The adrenal glands are important endocrine organs that play a major role in the stress response. Some adrenal glands abnormalities are treated with hormone replacement therapy, which does not address physiological requirements. Modern technologies make it possible to develop gene therapy drugs that can completely cure diseases caused by mutations in specific genes. Congenital adrenal hyperplasia (CAH) is an example of such a potentially treatable monogenic disease. CAH is an autosomal recessive inherited disease with an overall incidence of 1:9500–1:20,000 newborns. To date, there are several promising drugs for CAH gene therapy. At the same time, it remains unclear how new approaches can be tested, as there are no models for this disease. The present review focuses on modern models for inherited adrenal gland insufficiency and their detailed characterization. In addition, the advantages and disadvantages of various pathological models are discussed, and ways of further development are suggested. The adrenal cortex is the most important site for the synthesis of steroid hormones. It is histologically and functionally divided into layers surrounding the central medulla. The zona glomerulosa (ZG), which produces mineralocorticoids , is located directly under the capsule that covers the entire gland. The zona fasciculata (ZF), which synthesizes glucocorticoids (cortisol in humans and corticosterone in rodents), is located behind ZG, as is the zona reticularis (ZR), which produces adrenal androgens . Mineralocorticoid production is controlled by the renin-angiotensin-aldosterone system, while glucocorticoids are under the control of the hypothalamic-pituitary-adrenal system. Glucocorticoids regulate glucose metabolism, inflammation, immune responses, muscular and skeletal mass, as well as cognition, well-being, and memory. Mineralocorticoids control extracellular fluid volume and sodium homeostasis and therefore have an important effect on blood pressure .StAR, CYP11A1, CYP11B1, HSD3B2, CYP17A1  is capable of self-renewing throughout life, replacing senescent cells, and maintaining or expanding functional areas to meet physiological needs for steroids, or in response to external pharmacological stimuli ,3,4. Adr CYP17A1  6,7]. T,7. TStARCurrently, the only available treatment for patients with adrenal insufficiency is hormone replacement therapy, which in most cases must be lifelong. The administration of exogenous glucocorticoids compensates for the lack of endogenous cortisol and acts through negative feedback from the hypothalamus and pituitary gland by suppressing the secretion of corticotropin-releasing hormone (CRH) and ACTH. The disadvantage of traditional replacement therapy is that choosing the correct dosage of medication is extremely difficult and does not reproduce the physiological rhythm of hormone release and an incorrect dosage can lead to negative consequences with prolonged treatment . DespiteCYP21A2 gene. CYP21A2 is an enzyme that is necessary for the synthesis of aldosterone and cortisol . Preclinical studies with BBP-631 in the Cyp21-/- mouse model and non-human primates showed strong rescue of steroidogenesis and the ability of the virus to transduce adrenal cells [There is a gene therapy approach for the treatment of CAH that is already in phase ½ of clinical trials. The strategy developed by the company Adrenas is based on the intravenous administration of AAV5 carrying the wild-type coding sequence of the al cells .The use of cell and gene therapy strategies to create or support functional adrenal tissue is a promising alternative to existing methods of treating primary adrenal insufficiency, regardless of disease etiology, as this approach allows the organ to function more physiologically. However, these approaches require species-specific disease models, as different animals differ physiologically (see below).Although the development of cell therapy strategies in the adrenal region is still in its infancy, it is important to recognize that some significant results have been achieved to date. The following sections discuss current approaches and advances, including the use of adrenal cells for transplantation, the use of functional steroidogenic cells derived from stem cells or reprogrammed somatic cells, and the creation of three-dimensional models of adrenal organoids .There are a few approaches to obtaining steroidogenic tissue that have been developed A. Most oCYP21A2 gene, causing a difference in the types of steroids produced by murine MSCs and human MSCs. Glucocorticoids were the major steroids produced by transformed human MSCs, while testosterone was the major product from the transformed murine MSCs. Furthermore, murine MSCs express the adipose tissue type of adrenocorticotropic hormone receptor (with low ACTH affinity), whereas human MSCs did not express a special isoform of the receptor that could respond to ACTH [MSCs are multipotent somatic stem cells that originate in the mesoderm, as is the case with steroidogenic organs. Although originally identified in bone marrow , similar to ACTH .Despite the evidence that human MSC from different sources can be induced to be steroidogenic, differences in the differentiation process of the initial tissues were observed ,18. For NR5A1), regulates steroidogenic pathways, including genes encoding cytochrome P450 enzymes such as CYP11A1 [CYP17A1 [CYP21A2 [CYP11B1 [3-HSD) [DAX1, AMH, SOX9, SRY, and INHA [LRH-1 [As mentioned above, for the most part, protocols for differentiating stem cells into the steroidogenic lineage are based on the forced expression of steroidogenic factor 1 (SF1). This gene, encoded by nuclear receptor subfamily 5, group A, member 1  , and othand INHA ,30,31,32A [LRH-1 ,34. LiveCYP21A2, CYP17A1 and CYP11B1 genes. The steroids produced by the differentiated cells carrying mutations had a pathogenic profile similar to those demonstrated during disease progression (high levels of progesterone and 11-deoxycorticosterone and low levels of cortisol). Treatment with lenti- or adeno-associated viruses carrying the normal corresponding gene copy restored normal steroidogenesis [The same differentiation method was used for CAH patient-specific cells  with different mutations in the ogenesis ,36 . Allen et al. used porcine adrenal glands as a biologic scaffold for human fetal (between 12 and 16 weeks gestation) adrenal gland cells. The simple mix of the scaffold and cells yielded the attachment of most cells onto the surface of the scaffold, with rare cell infiltration events. It is interesting that the decellularization process removed the laminin molecules from the ECM instead of collagen IV and fibronectin, but manual addition of laminin (known to be an important component of the stem and differentiated cells niche) had no effect on the efficiency of the cellurization or cortisol production of the obtained artificial organ . Five yeIt should be noted that SF1-driven differentiated cells embedded in alginate showed completely opposite behavior in comparison to those obtained from organs . Human iModern approaches in molecular biology give scientists the opportunity to work with human stem cells and differentiate them into almost any somatic cell. Developing human adrenal glands from stem cells using various differentiation methodologies is the future of adrenal disease modeling and insufficiency treatment.The aforementioned models could be used to model normal adrenal tissue. At the same time, there are several approaches to inhibit steroidogenesis in hormonal hypersecretion . It may As long as 3D human organ structures are not perfectly developed, there are several in vivo models of CAH that exist and are usable C.The mechanical model of adrenal insufficiency used in the past to test different therapeutic approaches is the adrenalectomized animals . This tyCyp21a2-p-Cyp21a1 hybrid gene with different mutations that completely switch off 21-hydroxylase activity. The mice have a pronounced CAH phenotype, which is manifested by a loss of corticosterone and aldosterone production, weight loss, and increased level of progesterone. According to the NCBI blast analysis, there is a 78% nucleotide and 73% aminoacid sequence difference between the mouse and human Cyp21a2 genes. For this reason, it remains unclear whether it is correct to treat mice with the human Cyp21 protein or gene. Restoration of 21-OH activity by the human gene in Cyp21A2-deficient mice has been shown to restore the normal phenotype [CYP21A2 gene functional compatibility between humans and mice, humanized mice were created [Cyp21A2 gene based studies.The first genetically modified model is the C57Bl/10SnSlc-H-2aw18 mouse strain, which carry the henotype . In orde created . No adveCyp11b1 gene. These mice represent an increased 11-deoxycorticosterone and progesterone to corticosterone ratio that can be rescued by intraadrenal AA9-Cyp11b1 induction [Another CAH mouse model was created by using CRISPR/Cas9 mediated genome editing that disrupted the 4th exon in mice with the nduction .StAR and CYP11A1  cells lies through the fetal stage and remain as fetal or can proceed to terminally differentiate. It was noted that the groups of fetal and definitive zone markers can be detected at low levels, even at the AGP stage of development . In studTherefore, single cell transcriptome sequencing methods form the basis of projects describing structure and development of the mammalian organs ,76. ResoIt is clear that the development of therapies for adrenal pathologies based on cellular technologies is an urgent scientific and technological task. At this stage of development, the fundamental possibility of obtaining cell lines with some adrenal characteristics, as well as volumetric organ-like structures , was shown. However, there are still many gaps both in the developed protocols and in understanding the molecular basis of the ongoing processes.For example, attempts to obtain steroidogenic cells in culture from various pluripotent cells have demonstrated that differentiation must consist of several stages, since it is difficult to change open ESC chromatin in one step to the expression of genes of the terminally differentiated state. Moreover, aggressive induction of SF1 in ESCs has been shown to be toxic . TherefoAt the same time, it is important to understand that terminally differentiated cells proliferate poorly, or even not at all. Although they remain viable for a long time in culture, it is not clear how this will work in vivo. The most obvious direction for further development is to focus on obtaining a culture of progenitors capable of proliferating and differentiating in vivo and in vitro in response to external signals.The scientific community has yet to gain a definitive understanding of the sources and pathways of differentiation of adrenal progenitors into cells that produce different types of hormones. For example, when differentiation is mediated by exogenous expression of SF1, cortisol is primarily measured, demonstrating differentiation into the zona fasciculata, which contradicts the current ideas of the differentiation pathway going through the zona glomerulosa in general. In some studies , no aldoSF1. Such attempts have been made by Ruiz through lentiviral expression of transcription factors thought to be regulators of SF1 [In general, forcing exogenous expression of SF1 to obtain adrenal steroidogenic cells raises many questions. Different authors have used various delivery methods for this factor, including lentiviral transduction. In one study, using adeno-associated virus as a vector endogenous gene was shown not to be triggered , and in s of SF1 . SteroidData from single cell transcriptome analysis can help to characterize more precisely the expression profile of progenitor cells in different tissues, making it possible to control the correctness of its implementation at different stages of in vitro differentiation. For example, the markers for hematopoietic stem cells are now well known and defined, and their presence in other stem cell populations is an important exclusion factor. Markers of progenitors of other cell types  are still poorly described, and this information is essential for cell type determination.In the absence of appropriate CAH models, it is difficult to evaluate the performance of different drug prototypes. Although CAH models are under development , it is c


As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios. Cells constantly exchange information with their surroundings, and external inputs are first received by their outermost layer, the plasma membrane (PM). This interface, far from being an inert wall, integrates and transmits incoming stimuli, ultimately impacting cell behavior. In this context, the traditional view of such stimuli as biochemical messengers has now changed to include the concept that physical perturbations are also of major importance . By sensHowever, changes in cell PM area upon mechanical perturbations are necessarily accompanied by changes in topography at the local scale. This is exemplified by caveolae flattening upon cell stretch  or creatHere, we studied this problem by using as a model the controlled compression of fibroblasts through the application and release of stretch. We show that upon cell compression, bud-shaped PM deformations of negative curvature (evaginations) on the 100 nm scale are formed and enriched by IRSp53, a negative curvature-sensing protein. This creates a local node where specific PM topography is selectively coupled through IRSp53  to activate actin polymerization mediated by Rac1 and Arp2/3. The activation of this cascade flattens the structure, recovering the PM shape to its initial state. Our findings demonstrate that a local mechanosensing mechanism controls PM homeostasis when perturbed through compression.To study how PM topography is regulated, we subjected normal human dermal fibroblasts (NHDFs) transfected with an EGFP-membrane marker to a physiologically relevant 5% equibiaxial stretch by using a custom-made stretch system composed by a PDMS stretchable membrane clamped between two metal rings, as previously described in –1, respectively .In cells, passive fold formation is followed by active resorption involving actin cytoskeleton rearrangements, allowing for topography equilibration within minutes . As prevell line . Here foAltogether, these data indicate that PM compression led primarily to the formation of evaginations of regular size and shape at the apical side, which are immediately resorbed by the cell in an active process to re-equilibrate PM topography and tension.In light of these results, we wondered if the PM evaginations formed upon compression could be sensed by the cell, triggering a mechanism to recover PM shape. Based on previous results showing that actin depolymerization by either latrunculin A or cytochalasin D blocked PM remodeling after stretch , we hypo–1. They also revealed that lower curvature values comparable to the ones obtained by TEM imaging of our evaginations led to a twofold enrichment of this domain with respect to a control membrane marker  or even directly bind actin monomers . Interese marker .-/- cells, and compared their decay curves with wild-type (WT) MEF cells , with the EGFP-tagged full length (FL) WT IRSp53 protein (EGFP-IRSP53-FL). As a control, we used a GFP-bound mitochondrial marker (Mito-GFP) instead of EGFP-IRSP53-FL. A strong APEX signal  was observed around the mitochondrial membrane for Mito-GFP-transfected cells . IRSp53 possesses multiple domains with multiple interactors  and dominant negative (T17N) forms of Rac1. Confirming the involvement of Rac1, the expression of Rac1-G12V accelerated evagination resorption significantly whereas Rac1-T17N slowed it down in NHDF .IRSp53 has been described to promote Arp2/3-mediated actin polymerization, which is driven by NPFs (WASP or WAVEs), both acting as an upstream  and downPrevious work on PM ruffling showed that IRSp53 couples Rac1 to the activation of the WRC, and the subsequent nucleation of branched actin filaments mediated by Arp2/3 .-/-R cells with different inhibitors. First, cell treatment with 10 µM of the N-WASP inhibitor Wiskostatin  are likely to play compensatory or redundant roles. IRSp53 in complex with WAVE has also been described to localize to saddle curvatures , which oBeyond the formation of evaginations, we previously showed that cell compression upon a stretch cycle also triggers endocytosis through the CLIC-GEEC (but not the clathrin-mediated) pathway . In turnThus, our findings unveil a novel mechanosensing mechanism: upon cell compression, cells are known to use caveolae formation  and the Drosophila embryos  and cultured in DMEM without pyruvate (Thermo Fisher 41965-039) supplemented with 10% FBS (vol/vol), 1% penicillin-streptomycin (vol/vol), and 1% insulin-transferrin-selenium (vol/vol) (Thermo Fisher 41400045). WT MEF2 were derived from IRSp53 embryos . IRSp53  embryos , leadingAAAAGCCTACTACGACGGCG-3’) was subcloned into expression plasmid pSpCas9(BB)–2A-Puro(PX459)V2.0 (Addgene plasmid ID: 62988) and sequences validated by sequencing. 24 hr after transfection, MEFs IRSp53-/- cells were selected for 5 days with culture medium containing 1 μg/ml puromycin (AdipoGen). Clones derived from single cells were obtained by selected population employing serial dilution protocol.Genome editing by CRISPR/Cas9 was more precisely as follows: IRTKs sgRNA (5’-2-independent media (Thermo Fisher 18045088) was used for microscopy imaging and was supplemented with 10 µg/ml of rutin (Sigma R5143) to prevent photobleaching (The culture was maintained in DMEM with 20% FBS (vol/vol) supplemented with 1% penicillin-streptomycin (vol/vol) and 1 µ/ml puromycin to selectively maintain cells expressing the selection vector. COleaching . mCherryleaching . mEmeralleaching , EGFP-IRleaching , and EGFleaching  containeleaching  were creleaching . Actin wOn the day prior to the experiment, cells were transfected by electroporation with the selected plasmids using the Neon Transfection System (Invitrogen) following the protocol provided by the company. CK-666 was purchased from Merck (Ref 182515), SMIFH2 was from Abcam (ab218296), Wiskostatin was bought from Sigma (W2270), and Para-Nitro-Blebbistatin was from Optopharma (DR-N-111). All compounds were diluted in DMSO and conserved according to the manufacturer’s instructions. On the day of the experiment, drugs were diluted in culture media, filtered through a 0.22 µm filter and warmed up to 37°C prior to addition to the culture. Cells were treated with 25 µM of CK-666 for 30 min, 10 µM of PNB for 30–40 min, and 10 µM Wiskostatin or 15 µM SMIFH for 1 hr prior to the experiment.-/-R MEF cells were seeded in a six-well plate (150k cells/well); next day, each well was transfected using lipofectamine  with 10, 25, or 50 nM of control siRNA  or siRNA against NCKAP1 to silence the Nap1 protein . Cells were used 72 hr after transfection occurred.IRSp53-/-, and IRSP53-/- C/C9IRTKs cells was extracted using the RNeasy Mini kit (QIAGEN) and quantified by NanoDrop to assess both concentration and quality of the samples. One µg of total RNA was subjected to reverse transcription using the High-Capacity cDNA Reverse Transcription Kit from Applied Biosystems. Gene expression was analyzed by using the TaqMan Gene expression Assay (Applied Biosystems). 0.1 ng of cDNA was amplified, in triplicate, in a reaction volume of 25 μl with 10 pmol of each gene-specific primer and the SYBR Green PCR MasterMix (Applied Biosystems). Real-time PCR was performed on the 14 ABI/Prism 7700 Sequence Detector System (PerkinElmer/Applied Biosystems) using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Specificity of the amplified products was confirmed by melting curve analysis  and by 6% PAGE. Preparations with RNA template without reverse transcription were used as negative controls. Samples were amplified with primers for each gene  and GAPDH as a housekeeping gene. The cycle threshold Ct values were normalized to the GAPDH curve. PCR experiments were performed in triplicate and standard deviations calculated and displayed as error bars. Primer assay IDs were: Gapdh, mm99999915_g1; Baiap2 (IRSp53), mm00499943_m1; Baiap2l1 (IRTKs), mm00508802_m1; Baiap2l2 (Pinkbar), mm00616958_m1; Mtss1 (MIM), mm00460614_m1; Mtss1l (ABBA), mm01244296_m1.Total RNA of WT, IRSP53-/-R cells transfected with control siRNA or siRNA against NCKAP1 were isolated with the RNAeasy kit (QIAGEN) or High Pure RNA Isolation Kit (Roche). One µg of total RNA was subjected to reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad). 0.1 ng of cDNA was amplified, in triplicate, in a reaction volume of 20 μl with 10 pmol of each gene-specific primer and the Fast SYBR Green MasterMix (Thermo Fisher). Real-time PCR was performed on the StepStonePlus Real-Time PCR System (Applied Biosystems) using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Samples were amplified with primers for each gene  and GAPDH as a housekeeping gene. The cycle threshold Ct values were normalized to both GAPDH and Rn18x curves. PCR experiments were performed in triplicate and standard deviations calculated and displayed as error bars. Primer assay IDs were: Nap1 , GAPDH (FW ATCCTGCACCACCAACTGCT and RV GGGCCATCCACAGTCTTCTG), and Rn18x (FW GCAATTATTCCCCATGAACG and RV GGCCTCACTAAACCATCCAA).Total RNA of IRSp53The mouse monoclonal anti-IRSp53 was generated in IFOM . The rabThe stretchable PDMS membranes were prepared as described in After overnight fibronectin coating, PDMS membranes were quickly washed and 3000 cells were seeded on top and allowed to spread for 45 min to 1 hr in the incubator. Then, rings were mounted on the stretch device coupled to the microscope stage, vacuum was applied for 3 min to stretch the membrane, and then vacuum was released to come back to the initial shape as described in Cells were prepared as explained in the previous section. Right after stretch release, the sample was fixed in 2.5% glutaraldehyde EM grade (Electron Microscopy Sciences 16220) plus 2% PFA (Electron Microscopy Sciences 15710S) diluted in 0.1 M phosphate buffer (PB) at 37°C for 1 hr. Samples were then washed 4× for 10 min in 0.1 M PB and imaged with epifluorescence microscopy as described below to acquire fluorescence images of the cell PM. PDMS membranes were then cut into 1×0.5 cm rectangles in which the pattern was centered. For confocal Airyscan imaging, cells were fixed as abovementionned and PDMS membranes were cut in rectangles and fitted in a glass bottom dish before fluorescent imaging. Samples were subsequently placed on top of 12 mm coverslips for further processing. Dehydration was carried out by soaking samples in increasing ethanol concentrations . After this, samples were critical point dried and covered with a thin layer of gold to be imaged.4 and 0.8% K3Fe(CN)6 for 1 hr at 4°C in the dark. Next, dehydration in increasing ethanol concentrations  was done. Samples were then embedded in increasing concentrations of Pelco EPONATE 12 resin (Pelco 18010) mixed with acetone. 1:3 infiltration was done for 1 hr then 2:2 for 1 hr and finally 3:1 overnight. On the next day, embedding was continued with EPON12 without catalyzer for 3×2 hr washes and then overnight. Last, samples were embedded in EPON12 plus catalyzer DMP-30 (Pelco 18010) for 2×3 hr. To finish, blocks were mounted and polymerized for 48 hr at 60°C. PDMS membrane was next peeled off and ultrathin sections were cut and mounted on grids for imaging.Cells were fixed, washed, and PDMS membranes were cut and mounted as for SEM imaging. After this, samples were postfixed with 1% OsO2O2, 31642, Sigma). The samples were washed three times with cold 0.1 M Cacodylate buffer, and subsequently incubated for 30 min with cold 1% OsO4. Dehydration, resin embedding, and block mounting were done as described in the TEM experiments section.Two days prior to the experiment, cells were co-transfected by electroporation with mKate2-P2A-APEX2-csGBP (Addgene #108875) and EGFP-IRSp53-FL in a 3:1 ratio, using the Neon Transfection System (Invitrogen) following the protocol provided by the company. Before seeding, cells were sorted for double positive mKate and GFP fluorescence, excluding very high and very low transfection levels. Cells were subsequently seeded and stretched in the same conditions as explained in the stretch experiments section. Right after stretch release, the sample was fixed in 2.5% glutaraldehyde EM grade (Electron Microscopy Sciences 16220) diluted in 0.1 M Cacodylate buffer at 37°C for 10 min, followed by incubation on ice for 50 min in the presence of the fixative. All subsequent steps were performed on ice. The sample was washed three times with cold 0.1 M Cacodylate buffer, and next cut into 1×0.5 cm rectangles containing the fixed cells. Cells were washed for 2 min with a fresh cold 1 mg/ml 3,3’-diaminobenzidine (DAB)  solution in 0.1 M Cacodylate buffer. Cells were immediately incubated with a fresh cold 1 mg/ml DAB solution in cold 0.1 M Cacodylate buffer supplemented with 5.88 mM hydrogen peroxidase (PERDROGEN 30% HFluorescence live cells images were acquired with Metamorph software using an upright microscope (Nikon eclipse Ni-U) with a 60× water dipping objective  and an Orca Flash 4.0 camera (Hamamatsu). Fluorophore emission was collected every 3 s. Cells were imaged in a relaxed state and then for 3 min at 5% stretch, and for 3 min during the release of stretch. Fixed cells images were acquired either in the abovementioned acquisition system except for the experiments related to WAVE protein enrichment. In this case, images were acquired in a Zeiss Airyscan microscope . Z-stack of single cells were acquired in full Airyscan mode to visualize the PM and the WAVE-C-GFP protein. SEM images were taken using the xTm Microscope Control software in a NOVA NanoSEM 230 microscope (FEI Company) under the high vacuum mode using ET and TL detectors to acquire high and ultra-high resolution images of the cell surface. TEM samples were observed in a Jeol 1010 microscope  equipped with a tungsten cathode in the CCiTUB EM and Cryomicroscopy Units. Images were acquired at 80 kV with a CCD Megaview 1kx1k.Ievag), the integrated fluorescence signal of a neighboring ROI of the same size and devoid of any structures (IPM), the integrated fluorescence signal of the entire cell (Icell), and the integrated fluorescence signal of a background region of the same size as the cell (IBG). Then, the final evagination signal Ifinal was computed as:All images used for time course analysis were aligned using the Template Matching plugin from Fiji to correct the drift. To assess the evolution of PM evaginations or the different marked proteins, their fluorescence was quantified. To ensure that we only considered the fluorescence of structures induced by stretch, the analysis was carried out in regions devoid of visible endomembrane structures before the application of stretch. For each evagination, we calculated the integrated fluorescence signal of a small region of interest (ROI) containing the evagination  and the rest of the data represented in the same graph were normalized to the control. Exceptionally, Actin and Ezrin curves were normalized to 0.5  for visualization purposes. To quantify the degree of resorption of the evaginations, as each experimental data from an evagination could not always be fitted with single exponential decay curve, we adopted the strategy of fitting the average decay curve from 10 evaginations of a single cell as a function of time k (s–1) representative of the resorption capacity of the cell, on which statistical analysis can be performed. Lag time was calculated by identifying the maximum intensity timepoints in the protein and PM channels, and subtracting them to obtain the time between the two events. Lag time was calculated for each individual evagination, and an average of the 10 evaginations of a cell was calculated to obtain a representative lag per cell. This data was used to plot lag time versus decay of each cell. When time of maximum fluorescence was plotted, a similar procedure was followed but maximum fluorescent peak averaged for each cell was displayed instead of lag, in both protein and membrane channels.We obtained a decay constant The enrichment analysis was performed on the z-stack images acquired in Airyscan mode of cells co-transfected with the mCherry-membrane marker and the WAVE-C-GFP protein. Though sample preparation was optimized, a single slide of a stack did not capture the best signal for an entire cell. Therefore, the following treatment was performed in both channels: the slice of maximum intensity was identified and a z-projection of the previous and post three slides (sum of seven slides) was performed. Enrichment was calculated using this projection in the following manner: evaginations were identified in the SEM image and 10 evaginations for which both membrane and WAVE protein displayed a fluorescent signal were quantified. We calculated the mean intensity of a small ROI containing the evagination in the protein channel (mf contained in evaginations was estimated as:Three regions of different parts of the cell were randomly chosen from every cell at the timepoint t0s (right after the release of stretch) and the number of evaginations was manually counted by comparing the analyzed images with the images of the cell during stretch, to discard PM structures not formed by stretch release. For stored area calculation, the membrane area fraction ize is the average fluorescence intensity of a cell zone (containing evaginations), izf is the average fluorescence intensity of a neighboring flat patch of membrane , and bg is the average intensity of background. For each cell, this was done for three random regions containing evaginations.where Images of the fixed sample were acquired in fluorescence and brightfield and positions of the imaged cells in the pattern were noted down. Sample was then processed for SEM imaging and the same cells were found by manually following their location on the pattern, and visual verification was done to check for correct matching. Fluorescent and SEM images were then aligned by using the BigWarp plugin on Fiji.N values can be found in each one of the graphs shown in the figures.In the case of data following a normal distribution, t-test or ANOVA was done depending on whether there were two or more datasets to compare. For data not following normal distributions, Mann-Whitney or Kruskal-Wallis tests were applied depending on whether there were two or more datasets to test. All data are shown as mean ± SEM. Specific p and To understand the physical mechanism leading to the active flattening of membrane evaginations caused by compression of the PM, we focused on a single evagination and described it mathematically under the assumption of axisymmetry. We modeled the membrane as locally inextensible thin sheet with bending rigidity N · s/µm .µ is a friction coefficient, which we took as 3  out-of-plane polymerization forces driving further extension of the evagination and (2) a lateral contractility gradient driving centrifugal actin flow, which drags and tends to flatten the evagination. This competition may lead to different outcomes depending on the relative strength. If (1) is negligible, then the centrifugal actin flow irons out the evagination very abruptly. If (2) is negligible, then polymerization forces may extend the evagination further. In an intermediate regime that agrees with observations, lateral actin flow controls the outcome, that is recovery of membrane cortex homeostasis, but out-of-plane polymerization forces affect the dynamics by delaying flattening. In this important paper, the authors characterize and model a novel self-organizing circuit used by cells to correct for nanoscale membrane evaginations that form upon a rapid drop in membrane area. The evidence supporting the conclusions is compelling and provides new insights on plasma membrane-cortex coupling. It is of interest to mechanobiologists and biophysicists and could be relevant in processes in developmental biology or in tissues where the cell area changes rapidly, such as in muscles, heart, and lungs. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including X as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Suzanne Pfeffer as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Milos Galic (Reviewer #3).Thank you for submitting your article "A mechanosensing mechanism mediated by IRSp53 controls plasma membrane shape homeostasis at the nanoscale" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers have considered that your work represents a well-executed study in a very defined reductionist model system. It provides new insights on plasma membrane-cortex coupling. After discussion, we think that your manuscript would nevertheless benefit from the following revisions:1) Strengthen the model:One issue of the paper is that the authors do not provide test of their model other than the absence of effect of Myo2 and do not discuss other mechanisms. The authors consider that the resorption is essentially related to actin production at the place where IRSp53 is localized, which induces an actin flow parallel to the plasma membrane, away from the evagination. Practically, this mechanism should also be present in any membrane protrusion, including filopodia. Thus, it would be important to compare the different actin fluxes at the evagination to determine the dominant contribution. What about the retrograde flow (perpendicular to the flow considered here) which would also lead to a resorption, and the protrusive forces that would counteract them? The authors mention the protrusive forces but do not explain why they are not strong enough.2) Probe for additional proteins at the evagination sites2.1. Other I-BAR proteins:The resorption effect does not disappear in the absence of IRSp53 . It becomes just slower , and actin is recruited but less . Considering their redundant function, additional I-BAR domain proteins may contribute to this mechanism as well. To address this possibility, the authors should monitor the localization of other candidate proteins  at membrane evagination. Optionally, protein function could be tested via knockdown to delineate the contribution of individual I-BAR domain proteins.2.2. Other N-BAR proteins:eLife 3 (2014)). Could it be the case here too?It was shown for filopodia that N-BAR domain proteins limit their growth , and lead to resorption of the bud. This is not considered in the paper and it the authors should test whether endocytosis plays a role.3) Better characterization of phenotype:3.1. Apical vs basal:The phenomenon seems to occur entirely on the apical side as claimed in Figure 1, but the authors do not show what happens on the ventral part. The authors should clarify this point and provides evidence that the dots in the fluorescence image in Figure 1 are all apical.3.2. Resorption time versus characteristic timeThe authors mention that they cannot always measure the characteristic time of the evaginations flattening, and thus measure their time of resorption. But it becomes more difficult to compare quantitatively between different conditions. In particular, when they compare the time of resorption for different constructs, since this time might depend on the level of transfection/expression of the different IRSp53 constructs, the authors should normalize their data by the expression levels. Moreover, if the initial bud size varies, the full resorption will take longer even with the same decay time. This seems to be the case when comparing data with and without IRSp53 : the decay time seems similar but with a higher excess area at time zero in the absence of IRSp53   which would correspond to larger buds. Thus, comparing the full duration of the processes rather than the characteristic time is probably not appropriate. Essential revisions:The reviewers have considered that your work represents a well-executed study in a very defined reductionist model system. It provides new insights on plasma membrane-cortex coupling. After discussion, we think that your manuscript would nevertheless benefit from the following revisions:1) Strengthen the model:One issue of the paper is that the authors do not provide test of their model other than the absence of effect of Myo2 and do not discuss other mechanisms. The authors consider that the resorption is essentially related to actin production at the place where IRSp53 is localized, which induces an actin flow parallel to the plasma membrane, away from the evagination. Practically, this mechanism should also be present in any membrane protrusion, including filopodia. Thus, it would be important to compare the different actin fluxes at the evagination to determine the dominant contribution. What about the retrograde flow (perpendicular to the flow considered here) which would also lead to a resorption, and the protrusive forces that would counteract them? The authors mention the protrusive forces but do not explain why they are not strong enough.We agree with the referees that our model in the original submission focused on the novel mechanism proposed to explain flattening of an evagination as a result of localized Arp2/3 actin polymerization. The literature amply discusses how actin polymerization exerts out-of-plane forces on the membrane, and hence favors membrane projections, whereas our experiments show that localized polymerization drives flattening. This motivated our proposed mechanism, based on a lateral gradient of cortical tension driving centrifugal flows. We agree that this mechanism is quite generic, and may also be present in other situations where localized polymerization drives membrane extensions. We note that the rationale of our model applies to Arp2/3 polymerization, and not to formin-based polymerization typical in filopodia, since Arp2/3 polymerization can push the membrane outwards, but can also compete with the contractile component of the actin network to locally reduce contractility.However, the question of what controls the competition between out-of-plane protrusive forces and the proposed in-plane flattening mechanism remains.Following the suggestions of the referees, in the revised manuscript we have extended our model. The reviewers mention retrograde flows and protrusive force. Regarding retrograde flows, we note that their length scales are larger than the 100 nm scale of our invaginations, and therefore could potentially displace but not likely reshape evaginations. Consistently, we did not observe any appreciable differences in the behavior of evaginations in the cell center, lamella, or lamellipodia, where retrograde flows should be very different. We therefore focused our model extension on protrusive forces. In our extended model, we accounted not only for the lateral tension gradients driving centrifugal actin flows that flatten the membrane, but also for out-of-plane protrusive polymerization forces. Based on quantitative in-vitro measurements of the relation between polymerization velocity and opposing pressure, we introduced a growing network perpendicular to the plane (coarse-grained as a filament at the center of the evagination), which upon contact with the membrane exerts a normal force on it, which can slow-down, stall, or reverse its growth velocity.For reasonable parameter estimations, we find that this force is not able to stop the resorption and recovery of membrane/cortex homeostasis, but it slows down significantly the process. In our previous version of the model, resorption of the evagination was very abrupt (much faster than in experiments) and related to a mechanical snapping of the membrane bud under tension. With the modified model, snapping is limited by the disassembly of the out-of-plane network under membrane force, and instead we find a progressive resorption that better agrees with the experiments.In the revised manuscript, figures and Methods section, we describe and motivate this more complete version of the model. We further consider two alternative scenarios to better appreciate individually the lateral and out-of-plane mechanical effects of localized polymerization. The first one corresponds to the model in our original submission, which leads to homeostasis but in an unrealistically abrupt manner. The second one considers only the out-of-plane part of the model. We show that depending on the strength of polymerization against the membrane, which can depend on a variety of actin regulators and cross-linkers, out-of-plane localized polymerization can result in a stable evagination or in its extension.2) Probe for additional proteins at the evagination sites2.1. Other I-BAR proteins:The resorption effect does not disappear in the absence of IRSp53 . It becomes just slower , and actin is recruited but less . Considering their redundant function, additional I-BAR domain proteins may contribute to this mechanism as well. To address this possibility, the authors should monitor the localization of other candidate proteins  at membrane evagination. Optionally, protein function could be tested via knockdown to delineate the contribution of individual I-BAR domain proteins.As the reviewers point out, the resorption effect is not total in the curves we show of the IRSp53 KO cell line, compared with the control cells reconstituted with IRSp53 proteins. As the reviewer suggested, this may come from the fact that I-BAR domain proteins may have redundant functions in this process. We verified the expression levels of the different proteins from the IBAR-domain family in the KO and reconstituted cell lines using RT-PCR. This revealed that IRTKs and MIM proteins were overexpressed (by 10 and 30 times fold respectively) in the IRSp53 KO cell line, and this overexpression was conserved in the reconstituted control  remain valid. After correcting our mislabeling, these experiments were carried out in IRSp53-reconstituted rather than KO cells, but the conclusions here remain valid. First, overexpressing WT IRSp53 has no effect, thereby showing that transient overexpression per se does not affect membrane resorption. Then, overexpression of mutant IRSp53 protein impairs resorption, likely through a dominant-negative rather than rescue effect. We have verified that the effect on resorption holds when decay times rather than fluorescence intensities are calculated. Further, the effect on decay times still correlates with the delay in protein recruitment, as we reported in our first submission.Apart from these results with transient mutant transfection, we have also verified localization of IRSp53 using the Apex technique and confirmed its enrichment in the evagination. Our results also show the role of Rac1 and Wave Regulatory Complex (WRC) in the resorption process (we now have given stronger evidence in the revised manuscript as explained in the Response to point 2.3). The synergy in IRSP53, Rac1 and WRC interactions leading to Arp2/3-mediated actin polymerization is very well described. Therefore, we think that our data show that IRSp53 participates in recognizing the curved structure of the evaginations, leading to WRC recruitment in a feedback loop promoting actin polymerization. After this revision, it is clear that IRTKs and MIM proteins may play similar or compensatory roles, and therefore the role of IRSp53 may not be unique. We have toned down the manuscript conclusions accordingly. We have also modified substantially Figure 2 and 3 to update for these changes, and introduced Figure 2—figure supplement 1 and Figure 3—figure supplement 2 with additional new results. We have also modified the text accordingly.2.2. Other N-BAR proteins:It was shown for filopodia that N-BAR domain proteins limit their growth ). Could it be the case here too?This is an interesting possibility. In the paper by Galic et al., an N-BAR protein is recruited through curvature sensing at the neck of nascent filipodia, which arise at previously formed actin patches. The negative curvature is generated through acto-myosin contraction from these patches, counterbalancing the protruding forces necessary for filipodia extension. The N-BAR protein acts as a Rac1 inhibitor therefore limiting growth of the filipodia. The mechanism described is very different from our findings, in which evaginations are passively formed at stochastic places of the plasma membrane to accommodate excess area. In our case, Rac1 activation promotes the flattening rather than growth of the evagination, so we do not think that this N-BAR-mediated mechanism can explain the response. Further, in an initial screening that we carried out at the beginning of this project, results of our preliminary experiments overexpressing some N-BAR or F-BAR domain containing proteins  did not show a slower resorption. We did not see either a faster resorption in stable cell lines where we silenced Amphyphisin, FCHO1, or GRAF1. Of course, we could test more proteins, and there could potentially be compensation mechanisms, such as the one described for IRSp53 above. However, in the manuscript we preferred to focus on I-BAR domain containing proteins and subsequent actin nucleating factor recruitment, due to their potential role in mediating the restoration of membrane homeostasis.To better clarify this issue, we now mention the potential contribution of other BAR proteins in the discussion of the manuscript (page 9).2.3. Actin dynamicsAlthough it seems likely that IRSP3 triggers Rac and then Arp2/3 via WAVE, this is not supported by direct evidence. Moreover, it is not clear that this happens really locally or if global effects contribute. Hence it is important to localize Arp2/3 and components of the WAVE complex at the evagination sites. It is also important to knock out or knock down the WAVE complex to nail down the importance in this context.As the reviewer asked, we have studied the localization of the Wave complex components at the evagination. This kind of study is not straightforward given that any overexpressed, membrane-bound fluorescent protein will show an increase in signal at the evagination due to the formation of a 3D-shape membrane. For IRSp53 we have used the Apex technique, which was very complicated and low yield. Therefore, for the Wave complex, we decided to overexpress pEGFP-C3 Wave2  and compared its enrichment relative to the plasma membrane fluorescent marker used for membrane labelling throughout the study. For this, we fixed the cells just after stretch release, and imaged then in Airyscan mode, which has improved resolution compared with classical confocal microscopy. To make sure we were restricting the analysis to evaginations, we prepared the same samples for electron microscopy imaging and used, as previously done, a pattern on the PDMS substrate to obtain SEM images of the same cells. For the structures clearly identified as evaginations in the SEM image, we calculated the ratio between the fluorescence intensity in the evagination, and in an adjacent area containing only flat membrane. This ratio was significantly higher for the WAVE-C-GFP signal than for the membrane signal, , which confirms enrichment of the complex at the evagination.2 of cell membrane. Cells silenced for Nap1 (thereby severing Wave complex functions) showed a significantly higher amount of evaginations , indicating that the resorption was impaired under this condition.We also proceeded to silence the Nap1 protein from the Wave complex (Nap1 is present in both Wave1 and Wave2 complexes) using siRNA. We subjected cells to 50nM siRNA targeting the Nap1 gene and using RT-PCR, we saw a robust decrease of Nap1 RNA levels when compared with 50nM of non-targeting siRNA control. We initially had the idea of co-transfecting the siRNA together with the plasma membrane marker to perform live experiments, unfortunately the yield of the fluorescence labelling in this context was extremely low. Therefore, we opted for an alternative experiment employing SEM imaging at a fixed time, as the images obtained by SEM very clearly show the evaginations without the need for fluorescence labelling. We fixed the sample 25 s after the release of the stretch to allow for some resorption to occur and compared the images of cells where Nap1 was silenced compared with the non-targeting siRNA control. We quantified the number of evaginations per µmOverall, we believe that the use of CK666 to inhibit Arp2/3 (which clearly showed impeded resorption) combined with this new evidence of WAVE localization and function at the evagination convincingly show that the actin polymerization event observed at the evagination is driven by this molecular cascade.We have now inserted these results in Figure 5 and Figure 5—figure supplement 1, and their corresponding explanation in the text (page 8).Next, if they confirm their hypothesis, the authors could also comment on why the machinery for protrusion growth is not active at these sites, for instance why formins are not recruited and only Arp2/3 dependent polymerization takes place.This is an interesting question. We have shown in the past  that mDia is not recruited at the Cdc42/VASP/IRSp53 machinery foci driving filopodia elongation in MEFs. This may be used to underline that different machinery may be recruited and function at different sites (not necessary in common) and that, at least, formins may be excluded by some IRSp53 driven actin regulatory processes. Additionally, treatment with the formin inhibitor SMIFH2 reduced the number of filopodia as expected but did not affect evagination resorption. These results are shown in Figure 4—figure supplement 1C and D. Supporting this notion, our modelling results show that membrane flattening can be explained by an isotropic actin flow, which would be mediated by branched rather than linear polymerization. We now comment on this fact in the discussion part of the revised manuscript.2.4. ClathrinThe process lasts over 3 min for the full resorption. This leaves time for other mechanisms that could influence lipid membrane total area, thus its tension (clathrin-independent endocytosis for instance that depends on actin), and lead to resorption of the bud. This is not considered in the paper and it the authors should test whether endocytosis plays a role.We agree with the reviewers that upon stretch release, endocytosis indeed occurs and plays a role in plasma membrane homeostasis. This was the central topic of one of our previous publications , where we characterized how membrane tension decrease upon stretch release affects CDC42-mediated CLIC-GEEC endocytosis (but not clathrin-mediated endocytosis). In the original submission of this manuscript, we acknowledged that endocytosis increases in response to decreased plasma membrane tension, and we cited the Thottacherry et al. study in our introduction. However, this cannot explain our results for two main reasons. First, the actin polymerization pathways involved are different . Second, endocytic buds do not colocalize with stretch-mediated evaginations, as also characterized by Thottacherry et al. We have now clarified this in the introduction and in the discussion (page 9), which we have modified with our revised findings.3) Better characterization of phenotype:3.1. Apical vs basal:The phenomenon seems to occur entirely on the apical side as claimed in Figure 1, but the authors do not show what happens on the ventral part. The authors should clarify this point and provides evidence that the dots in the fluorescence image in Figure 1 are all apical.We are aware, as the reviewers correctly point out, that we only show evaginations of the apical side of the cell. We have attempted to characterize what happens on the ventral part using electron microscopy as well but were not successful. Using TEM to prepare the samples, we must peel the cells from the PDMS membrane to proceed with serial cuts, but in this process the ventral membrane is torn. We have tried to unroof cells just after stretch release and perform SEM imaging, but the whole protocol was quite slow compared with the resorption time scale, and also rendered dirty samples that could not resolve the structure of basal invaginations. Still, alignment of the SEM images with the fluorescence stainings  show that a large part of the fluorescence dots come from evaginations at the apical membrane. We do know that curved membrane structures appear at the ventral part as well  and we hypothesize that invaginations will form at the ventral part case given the high rigidity of the PDMS substrate. We also hypothesize that in physiological situations, invaginations will emerge if it is more favorable for the membrane to push inward the cytoskeleton, which would be the case of very rigid and non-porous substrates. In many other cases, evaginations will emerge if is it more favorable to bend the membrane outwards, as happens here at the apical side of the cell. We only studied the machinery involved in the resorption of the evaginations, which in our case was the majority of the structures formed, and that we think are highly physiologically relevant. We now clarified this aspect in the introduction (page 2).3.2. Resorption time versus characteristic timeThe authors mention that they cannot always measure the characteristic time of the evaginations flattening, and thus measure their time of resorption. But it becomes more difficult to compare quantitatively between different conditions. In particular, when they compare the time of resorption for different constructs, since this time might depend on the level of transfection/expression of the different IRSp53 constructs, the authors should normalize their data by the expression levels. Moreover, if the initial bud size varies, the full resorption will take longer even with the same decay time. This seems to be the case when comparing data with and without IRSp53 : the decay time seems similar but with a higher excess area at time zero in the absence of IRSp53   which would correspond to larger buds. Thus, comparing the full duration of the processes rather than the characteristic time is probably not appropriate.We thank the reviewer for insisting on that point and agree with them that the decay constant  is more informative for our study. The reason for not measuring decay constants in our original submission was that fluorescence curves from individual reservoirs were often noisy, and not well fitted by an exponential decay curve. To circumvent this, we have now calculated an average decay curve for all the reservoirs within a given cell, and then fitted this average curve. In this way, noise is largely averaged out, and the curves are well fitted to an exponential decay. Overall, the fit does not drastically change the results, except for the shRNA data, as discussed in detail in response to reviewer question 2.1. All the data have now been fitted with an exponential decay curve and comparison of the decay constants have been used in the analysis.


Reasoning is a complex form of human cognition whose nature has long been debated. While a number of neurocognitive mechanisms for deductive reasoning have been offered, one of the most prominent accounts is Mental Model Theory (MMT). According to MMT, humans are able to manipulate and represent information for reasoning and problem solving by leveraging the brain’s evolved visuospatial resources. Thus, when solving deductive reasoning problems, reasoners build “mental models” of the essential pieces of information conveyed in the premises, with their relations to each other represented spatially—even when the information contained within a reasoning problem is not intrinsically spatial. Crucially, taking a spatially-based approach, such as building mental models, supports higher accuracy on deductive reasoning problems. However, no study has empirically tested whether explicitly training this mental modeling ability leads to improved deductive reasoning performance.https://osf.io/4b7kn), we conducted a between-subjects experiment (N = 301) which compared the Mental Models Training App to 3 distinct control conditions in order to examine which specific components (if any) of the training were causally responsible for improved reasoning performance.Therefore, we designed the Mental Models Training App, a cognitive training mobile application which requires participants to complete increasingly difficult reasoning problems while using an external mental modeling tool. In this preregistered study , in the hope that this translational research may be utilized by the general public to improve their reasoning ability.Therefore, while the present results demonstrate the ability of the Mental Models Training App to enhance verbal deductive reasoning, they do not support the hypothesis that directly training participants mental modeling ability yields improved performance beyond the effects of adaptive practice of reasoning. Future research should examine the long-term effects of repeated usage of the Mental Models Training App, as well as transfer effects to other forms of reasoning. Finally, we present the Mental Models Training App as a free mobile application available on the Apple App store ( The primate brain evolved to interact with objects in space rather than interact with complex logic structures, so a great deal of the cerebral cortex is devoted to visuospatial and motor processing . Accordiabove a worse option, “spatializing” the concept of goodness, which is not inherently spatial. Several theories of human reasoning suggest that these sorts of problems, often called linear syllogisms, are solved using internal representations which are spatially ordered  and simply completed the MRRT posttest 24 h after they completed the pretest.2.4.2.All training conditions (Conditions 1–3) were completed by participants on their iPhones through the TestFlight application, which allowed participants to download a specific version of the training app using a condition-specific password provided by the researchers. Upon opening the app, participants entered their Prolific ID number along with the condition-specific password. The title of the app (“Reasoning Training”), the instructions provided about the reasoning problems , and the overall structure of the app (adaptive reasoning training with increasingly difficult problems) was kept the exact same across all conditions see  to creathttps://osf.io/a8zyn/.In all training conditions, participants completed the same 4 levels of increasingly difficult MRRT problems see . The ver2.4.3.In order to control for the effects of practicing verbal deductive reasoning problems in a mobile application, Condition 1 was designed the same as Conditions 2 and 3, except that there was no spatial tool included in the training. Participants still received instructions for solving reasoning problems, the problem timing remained the same, correct/incorrect feedback was still provided after each problem, and the levels still advanced in the same increasingly difficult manner. However, the cycles within each level only included 2 successive reasoning problems see  and ther2.4.4.In order to control for the visual, spatial, and motor processes engaged by using a spatial tool during the reasoning training, Condition 2 matched the design of Condition 3, but provided participants with a spatial alphabetization tool  instead after each reasoning problem using a different set of names than those shown in the previous problem. This design ensured that participants were not distracted during the reasoning problem  and that they could not use the alphabetization tool in order to create mental models during the reasoning problems or retrospectively after solving reasoning problems. Relatedly, participants in Condition 2 completed cycles with the following components: 1) complete a reasoning problem without a tool, (2) alphabetize a separate list of names in the specific spatial configuration and alphabetical direction, (3) for non-spatial problems, verbally explain how they used the spatial alphabetization tool to arrange the names to form the alphabetized shape (4) complete a new reasoning problem, (4) alphabetize a separate list of names in the specific spatial configuration and alphabetical direction  was that it provided participants with a spatial modeling tool to create external mental models while solving increasingly difficult reasoning problems in the app’s levels. The spatialization tool was introduced during the instructions section of the app, wherein participants were shown (1) a visual example of how the tool could be used to represent reasoning problems in a spatial manner, (2) how to tap in the workspace to create pre-labeled tokens for each of the names in a reasoning problem, (3) how to move the tokens around within the workspace to create a mental model for a reasoning problem, and (4) an example reasoning problem in which they could use the tool to create a mental model and solve the problem. After completing the instructions, participants began level 1 of the training.Within each level of the Mental Models Training App (Condition 3), participants completed cycles with the following structure: (1) complete a problem using the spatialization tool to create mental models of the names in the premises, (2) for non-spatial problems, verbally explain how they solved the problem using the spatialization tool, (3) complete a new problem without the use of the spatialization tool, and (4) use the tool to spatially explain how they solved the previous problem see . The goaTypical responses to the verbal explanation prompt for non-spatial problems in Condition 3 included: “I used the tool similar to above and below to rank the level of excitement,” “I placed those who were more patient further to the right than those who were less patient,” and “I used the visual tool to show the hierarchy.” The prevalence of these sorts of responses suggested that the spatialization tool was generally used as intended. In addition, thorough visual inspection of the mental models created throughout the training by participants in this condition confirmed that the mental modeling tool was utilized as intended.2.5.via mixed-effects linear regression. All models estimated fixed effects, given that the high number of variables included made random slope estimations computationally infeasible  from pretest to posttest, we conducted a series of mixed-effects models testing for condition-by-time interactions. Mixed-effects models are appropriate when several repeated measurements or observations (Level 1) are nested within a higher level of data Level 2; . In the feasible . All mixfeasible . Signifi3.3.1.Descriptive statics for all variables measured at pretest (separated by condition) can be found in 3.2.p > 0.38) or RT .We ran two mixed-effects models  to examine whether each of the training conditions (1–3) significantly improved MRRT performance from pretest to posttest, using the passive control condition with no intervention (condition 0) as the reference factor level. All models controlled for stimulus properties of the MRRT problems  and demographic characteristics of the participants . Results indicated significant condition-by-time effects of all three conditions (1–3) on MRRT accuracy  and RT . Conditi3.3.Next, we examined differences in performance within the training app across conditions 1–3 (condition 0 was not included as it did not include the app intervention). Participants in condition 1 (adaptive training) completed the training in an average of 21.93 min, which was significantly shorter  than the average completion time in condition 2  and condition 3 , Condition 1 , or Condition 2 .Based on the finding that participants in Condition 3 showed improved performance on 3-premise problems in level 3 of the mobile training app (compared to Conditions 1 and 2), we conducted exploratory analyses testing for a significant three-way interaction between Condition-Time-Premises on reasoning performance (examining the posttest training effects in Condition 3 as compared to the other conditions). Results indicated no significant Condition-Time-Premises interaction for Condition 3 compared to: Condition 0  significantly improved verbal deductive reasoning performance, as indicated by increased accuracy and reduced reaction time on the MRRT , compareWe did not find evidence for an additive benefit of the spatialization tool, nor a closely matched control version of that tool, for improving reasoning performance after the training. In line with prior research on cognitive training , it is pduring the mobile training app. Specifically, participants in the Mental Models Training App training completed level 3 of the training  with significantly fewer total attempts (an average of 8 problems completed compared to 12 problems in both of the control conditions). Previous research on deductive verbal reasoning has found that the single most impactful stimulus factor on problem difficulty is the number of premises .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The data and code for this study can be found in the Open Science Framework  and the John Temple Foundation for AG and AW [ID 61114]. RC was supported by a National Science Foundation Graduate Research Fellowship and by the Patrick Healy Graduate Fellowship from Georgetown University.RC and AG are the developers of intellectual property owned by Georgetown University related to the Mental Models Training App technology that is described in this manuscript. Although the app is free to download, it includes advertisements and in-app purchases that have the potential to generate revenue for RC, AG, and Georgetown University. Google manages all aspects of the advertisements including content and placement. Furthermore, in-app advertisements do not necessarily represent the data presented in the app.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MAGEC rods (NuVasive) provide distraction growth in early-onset scoliosis. Pedicle screw use with MAGEC rods can lead to anchor failure. Sublaminar bands offer superior fixation points for the MAGEC system while preserving pedicles and facets, avoiding spinal cord injury, and eliminating the need for fluoroscopy. Sublaminar bands can be safely used up to cervical vertebra four (C4), substantially decreasing the risk of complications such as anchor pull-out, rod breakage, and proximal junctional kyphosis that typically occurs with pedicle screws and hooks. This case demonstrates the viable option of sublaminar band fixation as an anchor system for MAGEC rods. This is a retrospective case review of one patient with early-onset scoliosis who underwent multiple osteotomies, spinal cord decompression, and placement of MAGEC rods with sublaminar bands. The patient had successful distraction procedures conducted routinely throughout a 44-month period with no associated implant complications or neurologic sequelae during that period. The patient had achieved maximal distraction with the implanted rods and thereafter underwent removal of the MAGEC rods and replacement implantation with longer MAGEC rods. The purpose of this case review was to demonstrate the superior fixation results provided with sublaminar band fixation for MAGEC rod distraction systems. Sublaminar band fixation can also reduce the incidence of proximal junctional kyphosis that typically occurs with pedicle screws and hooks.1 Up to six levels of anchors, three on the proximal end and three on the distal end, can be used without arthrodesis of these normal spinal segments. We hypothesized that it is possible to reduce complications related to anchor failure in the MAGEC rod distraction system with the use of sublaminar band fixation.MAGEC rods (NuVasive) have been shown to provide distraction growth in early-onset scoliosis (EOS).A four-and-a-half-year-old girl with a history of paraspinal embryonal rhabdomyosarcoma diagnosed at birth and status post tumor resection at 14 months of age presented to our clinic for management of early-onset thoracogenic scoliosis. The neoplasm had spanned from C6-L2 and involved the right erector spinae muscle, right posterior medial chest wall, right paramedian spine, and upper thoracic spinal canal, and the patient had reverse S-shaped thoracolumbar scoliosis. Extensive resection of the right erector spinae muscles was done at the time of tumor resection, along with resections of right T1-T9 rib heads, right T2-T6 posterior medial rib, right T1-T6 posterior medial intercostal muscles, right C7-T9 transverse process, right T1-T3 deep paraspinous mass, right T2-T3 neurovascular bundles, and right T2-T3 pedicles and decompression at right T2-T3, and a latissimus dorsi flap was used to fill the defect of the absent erector spinae musculature. She completed chemotherapy and was treated in a thoracic lumbar sacral orthosis because of her substantially weakened spine from the large resection that was done to help clear her tumor burden. Over the span of 3 years, the patient's spine progressed to a 77° dextroscoliotic curve in the T1-T10 region and a 39° levoscoliotic curve in the T10-L4 region. Throughout that time, she developed severe restrictive pulmonary disease because of her chest wall deformity and scoliosis. At 4 years of age, progressive myelopathy with bowel and bladder incontinence was noted. She developed lower extremity pain and weakness, which led to inability to independently ambulate. MRI with contrast showed no recurrent neoplasm but severe scoliosis and spinal stenosis at T3-T4. To alleviate her symptoms and EOS, we deemed her a candidate for decompression and osteotomies with MAGEC rod placement with sublaminar band fixation . Similar corrective maneuvers were used on the right side with a 5.5-mm MAGEC reverse lengthening rod cut to the appropriate length and contoured to the appropriate sagittal alignment. The right MAGEC rod was connected to the spine with three superior and three inferior LigaPASS connectors from C7-L2 and sequentially tightened to correct the spinal deformity (Figure The patient initially had weekly 1-mm MAGEC distractions for the first 4 weeks postoperatively, followed by monthly 1-mm MAGEC distractions for a total of 44 spinal lengthening procedures over the span of 44 months to achieve maximal distraction with the MAGEC rods. Maximal distraction was noted with a clicking sound on attempting additional distraction and confirmed on radiograph Figure . The wouThe initial implanted rods did not show any notable signs of wear or breakage. They had been able to maintain correction of the spinal deformity throughout the time the rods had remained in the patient. The patient is routinely followed, and distraction is done regularly with a goal of 1.2-centimeters distraction per year.2-4Although the MAGEC rod system is an approved method for correcting EOS, many studies have highlighted the high complication rates that can occur with this system. Many of the complications that occur are unplanned revision surgeries, anchor pull-out, implant failure, and rod breakage.2 As suggested by Agarwal et al, the patient's spine was distracted at a rate of 1 mm biweekly over the course of 4 weeks immediately after the first set of MAGEC rods were implanted and then at a rate of 1 mm per month. The patient did not have any complaints with this method of treatment, and no problems were noted on physical examination of the patient.2 The sagittal plane was also maintained after the exchange of MAGEC rods.A common reason for implant failure of MAGEC rods is failure of the distraction mechanism. This kind of failure may be due to accelerated distraction over a short period of time.6 Two or three-level sublaminar band fixation in the cervical, thoracic, and/or lumbar spine can be used as anchors with the MAGEC rod without disrupting the pedicle or facet joint and without arthrodesis, eliminating the need for pedicle screws, hooks, and/or rib anchors.5 These other methods of fixation of MAGEC rods to the vertebrae typically require two to three levels of fixation as well. We were able to use sublaminar band fixation safely up to the C4 level, avoiding proximal junctional kyphosis, a complication that can occur with the use of pedicle screws or hook and pedicle screw constructs.8 MAGEC rods are typically fixated in the upper thoracics, no further than T1 or T2 because of concerns of the occurrence of proximal junctional kyphosis and thus requiring correction later in life.9 Sublaminar bands provide “bicortical” fixation without arthrodesis at the anchor sites and are safer to place than pedicle screws or hooks in the immature, deformed spine. The bands are less prominent than rib hook fixation and can also be used for rib anchors. The sagittal plane of the spine can be addressed by placing the end-to-end connectors adjacent to the anchors or adjacent to the MAGEC rod. Sublaminar bands also do not require fluoroscopy during fixation, thus reducing radiation exposure. We demonstrate that sublaminar bands are a safe method of fixation and a viable option for successful MAGEC rod fixation in a young, skeletally immature patient.Sublaminar bands can be used to successfully anchor rods and correct the deformity of scoliotic spines, and even in neuromuscular scoliosis.11 This case report is one of many examples that show the potential of sublaminar bands’ success in scoliosis correction and now with the use of MAGEC rods.In conclusion, we present a case of a young patient who had successful implantation and maintenance of MAGEC rods fixated with sublaminar bands for over 4 years, and this patient has been followed for 51 months to date, which is one of the longest average follow-ups reported in the literature for patients implanted with MAGEC rods for EOS.


Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD), characterized by atrophic lesions that first start in the outer retina and progressively expand to cover the macula and the fovea, the center of the macula, leading to irreversible loss of vision over time. GA is distinct from wet or neovascular AMD (nAMD), the other form of advanced AMD. Neovascular AMD is characterized by new invading leaky blood vessels in the macula that can lead to acute vision loss. GA and nAMD may coexist in the same eye.The underlying pathophysiology of GA is complex and thought to involve chronic inflammation due to overactivation of the complement system that leads to the loss of photoreceptors, retinal pigment epithelium (RPE), and the underlying choriocapillaris. The disappearance of these structures appears as sharply demarcated atrophic lesions that are typical of GA.Researchers have reported about 1 million reported cases of GA in the United States, and about 160,000 cases occur per year. The most important risk factors for GA are increasing age and family history.Diagnosis of GA is usually made by using multimodal imaging techniques. Lesions associated with GA are highly heterogeneous, and the growth rate may differ from patient to patient. Despite the progressive nature of GA, the fovea may be spared until much later in the disease, thereby retaining central vision in patients. With time, atrophic lesions may progressively grow to involve the fovea, thereby severely impairing central vision. Vision loss can happen rapidly once the lesions reach the fovea. However, even without the involvement of the fovea, ongoing vision impairment impacting daily life may be present. Median time from GA not involving the center of the fovea  to GA with lesion boundary affecting the foveal center  ranges from 1.4 to 2.5 years.GA can greatly impact patients’ functioning and quality of life and limit their independence by interfering with activities of daily living, including difficulties with reading, driving, watching television, recognizing faces, and being unable to do household chores.No treatments have been available until intravitreal pegcetacoplan was recently approved by the US Food and Drug Administration for GA secondary to AMD. Smoking and genetic mutations in the complement system are additional risk factors.4 Excessive drusen accumulation and components of drusen, such as cellular debris, lipids, and lipoproteins, may trigger chronic inflammation via multiple pathways, including the complement cascade.4 Chronic inflammation can eventually lead to photoreceptor, RPE, and choriocapillaris cell death, causing the appearance of sharply defined atrophic lesions, visually resembling geographic areas on a map, that are characteristic of GA and the appearance of choroidal vessels due to the missing RPE layer.With aging, the retinal pigment epithelium (RPE) is exposed to intrinsic and extrinsic oxidative stressors as well as environmental stressors such as cigarette smoke. Oxidative damage accumulates, resulting in the formation of drusen, yellow deposits of lipids between the RPE and Bruch’s membrane (early to intermediate AMD). The appearance and progression of drusen deposits are prognostic features of GA.15 Overactivation of the complement system is strongly associated with lesion development and progression in GA. In particular, it is associated with the loss of photoreceptors, RPE, and choriocapillaris.16 Photoreceptors are specialized sensory cells that convert light into electrical impulse.17 The outer segment of the photoreceptors is connected to the RPE, which is a single cell layer in the outer layers of the retina that plays a critical role in the maintenance and survival of the photoreceptor cells, in clearing cellular debris, and in regulating the integrity of the choroidal capillaries.19 Choriocapillaris supplies blood to the outer layer of the retina and nourishes the retinal nerve cells.18 When functioning normally, the complement system is an important part of the body’s natural immune response. Under steady-state conditions, complement proteins are deployed to rapidly respond to microbes and stress signals. However, overactivation or loss of regulation of this host response can have detrimental consequences by perpetuating a vicious cycle of tissue injury and inflammation. Complement activity is found in both GA lesions and areas just outside the lesion. In areas outside the lesion, complement activation may accelerate cell damage, thereby increasing the risk of lesion growth.Genome-wide association studies have found genetic variants of factors of the complement pathway to be associated with increased risk of AMD. A genetic variant in the complement C3 gene was found to be associated with AMD, including GA.20  converge at C3, with convertase-mediated cleavage of C3 into C3a/C3b, which is associated with downstream effects, and C3b, which is associated with amplification of complement activation. Activation of both C3 and further downstream C5 is necessary to initiate the assembly of the membrane attack complex (MAC), which causes cell membrane destruction and, hence, cell death.Overactivation of the complement system leads to excess phagocytosis, inflammation, and cell lysis —all of wC3a and, further downstream, C5a activate and recruit inflammatory cells to the site of activation.4 Current mechanism and pathophysiology being targeted include complement factor C5 with the agent avacincaptad pegol/ARC1905 , which is a pegylated RNA aptamer that binds and inhibits C522 and cell therapy by transplanting RPE cells into the subretinal space to replace damaged RPE cells (phase 2a).23 Pegcetacoplan, recently approved by the FDA for intravitreal injection for GA secondary to AMD, is a complement inhibitor that binds to complement protein C3 and its activation fragment C3b to regulate the cleavage of C3 and the generation of downstream effectors of complement activation.9The complement system is involved in drusen formation, and C3 and C5 fragments have been detected in drusen in histopathological probes of patients with AMD. Activation of the complement cascade results in cleavage of factor C5 into C5a and C5b fragments. The C5a fragment is an important inflammatory activator that is deposited in the drusen of patients with AMD. C5a recruits inflammatory cells and stimulates RPE cells to produce VEGF. The C5b fragment leads to the formation of MAC, which precipitates in Bruch’s membrane and choriocapillaris, the degree of which is age and AMD dependent. The deposition of MAC in choriocapillaris and its presentation to choroidal endothelial cells trigger cell lysis, likely contributing to RPE atrophy in atrophic AMD and nAMD, respectively. Because of the complement cascade’s role in the pathology of AMD, complement inhibition has been identified as a key candidate for therapeutic intervention.5 These atrophic lesions in the outer retina, referred to as lesions without subfoveal involvement , appear early in the disease and may cause vision loss early in the progression of the disease. Often, the fovea can be spared until later in the course of the disease, resulting in visual acuity that remains nearly normal. However, contrast sensitivity and ability to read may suffer. Once lesions encroach on the center point of the fovea with progressive disease, they are referred to as lesions with subfoveal involvement . With subfoveal involvement, central vision is severely impaired and the patient experiences rapid loss of visual acuity function. Median time from nonsubfoveal to subfoveal involvement of GA ranges from 1.4 to 2.5 years.GA leads to scotomas, also known as visual field deficits or blind spots. GA encompasses the degeneration of the macula, which is the most central area of the retina and responsible for detailed central vision. The fovea, located in the center of the macula, contains a high density of cone photoreceptors and is responsible for central and color vision. However, even without the involvement of the fovea, lesions outside the fovea may cause visual disabilities in daily life, including reading and recognizing faces. The hallmark sign of GA—sharply demarcated atrophic lesions due to loss of photoreceptors, RPE, and choriocapillaris—which first appears in the outer retina, causes progressive visual impairment by expanding toward the fovea and eventually leading to irreversible vision loss .4,5 ThesGA is not diagnosed by laboratory or genetic testing. Diagnosis of GA is made with the use of visual function tests to assess visual impairments and imaging tests to detect clinical features.24:Impairments in visual function in patients with GA can be measured using the following methods5- Best-corrected visual acuity (BCVA) is a standard vision assessment that employs the Snellen chart or the Early Treatment Diabetic Retinopathy Study chart to measure visual acuity. Visual acuity is measured by how many letters a patient can read at a distance. Depending on the degree of foveal sparing, patients may continue to be able to read individual letters and maintain BCVA.5 Compared with patients with intermediate AMD, patients with GA and BCVA of 20/50 or more read significantly slower.24 In a prospective natural history study of patients with GA, the median maximum reading rate decreased significantly from 110 words per minute (wpm) at baseline to 51 wpm at 2 years. In the comparison cohort of patients who had drusen only, the maximum reading rate decreased from 130 wpm at baseline to 117 wpm at 2 years, which was not significant.25- Reading speed, which can be quantified by the number of correctly read words over a prespecified period, provides information about how much of the visual field is sufficiently preserved to recognize entire words or sentences as opposed to individual letters. With progression of GA, reading speed declines.Reading charts may be used to measure visual acuity; however, these frequently provide poor information on actual retina function due to foveal sparing and parafoveal scotomas. Reading ability may be evaluated by the following 2 types of tests:26 Low-luminance deficit is the difference between a patient’s BCVA and LLVA scores and is predictive of subsequent vision loss and lesion enlargement in GA.27Other tests for assessing visual acuity include low-luminance visual acuity (LLVA) and low-luminance deficit. LLVA measures visual function in low-light and the process by which it is conducted is similar to that for BCVA but with one eye covered with a filter to reduce light exposure.28The Amsler grid is used to examine impairments in a patient’s central visual field and line detection. Healthy eyes see the graph lines sharply, in focus and with no distortion or blind spots. Patients with AMD may see the grid lines as wavy, blurred, or distorted, and where the visual field is impaired, the grid may appear to have “holes,” dark areas, or scotomas.26Microperimetry is an alternative to reading charts and may be used to measure retinal sensitivity in specific parts of the retina to identify functional and nonfunctional areas. This technique uses varying intensity of light to stimulate the macula in multiple locations to identify functional and nonfunctional areas; the result depends on the patient’s ability to report recognition of the stimuli. With the use of an infrared camera, the fundus is monitored and the fixation point tracked, thus mapping the sensitivity of the visual field to the fundus photograph.5 Common multimodal imaging techniques that enable visualization of the fundus and surrounding vasculature and are used to diagnose GA and define areas of atrophy include the following:Clinical features of GA are detected by using ophthalmoscopy and imaging in which it is defined by cell layer loss with sharply defined borders and by loss of RPE, photoreceptors, and underlying choriocapillaris.5Color fundus photography (CFP): This is the historical standard for imaging GA and defines GA lesions as sharply demarcated areas of RPE hypopigmentation, with clearly visible underlying choroidal vessels, but CFP cannot be used to visualize characteristics of lesions associated with progressive GA.5Optical coherence tomography (OCT): An imaging modality that identifies GA lesions by the loss of retinal layers through cross-sectional and/or en face images. It is emerging as a preferred modality to assess GA lesion features.5Fundus autofluorescence (FAF): Use of short-wavelength light to visualize loss of RPE cells. Areas of GA lesions appear hypoautofluorescent (decreased autofluorescence) due to loss of RPE cells containing intrinsic fluorophores, such as lipofuscin. This is the primary modality for assessing GA lesion size and growth in clinical trials.29 They suggested that FAF be used for cases in which the diagnosis is unclear. A typical trajectory of diagnostic testing would entail BCVA followed by OCT. If the relevant equipment is available, then FAF would be acquired as needed, and in some cases, also CFP. GA and nAMD have unique sets of International Classification of Diseases, Tenth Revision, Clinical Modification codes30: GA lesions by laterality  and by foveal involvement .30The Classification of Atrophy Meetings group is an international group of experts who have coined the currently accepted classification system for GA based on imaging. They recommended that OCT should serve as the reference method for defining GA and its stages, given its ability to demonstrate different retinal layers in 3 dimensions, its wide availability, and the ease of OCT image acquisition.8A retrospective cohort analysis in the United Kingdom of 1,901 patients with bilateral GA found that 66.7% of patients with bilateral GA who were eligible to drive at baseline progressed to vision loss that left them ineligible to drive, with a median time to progression of 1.6 years (IQR = 0.7-2.7 years). Furthermore, 16% of patients who were not blind at baseline  became legally blind .2 Having GA in the fellow eye was also associated with higher risk of incident GA among US patients in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT) study after 5 years of follow-up .In another UK retrospective cohort analysis using electronic medical records, GA in the fellow eye was associated with greater rate of progression to GA in the study eye (11.2 per 100 person-years) than choroidal neovascularization in the fellow eye (4.1 per 100 person-years) or early/intermediate AMD in both eyes (2.0 per 100 person-years).5 A pooled analysis of data from 2 phase 3 clinical trials of GA failed to find strong correlations between GA lesion size and a range of functional end points.31 Reading speed can give information on the extent of the visual field and whether it is preserved enough to read entire words or sentences instead of individual letters.5 Therefore, disease progression is best determined by monitoring anatomical progression using multimodal imaging approaches (as described above).The impact that lesion growth—an indicator of disease progression—has on functional vision is often not captured by BCVA, a standard vision assessment used to measure visual acuity. Many patients with a good BCVA score have difficulties with reading, indicating that BCVA assessments may underestimate functional deficits.5 Various characteristics of the lesions affect the rate of progression, which varies from patient to patient. Lesions will eventually expand and grow toward the fovea to coalesce with other lesions to cover the fovea and affect vision.5Lesions associated with GA are heterogeneous with several different presentations.Characteristics of GA lesions such as focality , location , and lesion size affect the speed of disease progression.2/year vs 1.05 [0.2] mm2/year; P < 0.001).32Eyes with multifocal lesions have significantly higher mean (SE) GA growth rates than eyes with unifocal lesions  and at month 12 .In the Geographic Atrophy Progression study (N = 603), mean (SE) atrophy progression was observed to be significantly greater among eyes without subfoveal lesions compared with eyes with subfoveal lesions at month 6 .35 Furthermore, a literature review and meta-analysis showed that larger baseline GA area (in mm2) was significantly correlated with larger mean GA growth rate . For every 1-mm2 increase in mean baseline GA area, the mean GA growth rate increased by 0.14 (95% CI = 0.08-0.21) mm2/year.36 The presence of reticular pseudodrusen  is associated with a more rapid progression of GA.39 In a clinical study, in the presence of reticular pseudodrusen the mean (95% CI) GA lesion growth rate was 0.32 (0.30-0.34) mm/year compared with 0.28  without the presence of reticular pseudodrusen.38Studies showed that larger baseline lesion size is associated with higher progression rates than smaller baseline lesion size.40 Moreover, patients with GA feared worsening vision and of going blind and were frustrated with difficulties performing simple tasks, all of which impacted their emotional well-being.40 Overall, quality of life in patients with GA, as assessed by the vision-specific 25-Item National Eye Institute Visual Function Questionnaire (with a higher score representing better functioning), has been shown to be worse than that in patients with early or intermediate AMD and nAMD. Analysis from a cross-sectional US study of 739 patients with AMD  showed that mean composite scores were highest among patients with early or intermediate AMD (89.9) and lowest for the groups with bilateral GA (71.3) and bilateral GA plus nAMD (68.5).41 In a separate study, most patients with GA with a driver’s license reported to mainly travel with a partner or friend, and the number one reason for giving up driving was eyesight. Driving is of particular concern, and inability to drive affects dependency.42 As reported in the retrospective cohort analysis using an electronic medical record database, 67% of patients with bilateral GA who were eligible to drive at baseline progressed to vision loss that left them ineligible to drive in a median time of less than 2 years.8GA can profoundly affect patient functioning and interfere with activities of daily living. The consequences can be devastating in ways that are meaningful to patients. In an ethnographic study including 16 patients with GA, the most reported difficulties were reading (100%); driving (75%); watching movies, television, or theater (69%); recognizing faces (63%); and being unable to perform household chores (63%) .40 Moreo43 Moreover, patients with AMD have a high level of emotional stress that can be compared with patients with disabling chronic illness .43 In addition to increased susceptibility to depression, fear, anxiety, and social isolation, patients with GA are more likely to fall.45 In a prospective cohort study including Medicare patients, those with a code for atrophic AMD  had an 11% increased risk  of hip fractures compared with patients without a code for AMD  over a 4-year follow-up period (1996 - 1999).44Patients with GA are at high risk of developing clinical depression because of their inability to care for themselves and the impact of visual loss on life in general. Patients with unilateral blindness due to AMD are more often affected by depression than those with bilateral disease, possibly because of the fear of losing vision in the unaffected eye.9 Potential drugs currently in development for the treatment of GA are focused on slowing lesion growth and long-term preservation of retinal tissue.47There has been no treatment for GA until intravitreal pegcetacoplan was approved recently by the FDA for GA secondary to AMD. As the vision loss experienced by patients with GA is irreversible, the goal of a treatment is to slow progression of lesion growth.48Several complement inhibitors targeting various points along the complement pathway are being investigated in GA, including avacincaptad pegol (C5 inhibitor).5 It is distinct from nAMD, which has different treatment goals, as it can be treated with anti-VEGF therapy; however, GA cannot be treated with anti-VEGF.6 GA is a disease that is characterized by an irreversible progression leading to vision loss. Atrophic lesions can invade the fovea in less than 2 years from the point at which they form in the nonsubfoveal retina. Therefore, early intervention preceding foveal involvement may slow lesion growth and preserve vision over the long term.5GA is the advanced form of dry AMD characterized by atrophic lesions in the outer retina.20 and could therefore present a therapeutic target. NGM-621, an anti-C3 antibody, has failed to meet the primary endpoint in the CATALINA trial.49 Additional therapeutic targets include mitochondrial dysfunction and oxidative stress; however, elamipretide, a mitochondria-targeting drug, has failed to meet its primary endpoint in the phase 2 ReCLAIM-2 trial.50The underlying pathology of GA lesion may be caused by the overactivation of the complement system. Genetic variants of complement factors are found to increase susceptibility to AMD. In the complement cascade, the complement protein C3 plays a central role in driving the downstream damaging effects of complement overactivation and progression of GA9Until intravitreal pegcetacoplan, which is a complement C3 inhibitor, was recently approved by FDA for GA secondary to AMD, there has been a long-standing unmet need for therapies that slow the growth of lesions.