface. Subsequently, at a relatively high thermal decomposition temperature (Td_zn ~237 °C), the ZnO amorphous network structure started to emerge as a ZnO aggregation layer. Consequently, an a-IZO semiconductor layer, which determines the electrical performance, was initially formed regardless of the Zn molar ratio. The excess ZnO aggregation became thicker with respect to the Zn molar ratio. Consequently, this ZnO aggregation layer acts as a series resistance RZnO, which prevents charge injection into the TFT channel. Additionally, the effective voltage between the gate and drain sources is reduced, and the threshold voltage is increased because of the effects of the series resistance. The decrease in the electrical performance of the TFT in the transfer curve and output current can be explained by the effect of the ZnO aggregation layer, as discussed.Based on the structural characteristics, chemical growth, and electrical characteristics of the a-IZO TFT with respect to the Zn molarity, and a chemical growth pathway of the a-IZO film was proposed. In the a-IZO film fabricated using the solution process, an a-IZO layer with a thickness of approximately 20 nm was initially formed regardless of the Zn molarity ratio. As the Zn concentration increased, the excess ZnO aggregated on the surface of the film as a multi-layer structure. The structural characteristics of the a-IZO film were confirmed through the measurement results of SEM, XRD, and XPS. Furthermore, the solution-processed a-IZO film was in a gel state uniformly dispersed after spin coating and was grown into a multi-layer having a heterogeneous structure through the annealing process. The characteristics of chemical growth of the a-IZO film were analyzed through the data of AES and TGA. The multi-layer structure was grown from a uniform gel state through annealing, and the properties of the In and Zn element rearrangements were stoichiometrically defined through a chemical reaction pathway. It is speculated that the multi-layer structure is originated from the different decomposition temperatures of each In and Zn material, and coulombic interaction between the fixed charge and dangling bond of the dielectric and In-O bonding structure. Finally, it was inferred that the lowered electrical performance of the a-IZO TFT depending on the Zn molarity was because the ZnO aggregation acted as a series resistance. Further detailed research on the effects of ZnO structure on the charge transport behavior in an a-IZO semiconductor can be developed by excluding the influence of film thickness itself on the threshold voltage and on-state current of TFTs. We believe that the structural and electrical characterizations of nitrate-based solution-processed AOS in this study can be applied to further improve the feature characteristics of solution-processed oxide semiconductors having multi-layer structures.In this paper, we highlighted the relationship between the structural characteristics and electrical behavior of solution-processed