Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids

Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids Karen Rakowiecki , Deepika Patel , Luis Aponte Cofresi , Orly Lazarov doi: https://doi.org/10.1101/2025.07.26.666926 Karen Rakowiecki 1 Department of Anatomy and Cell Biology, University of Illinois at Chicago , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Deepika Patel 1 Department of Anatomy and Cell Biology, University of Illinois at Chicago , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Luis Aponte Cofresi 1 Department of Anatomy and Cell Biology, University of Illinois at Chicago , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Orly Lazarov 1 Department of Anatomy and Cell Biology, University of Illinois at Chicago , Chicago, IL, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: olazarov{at}uic.edu Abstract Full Text Info/History Metrics Preview PDF Summary Down syndrome (DS), due to trisomy 21 (T21), occurs in approximately 14.14 per 10,000 live births in the United States. Reduced neural progenitor cell (NPC) proliferation, delayed neurogenesis, impaired cortical lamination and altered cell fate specification are thought to contribute to cognitive impairments in DS individuals. The molecular mechanisms underlying these deficits are not fully understood. Notably, Amyloid precursor protein (APP), located on human chromosome 21 (HSA21), has been extensively implicated in these processes. Mouse models only partially recapitulate DS phenotypes due to genetic, developmental, and species-specific differences. Recent advances in induced pluripotent stem cell (iPSC) derived 3D cortical organoids allow for the study of DS cortical development in a human model system. Here, we show that normalizing APP gene copy number in DS cortical organoids ameliorated deficits in NPC proliferation, neuronal differentiation, and transcriptional programs. Our results demonstrate the value of cortical organoids in uncovering gene-specific roles in DS pathogenesis and identify APP as a promising target for addressing early neurodevelopmental impairments. Introduction DS is one of the most prevalent developmental disorders occurring in 14.14 out of every 10,000 live births in the United States 1 . Studies in the fetal frontal cortex have uncovered reduced NPC proliferation, delayed neurogenesis, and impaired cortical lamination as DS phenotypes 2 – 6 . Mouse models have recapitulated a number of these deficits 7 – 11 . Nevertheless, these models are limited in their ability to recapitulate the effect of HSA21 triplication on temporal alterations in neurogenesis 12 . The advent of patient derived iPSCs has allowed for improved elucidation of mechanisms underlying neurodevelopmental impairments in DS. A number of studies in DS NPCs reported deficits in proliferation and cell fate likely stemming from oxidative stress-induced senescence 5 , 13 – 22 . How these NPC phenotypes affect downstream cell lineages remains to be fully established. 3D cortical organoids effectively recapitulate aspects of cortical development including generation of NPC layering and diverse neuronal populations 23 – 26 . Thus, the effect of HSA21 triplication on specific cell populations through corticogenesis can be further elucidated in this model. Limited studies report that DS cortical organoids exhibit reduced expansion, RGC proliferation, and counts of deep and upper layer neurons 27 , 28 . In one study, transcriptional analysis indicated that layer IV neocortical excitatory neurons are a particularly vulnerable population 28 . Importantly, this model can be utilized to determine how individual HSA21 genes manifest these phenotypes. Knockout of 1 of 3 DSCAM alleles ameliorated a number of deficits 27 . APP is a ubiquitously expressed, single-pass transmembrane protein generated from HSA21 29 . Extensive literature implicates APP in proliferation, cell fate, and migration during cortical development 30 – 34 . APP gene overdose has been shown to mediate a number of DS CNS phenotypes 35 – 39 . However, its role in early corticogenesis remains to be investigated. Here, we generated DS cortical organoids to model deficits in early cortical development. We show that DS cortical organoids exhibited deficits in proliferation, neuronal differentiation, and transcription relative to isogenic controls. Notably, silencing of one APP allele mitigated these phenotypes in DS cortical organoids. Taken together, our studies better inform the development of therapeutics targeting cortical development in DS. Results DS cortical organoids exhibit increased APP protein expression and processing iPSC lines derived from DS patients and corresponding isogenic controls were obtained from WiCell (Ctrl #1 and DS #1) and National Institute of Neurological Disorders and Stroke (NINDS) Cell and Data Repository (Ctrl #2 and DS #2) 40 , 41 . To model corticogenesis, iPSC lines were differentiated into dorsal forebrain organoids utilizing a commercially available kit adapted from a previously characterized protocol 42 . Cortical organoids were collected at d25, d50, and d75 to analyze neurogenesis temporally. To determine if HSA21 triplication resulted in increased APP protein expression in cortical organoids, we performed immunoblotting for APP full length (APP-FL). Relative to Ctrl, DS organoids did not exhibit significantly increased APP-FL protein levels at d25 ( Figure 1A-B ). However, DS organoids did exhibit significantly increased APP-FL protein expression at d50 and d75 ( Figure 1C-D ). To determine if APP gene overdose leads to increased APP processing, we performed ELISAs for Aβ in organoid conditioned media. No alterations in Aβ-40 and Aβ-42 levels were observed across genotypes in d25 organoid conditioned media ( Figure 1E-F ). However, d50 and d75 DS cortical organoids exhibited significantly increased secreted Aβ-40 and Aβ-42 relative to Ctrl ( Figure 1E-F ). No alterations in Aβ-42 and Aβ-40 ratio were observed across genotypes ( Figure 1G ). Download figure Open in new tab Figure 1. DS cortical organoids exhibit increased expression and processing of APP (A) Representative immunoblots of APP-FL from d25, d50, and d75 Ctrl and DS dorsal forebrain organoids. (B-D) Quantification of protein expression normalized to β-actin. DS/Ctrl (n = 15, 10 orgs per n, 3 rounds). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (E-G) Levels of secreted Aβ-40 and Aβ-42 and Aβ-42/Aβ-40 ratio from d25, d50, and d75 Ctrl and DS dorsal forebrain organoid conditioned media measured by ELISA. A linear mixed model (LMM) was used to test for differences in Aβ-40, Aβ-42, or Aβ-42/Aβ-40 ratio. Specifically, day (d25, d50, and d75) and line (Ctrl and DS) were fixed effects and individual and round of differentiation were random effects. Post hoc comparisons were performed comparing levels of differences between line within each time point. Multiple comparisons were corrected for using Tukey’s adjustment. DS/Ctrl (n = 9-15, 5-10 orgs per n, 2-3 rounds). Data represented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. DS cortical organoids exhibit reduced expansion and neurogenic protein expression At d25, proliferative zones model the expanding ventricular zone (VZ) predominantly composed of SRY (sex determining region Y)-box 2 (SOX2)+ radial glia cells (RGCs) 43 , 44 . To determine the effect of HSA21 triplication on RGC proliferation and organization, immunostaining was performed on Ctrl and DS organoid cryosections for SOX2 and KI67, expressed in higher quantities by proliferating cells 45 . DS proliferative zones exhibited significantly decreased proliferating SOX2+ RGCs relative to Ctrl ( Figure 2A-B ). In continuity, DS proliferative zones were reduced in area and perimeter relative to Ctrl ( Figure 2C-D ). To begin interrogating alterations in neurogenesis, we ran immunoblots for the stemness protein SOX2 and neurogenic proteins doublecortin (DCX) and β-3 tubulin. At d25, DS cortical organoids exhibited significantly increased SOX2 and decreased DCX and β-3 tubulin protein expression relative to Ctrl ( Figure 2E-H ). To determine if deficits in neurogenesis endured, we performed the same immunoblotting at d50. DS cortical organoids exhibited decreased DCX but no alterations in SOX2 and β-3 tubulin protein expression relative to Ctrl suggesting persistent impairments in neuroblasts ( Figure 3A-D ). Download figure Open in new tab Figure 2. DS proliferative zones exhibit reduced expansion (A) Representative immunostaining of SOX2 and KI67 from d25 Ctrl and DS dorsal forebrain organoid cryosections. (B-D) Quantification of proliferating cell count (KI67+/SOX2+), neural stem cell count (SOX2+), area (μm 2 ), and perimeter (μm) of proliferative zones (pz). (DS/Ctrl (n = 70, 1pz per n, 10-13 orgs, 1 round). Data represented as mean ± SEM. Data analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bar, 50 µm. (E) Representative immunoblots of β-3 tubulin, DCX, and SRY SOX2 from d25 Ctrl and DS dorsal forebrain organoids. (F-H) Quantification of protein expression normalized to actin. DS/Ctrl (n = 15, 10 orgs per n, 3 rounds). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Download figure Open in new tab Figure 3. d50 DS cortical organoids exhibit reduced neuroblast protein expression (A) Representative immunoblots of β-3 tubulin, DCX, and SOX2 from d50 Ctrl and DS dorsal forebrain organoids. (B-D) Quantification of protein expression normalized to actin. DS/Ctrl (n = 15, 10 orgs per n, 3 rounds). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Normalization of APP gene copy number reduced APP-FL protein expression and processing in DS cortical organoids To address the role of increased APP gene dosage in DS corticogenesis, we obtained a genetically modified DS #2 line in which CRISPR-Cas9 was utilized to introduce a stop codon prior to the final exon of a single APP allele with the intent to induce nonsense mediated decay (DS #2 APP +/+/-). A corresponding DS #2 “mock CRISPR-Cas9” (DS#2 MC) line was developed in parallel as a control. Next, we asked if normalization of APP gene copy number is sufficient to reduce APP-FL protein expression in DS cortical organoids. DS #2 APP +/+/- organoids exhibited decreased APP-FL protein levels across all examined time points relative to DS #2 MC ( Figure 4A-D ). In continuity, DS #2 APP +/+/- organoid conditioned media exhibited a significant decrease in Aβ-40 and Aβ-42 levels at d50 and d75 relative to DS #2 MC ( Figure 4E-F ). No alterations in Aβ-42 and Aβ-40 ratio were observed across genotypes ( Figure 4G ). Download figure Open in new tab Figure 4. Normalization of APP gene copy number reduced APP-FL protein expression and processing in DS cortical organoids. (A) Representative immunoblots of APP-FL from d25, d50, and d75 DS #2 MC and DS #2 APP +/+/- dorsal forebrain organoids. (B-D) Quantification of protein expression normalized to β-actin. DS #2 MC/APP +/+/- (n= 3-6, 10 orgs per n, 1-2 rounds). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (E-G) Levels of secreted Aβ-40 and Aβ-42 and Aβ-42/Aβ-40 ratio from d25, d50, and d75 DS #2 MC and DS #2 APP +/+/- dorsal forebrain organoid conditioned media measured by ELISA. A linear mixed model (LMM) was used to test for differences in Aβ-40, Aβ-42, or Aβ-42/Aβ-40 ratio. Specifically, day (d25, d50, and d75) and line (DS #2 MC and DS #2 APP +/+/-) were fixed effects and individual and round of differentiation were random effects. Post hoc comparisons were performed comparing levels of differences between line within each time point. Multiple comparisons were corrected for using Tukey’s adjustment. DS #2 MC/APP +/+/- (n= 3-6, 5-10 orgs per n, 1-2 rounds). Data represented as mean ± SEM. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Reduced expansion and neurogenic protein expression mediated by APP gene overdose in DS cortical organoids To determine the role of increased APP gene dosage in impaired RGC proliferation and organization, immunostaining was performed on DS #2 MC and DS #2 APP +/+/- organoid cryosections for SOX2 and KI67. DS #2 APP +/+/- proliferative zones exhibited significantly increased proliferating SOX2+ RGCs relative to DS #2 MC ( Figure 5A-B ). In continuity, DS #2 APP +/+/- proliferative zones exhibited increased area and perimeter relative to DS #2 MC ( Figure 5C-D ). To begin interrogating the role of increased APP gene dosage in impaired neurogenesis, we ran immunoblots for the stemness protein SOX2 and neurogenic proteins DCX and β-3 tubulin. At d25, DS #2 APP +/+/- cortical organoids exhibited decreased SOX2 and increased DCX and β-3 tubulin protein expression relative to DS #2 MC ( Figure 5E-H ). To determine if this rescue persisted, we performed the same immunoblotting at d50. DS #2 APP +/+/- cortical organoids exhibited no alterations in SOX2, DCX, and β-3 tubulin protein expression relative to DS #2 MC ( Figure 6A-D ). Download figure Open in new tab Figure 5. Normalization of APP gene copy number increased proliferative zone expansion in DS cortical organoids. (A) Representative immunostaining of SOX2 and KI67 from d25 DS #2 MC and DS #2 APP +/+/- dorsal forebrain organoid cryosections. (B-D) Quantification of proliferating cell count (KI67+/SOX2+), neural stem cell count (SOX2+), area (μm 2 ), and perimeter (μm) of proliferative zones (pz). DS #2 MC/APP +/+/- (n= 50, 1 pz per n, 10 orgs, 1 round). Data represented as mean ± SEM. Data analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Scale bar, 50 µm. (E) Representative immunoblots of β-3 tubulin, DCX, and SRY SOX2 from d25 DS #2 MC and DS #2 APP +/+/- dorsal forebrain organoids. (F-H) Quantification of protein expression normalized to actin. DS #2 MC/APP +/+/- (n= 3, 10 orgs per n, 1 round). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Download figure Open in new tab Figure 6. Normalization of APP gene copy number does not alter stem or neurogenic protein expression in d50 DS cortical organoids. (A) Representative immunoblots of β-3 tubulin, DCX, and SOX2 from d50 DS #2 MC and DS #2 APP +/+/- dorsal forebrain organoids. (B-D) Quantification of protein expression normalized to actin. DS #2 MC/APP +/+/- (n= 3, 10 orgs per n, 1 round). Data represented as mean ± SEM. Data analyzed by unpaired two-tailed Student’s t-test. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Altered transcription mediated by APP gene overdose in DS cortical organoids Overdose of APP gene copy number has been shown to contribute to transcriptional changes in 2D DS cortical neuron cultures89,114. However, its impact on cell populations of early neurogenesis in DS remains to be investigated. To that end, we performed single-cell RNA sequencing on d25 and d50 cortical organoids across genotypes. Significantly differentially expressed genes (DEGs) with the highest average log 2 fold change when comparing DS #1/Ctrl #1 and DS #2/Ctrl #2 were identified at d25 in RGCs, intermediate progenitor cells (IPCs), and excitatory neurons (ENs) ( Figure 7A-C ). The same analysis was performed at d50 ( Figure 8A-C ). Significant DEGs with the highest average log 2 fold change were identified when comparing DS #2 APP +/+/-/DS #2 MC at d25 in RGCs, IPCs, and ENs ( Figure 9A-C ). The same analysis was performed at d50 ( Figure 10A-C ). Next, we performed Gene Ontology (GO) pathway analysis and cluster mapping of significant DEGs. At d25, DS cortical organoids exhibited significantly altered pathways related to RNA processing and cellular respiration ( Figure 7D-F ). These alterations persisted out to d50 ( Figure 8D-F ). At d25, DS #2 APP +/+/- cortical organoids exhibited significantly altered pathways related to RNA processing and autophagy ( Figure 9D-F ). At d50, DS #2 APP +/+/- cortical organoids exhibited significantly altered pathways related to RNA processing, ER stress, and oxidative phosphorylation ( Figure 10D-F ). Download figure Open in new tab Figure 7. d25 DS cortical organoids exhibit altered RNA processing and cellular respiration pathways (A-C) Bar plot showing DEGs with a significant cut off value of p < 0.05 and highest average log 2 fold change when comparing d25 DS#1/Ctrl #1 and DS#2/Ctrl #2 cortical organoids. (D-F) Cytoscape cluster mapping of altered GO pathways for d25 DS and Ctrl cortical organoids. Analysis based on log 2 fold change ratio of DEGs with a significant cut off value of p < 0.05. Download figure Open in new tab Figure 8. d50 DS cortical organoids exhibit altered RNA processing and cellular respiration pathways (A-C) Bar plot showing DEGs with a significant cut off value of p < 0.05 and highest average log 2 fold change when comparing d50 DS#1/Ctrl #1 and DS#2/Ctrl #2 cortical organoids. (D-F) Cytoscape cluster mapping of altered GO pathways for d50 DS and Ctrl cortical organoids. Analysis based on log 2 fold change ratio of DEGs with a significant cut off value of p < 0.05. Download figure Open in new tab Figure 9. Normalization of APP gene copy number altered RNA processing and autophagy pathways in d25 DS cortical organoids (A-C) Bar plot showing DEGs with a significant cut off value of p < 0.05 and highest average log 2 fold change when comparing d25 DS #2 MC and DS #2 APP +/+/- cortical organoids. (D-F) Cytoscape cluster mapping of altered GO pathways for d25 DS #2 MC and DS #2 APP +/+/- cortical organoids. Analysis based on log 2 fold change ratio of DEGs with a significant cut off value of p < 0.05. Download figure Open in new tab Figure 10. Normalization of APP gene copy number altered RNA processing and autophagy pathways in d50 DS cortical organoids (A-C) Bar plot showing DEGs with a significant cut off value of p < 0.05 and highest average log 2 fold change when comparing d50 DS #2 MC and DS #2 APP +/+/- cortical organoids. (D-F) Cytoscape cluster mapping of altered GO pathways for d50 DS #2 MC and DS #2 APP +/+/- cortical organoids. Analysis based on log 2 fold change ratio of DEGs with a significant cut off value of p < 0.05. Discussion In this study, we found that increased APP-FL protein expression in DS cortical organoids relative to Ctrl coincided with the appearance of IPCs and neurons. This is consistent with previous studies showing that APP mRNA expression increased as neurogenesis progressed peaking at neuronal differentiation and neurite outgrowth 46 . Interestingly, limited analysis of DS fetal tissue has shown only an increase in APP mRNA but not APP-FL protein expression 47 – 50 . It is of note that protein analysis was done utilizing only immunoblotting of 17 to 19 gestational week DS fetal brains. However, cultured DS NPCs have exhibited significantly increased APP-FL protein expression 51 , 52 . Given the numerous mechanisms through with APP regulates neurogenesis, rigorous studies delineating which cellular subtypes overexpress APP at what gestational week in DS fetal tissue are needed 31 , 46 , 51 , 53 , 54 . Our data show that silencing of one APP allele utilizing CRISPR-Cas9 in a DS iPSC line is sufficient to reduce APP-FL protein expression in derived cortical organoids at all examined time points. These results are consistent with previous studies in DS iPSC derived neuronal cultures 35 , 36 . Notably, silencing of one allele was sufficient to reduce APP-FL protein expression in d25 DS cortical organoids that exhibited similar levels to Ctrl. Given that APP mRNA is overexpressed in DS NPCs, it is likely post-transcriptional or post-translational mechanisms are regulating APP-FL protein expression in early neurogenesis 55 , 56 . Further investigation of how microRNAs (miRNAs) regulate APP expression in DS may provide insight to the discordant mRNA and protein levels at this time point 57 – 59 . HSA21 triplication results in an early, pervasive accumulation of amyloid pathology. Diffuse Aβ-42 deposits have been reported as early as 8 up to 27 years old, an earlier deposition than in AD 60 – 63 . Increased soluble Aβ precedes this plaque deposition 64 – 66 . Higher levels of soluble Aβ-42 were observed in one study at 21 gestational weeks 67 . Rigorous characterization of accumulation of amyloid pathology is necessary for appropriately timed therapeutic interventions. We found that DS cortical organoids as young as d50 secrete increased levels of Aβ-40 and Aβ-42 relative to Ctrl. These results are in line with previously published organoid experiments from additional DS individuals 68 , 69 . Therefore, increased soluble Aβ may occur as early as mid-gestation in DS. Importantly, we show silencing of one APP allele in DS cortical organoids resulted in a reduction in secreted Aβ. Thus, though multiple HSA21 genes have been shown to modulate amyloid pathology, APP expression likely plays a fundamental role in early increased Aβ secretion 70 . To investigate NPC pool formation, we characterized proliferative zone cell composition and morphology at d25. We showed that d25 DS proliferative zones contain fewer proliferating RGCs and overall decreased perimeter and area relative to Ctrl. Our results agree with a previously published characterization of DS organoids derived from additional individuals 27 . Limited studies reported decreased proliferation of cells populating both the VZ and SVZ and count of oSVZ SOX2+ RGCs in mid-gestation DS fetal tissue 2 , 3 . The VZ and SVZ of Ts65Dn mice have exhibited decreased thickness, reduced cell proliferation, and prolonged cell cycle 8 . Thus, reduced proliferation of VZ and SVZ RGCs is a hallmark DS phenotype. We found that silencing one APP allele restored expansion of proliferative zones in d25 DS cortical organoids. Next, we profiled the progression of neurogenesis in cortical organoids. We demonstrated that d25 DS cortical organoids expressed increased stem and decreased neurogenic proteins relative to Ctrl. The decrease in the neuroblast protein DCX persisted out to d50. Increased expression of SOX2, implicated in maintaining self-renewing RGCs, in concert with decreased expression of DCX and β-3 tubulin, implicated in IPC migration and differentiation, suggests deficits in formation and maintenance of the IPC pool in DS cortical organoids 43 , 44 , 71 – 74 . Several groups have shown the DS NPCs suffer from oxidative stress-induced senescence that significantly impacts proliferation and differentiation 5 , 13 – 16 . Silencing of one APP allele in d25 DS cortical organoids was sufficient to restore levels of these stem and neurogenic proteins. Notably, we show that normalization of APP gene copy number alters RNA processing, autophagy, ER stress, and oxidative phosphorylation pathways in RGCs and IPCs. Taken together, these results suggest that reduction in APP expression ameliorated NPC deficits in DS cortical organoids. In conclusion, we show that APP gene overdose mediates deficits in corticogenesis in DS cortical organoids. Future experiments should aim to determine the mechanisms by which APP mediates these deficits. Author Contributions KR performed the experiments, analyzed the data and wrote the manuscript. DP and LAC assisted in performing experiments. OL supervised the project, revised the manuscript, and provided funding sources (NIA AG079002, AG057468, AG076940, and AG033570). Declarations of Interests The authors declare no conflict of interests. Methods iPSC Culture Lines Ctrl #1 (UWWC1-DS2U) and DS #1 (UWWC1-DS1) were obtained from WiCell. Lines Ctrl #2 (ND50027) and DS #2 (ND50026) were obtained from National Institute of Neurological Disorders and Stroke (NINDS) 40 , 41 . Lines DS #2 MC and DS #2 APP +/+/- were donated by the Young-Pearse lab and have been previously described 75 . iPSCs were cultured on Matrigel hESC-Qualified Matrix (Corning, 354277) in mTeSR Plus (STEMCELL Technologies, 100-0276). Passaging was performed utilizing ReLeSR (STEMCELL Technologies, 100-0483). Organoid Culture Organoids were generated from iPSCs utilizing the STEMdiff Dorsal Forebrain Organoid Differentiation Kit (STEMCELL Technologies, 08620) and STEMdiff Neural Organoid Maintenance Kit (STEMCELL Technologies, 100-0120). Briefly, iPSC colonies were dissociated into single cells utilizing Gentle Cell Dissociation Reagent (STEMCELL Technologies, 100-0485) and 3 million cells were seeded per well into AggreWell 800 24-well plates (STEMCELL Technologies, 34850). Organoids were subsequently maintained according to kit instructions. Western Blotting Organoids were rinsed 3 times for 10 minutes with Dulbecco’s phosphate-buffered saline (DPBS) with no calcium or magnesium (Thermo Fisher Scientific, 14190144) to remove excess media. DPBS was aspirated completely, and organoids were snap frozen in liquid nitrogen. 10 day 25 or 5 day 50 or 75 organoids were pooled together per sample. Organoids were lysed in radio-immunoprecipitation assay (RIPA) buffer (Boston Bio Products, BP-115) with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78440) by running 10 times through a syringe (Fisher Scientific, BD 305620) and sonicating on ice at 20% power 3 times for 15 s with 5 s rest between. Lysate was centrifuge at 16 thousand g for 15 minutes at 4°C, and the supernatant was transferred to a fresh tube. The concentration of the protein lysate was determined utilizing the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23235). Lysates were prepared in Bolt LDS Sample Buffer (Thermo Fisher Scientific, B0007) and Bolt Sample Reducing Agent (Thermo Fisher Scientific, B0009). Western blot samples were boiled for 5 minutes at 95°C and separated on Bolt Bis-Tris Plus polyacrylamide gels (Thermo Fisher Scientific, NW04125BOX) in Bolt MES SDS Running Buffer (Thermo Fisher Scientific, B0002). The iBlot 2 Dry Blotting System (Invitrogen, IB21001) was utilized to transfer protein from gels to iBlot 2 Transfer Stacks (Thermo Fisher Scientific, IB23002). Nitrocellulose membranes were blocked for 1 hour in 5% non-fat dry milk (milk) in tris-buffered saline containing 0.1% Tween-20 (TBST). Blots were incubated overnight at 4°C in primary antibodies diluted in 5% milk in TBST and subsequently washed 3 times for 15 minutes in TBST. Blots were then incubated for 2 hours in secondary antibodies diluted in 5% milk in TBST and subsequently washed 3 times for 15 minutes in TBST. Membranes were developed utilizing SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34577) and imaged on Azure Biosystems 300q Image Western Blot Imaging System (Azure Biosystems, AZI300-01). Fiji (NIH) was utilized to quantify band intensities. Antibodies utilized in experiments are listed in Appendix A. Immunocytochemistry Organoids were rinsed 3 times for 10 minutes in DPBS to remove excess media and incubated overnight in 4% PFA at 4°C. They were then rinsed 3 times for 10 minutes in DPBS and incubated with 30% sucrose for approximately 24 to 48 hours at 4°C until they sunk to the bottom of the Eppendorf tube. Organoids were incubated for 1 hour at 37°C and snap frozen in a mold utilizing an isopentane bath chilled with liquid nitrogen in a solution of 10% sucrose and 7.5% gelatin in DPBS. Molds were cryosectioned into 20 μm sections and mounted on Superfrost Plus Microscope Slides (Fisher Scientific, 12-550-15). Sections were washed with phosphate-buffered saline containing 0.1% Tween-20 (PBST) for 30 minutes at 37°C to remove excess gelatin and blocked in 0.3% Triton X-100, 2% bovine serum albumin (BSA), and 5% normal donkey serum (NDS) diluted in PBS for 1 hour. After blocking, they were incubated in primary antibodies diluted in blocking buffer overnight at 4°C in a humidity chamber. Sections were washed 3 times for 5 minutes each in PBST and incubated in secondary antibodies diluted in PBST for 2 hours. Sections were then washed 3 times for 5 minutes each in PBST and coverslips were mounted onto slides with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, P36931). Antibodies utilized in experiments are listed in Appendix A. Images were acquired at 40x magnification utilizing confocal microscopy (Zeiss LSM 710). ELISA Organoids were rinsed 3 times in DPBS and placed in 1 mL of fresh media. 10 organoids for day 25 or 5 organoids for day 50 or 75 were pooled per well. Organoids were incubated in media for 48 hours. Resulting organoid conditioned media was centrifuged at 2 thousand rpm for 10 minutes at 4°C and supernatant was snap frozen in liquid nitrogen. Protein was extracted from organoids in corresponding wells and total protein concentration was determined as described in Section 2.2.3. To measure Aβ isoforms in organoid conditioned media, Human β Amyloid (1-40) ELISA Kit Wako II (Fujifilm Irving, 298-64601) and Human β Amyloid(1-42) ELISA Kit Wako (Fujifilm Irving, 298-62401) were utilized. Samples were prepared according to kit instructions at a dilution of 1:3 for Aβ-40 and 1:2 for Aβ-42. Resulting concentrations were normalized to total protein extracted from organoids in corresponding well. To measure tau isoforms in organoid protein lysate, PathScan RP Phospho-Tau (Ser396) Sandwich ELISA Kit (Cell Signaling, 43932) and FastScan Total Tau ELISA Kit (Cell Signaling, 57519) were utilized. All lysates were prepared according to kit instructions at a standard protein amount. Single-cell RNA Sequencing Cell number and viability were analyzed using Nexcelom Cellometer Auto2000 with AOPI fluorescent staining method. Sixteen thousand cells were loaded into the Chromium iX Controller (10X Genomics, PN-1000328) on a Chromium GEM-X Single cell 3’ Chip Kit v4 (10x Genomics, PN-1000215), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer’s protocol. The cDNA and library were generated using the GEM-X Single cell 3’ Kit v4 (10x Genomics, PN-1000691) and Dual Index Kit TT Set A (10X Genomics, PN-1000215) according to the manufacturer’s manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626) and Qubit DNA HS assay kit for qualitative and quantitative analysis respectively. The multiplexed libraries were pooled and sequenced on Illumina Novaseq X Plus sequencer with 100 cycle kits using the following read length: 28 bp Read1 for cell barcode and UMI, and 90 bp Read2 for transcript. The single cell library preparation and sequencing was done at Northwestern University NUseq facility core. Preprocessing Single-cell gene expression was quantified using CellRanger (10X Genomics). Single cells were filtered based on the following criteria: at least 500 genes expressed, at least 2000 UMI counts over all genes, and less than 15% mitochondrial gene expression. Gene expression was normalized using NormalizeExpression in Seurat 76 . To identify cell types, we used the Seurat label transfer functions FindTransferAnchors and TransferData with 30 principal components, using single-cell data published by Kanton et al. as the reference data set with the following modifications: astrocytes and microglia were removed from the reference prior to label transfer, and after label transfer all cells with “Glyc” (glycolysis) labels were removed from our data set. Integrated UMAP coordinates were computed using the IntegrateLayers function from Seurat with the CCAIntegration method 76 , 77 . Differential Expression Analysis Differential expression statistics between treatment groups within each cell line were computed using FindMarkers from Seurat with the Wilcox test 76 . DS vs Ctrl comparisons were run separately for our two cell lines (Ctrl #1/DS #2 and Ctrl #2/DS #2), and we then performed a correspondence analysis to identify concordant results from the two comparisons: a correspondence statistic was computed as the geometric mean of the -log10 adjusted p-values, times the sign of the product of the log2 fold-changes. Adjusted p-values 4, ensuring that we only preserved effects that were statistically significant in both cell lines, and changing in the same direction. To further compare with genes altered in the DS #2 APP +/+/- vs DS #2 MC, we identified genes with opposite effects in DS #2 APP +/+/-/DS #2 MC relative to the DS/Ctrl comparison. Pathway enrichment statistics were computed against Gene Ontology Biological Process (GO BP) pathways obtained from MSigDB for statistically significant or concordance-filtered genes using Fisher’s Exact test in R 76 . Statistics Statistical analysis was performed utilizing R version 4.3.1 (2024-06-16). One-way analysis of variance (ANOVA) and linear mixed model (LMM) analysis were performed utilizing emmeans and lmerTest packages in R respectively. Plots were visualized utilizing ggplot2 package in R and modified utilizing Inkscape version 1.2.1 (2022-07-14). In all graphs, data is shown as mean ± SEM. The following was used for p values: ns > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Acknowledgments We thank Dr. Peter Toth and Dr. Ke Ma at the UIC Fluorescence Imaging Core for guidance on microscopy imaging. We thank Dr. Jalees Rehman for guidance on analysis of sequencing data. We thank Dr. Mark Maienschein-Cline and Pinal Kanabar at the UIC Research Informatics Core for analysis of sequencing data. We thank Dr. Zachery Morrissey for guidance on data analysis. This work was financially supported by National Institutes of Health (NIH), National Institute on Aging (NIA) AG079002, AG057468, AG076940, and AG033570 (OL). Funder Information Declared National Institute on Aging, https://ror.org/049v75w11 , AG079002 , AG057468 , AG076940 , AG033570 Footnotes ↵ 2 Lead Contact References 1. ↵ Mai , C. T. et al. National population-based estimates for major birth defects, 2010– 2014 . Birth Defects Res 111 , 1420 – 1435 ( 2019 ). OpenUrl CrossRef PubMed 2. ↵ Lu , J. et al. OLIG2 over-expression impairs proliferation of human down syndrome neural progenitors . Hum Mol Genet 21 , 2330 – 2340 ( 2012 ). OpenUrl CrossRef PubMed 3. ↵ Baburamani , A. A. et al. Assessment of radial glia in the frontal lobe of fetuses with down syndrome . Acta Neuropathol Commun 8 , ( 2020 ). 4. Golden , J. A. & Hyman , B. T . Development of the Superior Temporal Neocortex Is Anomalous in Trisomy 21 . J Neuropathol Exp Neurol 53 , 513 – 520 ( 1994 ). OpenUrl CrossRef PubMed 5. ↵ Utagawa , E. C. et al. Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome . Acta Neuropathol Commun 10 , ( 2022 ). 6. ↵ Zhou , Y. , Tao , L. & Zhu , Y . TempShift Reveals the Sequential Development of Human Neocortex and Skewed Developmental Timing of Down Syndrome Brains . Brain Sci 13 , ( 2023 ). 7. ↵ Contestabile , A. et al. Cell cycle alteration and decreased cell proliferation in the hippocampal dentate gyrus and in the neocortical germinal matrix of fetuses with down syndrome and in Ts65Dn mice . Hippocampus 17 , 665 – 678 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ Chakrabarti , L. , Galdzicki , Z. & Haydar , T. F . Defects in embryonic neurogenesis and initial synapse formation in the forebrain of the Ts65Dn mouse model of Down syndrome . Journal of Neuroscience 27 , 11483 – 11495 ( 2007 ). OpenUrl Abstract / FREE Full Text 9. Tyler , W. A. & Haydar , T. F . Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome . The Journal of Neuroscience 33 , 5106 – 5119 ( 2013 ). OpenUrl Abstract / FREE Full Text 10. Kurabayashi , N. , et al. Neocortical neuronal production and maturation defects in the TcMAC21 mouse model of Down syndrome . iScience 26 , ( 2023 ). 11. ↵ Klein , J. A. & Haydar , T. F . Neurodevelopment in Down syndrome: Concordance in humans and models . Front Cell Neurosci 16 , ( 2022 ). 12. ↵ Shaw , P. R. , Klein , J. A. , Aziz , N. M. & Haydar , T. F . Longitudinal neuroanatomical and behavioral analyses show phenotypic drift and variability in the Ts65Dn mouse model of down syndrome . DMM Disease Models and Mechanisms 13 , ( 2020 ). 13. ↵ Prutton , K. M. , Marentette , J. O. , Maclean , K. N. & Roede , J. R . Characterization of mitochondrial and metabolic alterations induced by trisomy 21 during neural differentiation . Free Radic Biol Med 196 , 11 – 21 ( 2023 ). OpenUrl CrossRef PubMed 14. Meharena , H. S. et al. Down-syndrome-induced senescence disrupts the nuclear architecture of neural progenitors . Cell Stem Cell 29 , 116 – 130 ( 2022 ). OpenUrl CrossRef PubMed 15. Halevy , T. , Biancotti , J. C. , Yanuka , O. , Golan-Lev , T. & Benvenisty , N . Molecular Characterization of Down Syndrome Embryonic Stem Cells Reveals a Role for RUNX1 in Neural Differentiation . Stem Cell Reports 7 , 777 – 786 ( 2016 ). OpenUrl PubMed 16. ↵ Mollo , N. et al. Human Trisomic iPSCs from Down Syndrome Fibroblasts Manifest Mitochondrial Alterations Early During Neuronal Differentiation . Biology (Basel ) 10 , ( 2021 ). 17. Czermiński , J. T. & Lawrence , J. B . Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation . Dev Cell 52 , 294 – 308 .e3 ( 2020 ). OpenUrl CrossRef PubMed 18. Jiang , J. et al. Translating dosage compensation to trisomy 21 . Nature 500 , 296 – 300 ( 2013 ). OpenUrl CrossRef PubMed Web of Science 19. Lu , H. E. et al. Modeling neurogenesis impairment in down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells . Exp Cell Res 319 , 498 – 505 ( 2013 ). OpenUrl CrossRef PubMed 20. Prutton , K. M. et al. Oxidative stress as a candidate mechanism for accelerated neuroectodermal differentiation due to trisomy 21 . Free Radic Biol Med 186 , 32 – 42 ( 2022 ). OpenUrl PubMed 21. Klein , J. A. et al. Sonic Hedgehog Pathway Modulation Normalizes Expression of Olig2 in Rostrally Patterned NPCs With Trisomy 21 . Front Cell Neurosci 15 , ( 2022 ). 22. ↵ Murray , A. et al. Brief report: Isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration . Stem Cells 33 , 2077 – 2084 ( 2015 ). OpenUrl CrossRef PubMed 23. ↵ Velasco , S. et al. Individual brain organoids reproducibly form cell diversity of the human cerebral cortex . Nature 570 , 523 – 527 ( 2019 ). OpenUrl CrossRef PubMed 24. Camp , J. G. et al. Human cerebral organoids recapitulate gene expression programs of fetal neocortex development . Proc Natl Acad Sci U S A 112 , 15672 – 15677 ( 2015 ). OpenUrl Abstract / FREE Full Text 25. Yakoub , A. M. & Sadek , M . Analysis of Synapses in Cerebral Organoids . Cell Transplant 28 , 1173 – 1182 ( 2019 ). OpenUrl CrossRef PubMed 26. ↵ Zourray , C. , Kurian , M. A. , Barral , S. & Lignani , G. Electrophysiological Properties of Human Cortical Organoids: Current State of the Art and Future Directions . Frontiers in Molecular Neuroscience vol. 15 Preprint at doi: 10.3389/fnmol.2022.839366 ( 2022 ). OpenUrl CrossRef 27. ↵ Tang , X.-Y. et al. DSCAM/PAK1 pathway suppression reverses neurogenesis deficits in iPSC-derived cerebral organoids from patients with Down syndrome . Journal of Clinical Investigation 131 , ( 2021 ). 28. ↵ Li , Z. et al. Asynchronous excitatory neuron development in an isogenic cortical spheroid model of Down syndrome . Front Neurosci 16 , ( 2022 ). 29. ↵ Müller , U. C. , Deller , T. & Korte , M . Not just amyloid: Physiological functions of the amyloid precursor protein family . Nat Rev Neurosci 18 , 281 – 298 ( 2017 ). OpenUrl CrossRef PubMed 30. ↵ Caillé , I. et al. Soluble form of amyloid precursor protein regulates proliferation of progenitors in the adult subventricular zone . Development 131 , 2173 – 2181 ( 2004 ). OpenUrl Abstract / FREE Full Text 31. ↵ Coronel , R. , López-Alonso , V. , Gallego , M. I. & Liste , I . Low Levels of Amyloid Precursor Protein (APP) Promote Neurogenesis and Decrease Gliogenesis in Human Neural Stem Cells . Int J Mol Sci 24 , ( 2023 ). 32. Young-Pearse , T. L. et al. A critical function for β-amyloid precursor protein in neuronal migration revealed by in utero RNA interference . Journal of Neuroscience 27 , 14459 – 14469 ( 2007 ). OpenUrl Abstract / FREE Full Text 33. Lazarov , O. & Demars , M. P. All in the family: How the apps regulate neurogenesis . Frontiers in Neuroscience 1 – 21 Preprint at doi: 10.3389/fnins.2012.00081 ( 2012 ). OpenUrl CrossRef 34. ↵ Coronel , R. et al. Role of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem Cells . Molecular Neurobiology vol. 55 7107 – 7117 Preprint at doi: 10.1007/s12035-018-0914-2 ( 2018 ). OpenUrl CrossRef PubMed 35. ↵ Ovchinnikov , D. A. , Korn , O. , Virshup , I. , Wells , C. A. & Wolvetang , E. J . The Impact of APP on Alzheimer-like Pathogenesis and Gene Expression in Down Syndrome iPSC-Derived Neurons . Stem Cell Reports 11 , 32 – 42 ( 2018 ). OpenUrl PubMed 36. ↵ Wu , C. I. et al. APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer’s pathology in trisomy 21 neurons . Mol Psychiatry 27 , 1970 – 1989 ( 2022 ). OpenUrl PubMed 37. Chen , X. Q. et al. Targeting increased levels of APP in Down syndrome: Posiphen-mediated reductions in APP and its products reverse endosomal phenotypes in the Ts65Dn mouse model . Alzheimer’s and Dementia 17 , 271 – 292 ( 2021 ). OpenUrl 38. Cataldo , A. M. et al. App Gene Dosage Modulates Endosomal Abnormalities of Alzheimer’s Disease in a Segmental Trisomy 16 Mouse Model of Down Syndrome . The Journal of Neuroscience 23 , 6788 – 6792 ( 2003 ). OpenUrl Abstract / FREE Full Text 39. ↵ Salehi , A. et al. Increased App Expression in a Mouse Model of Down’s Syndrome Disrupts NGF Transport and Causes Cholinergic Neuron Degeneration . Neuron 51 , 29 – 42 ( 2006 ). OpenUrl CrossRef PubMed Web of Science 40. ↵ Weick , J. P. et al. Deficits in human trisomy 21 iPSCs and neurons . Proc Natl Acad Sci U S A 110 , 9962 – 9967 ( 2013 ). OpenUrl Abstract / FREE Full Text 41. ↵ McGowan , H. et al. hsa-let-7c miRNA Regulates Synaptic and Neuronal Function in Human Neurons . Front Synaptic Neurosci 10 , ( 2018 ). 42. ↵ Birey , F. et al. Assembly of functionally integrated human forebrain spheroids . Nature 545 , 54 – 59 ( 2017 ). OpenUrl CrossRef PubMed 43. ↵ Bylund , M. , Andersson , E. , Novitch , B. G. & Muhr , J . Vertebrate neurogenesis is counteracted by Sox1-3 activity . Nat Neurosci 6 , 1162 – 1168 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 44. ↵ Graham , V. , Khudyakov , J. , Ellis , P. & Pevny , L . SOX2 Functions to Maintain Neural Progenitor Identity . Neuron 39 , 749 – 765 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 45. ↵ Gerdes , J. et al. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67 . The Journal of Immunology 133 , ( 1984 ). 46. ↵ Nicolas , M. & Hassan , B. A . Amyloid precursor protein and neural development . Development (Cambridge ) 141 , 2543 – 2548 ( 2014 ). OpenUrl Abstract / FREE Full Text 47. ↵ Griffin , W. S. T. et al. Life-long overexpression of S100β in Down’s syndrome: implications for Alzheimer pathogenesis . Neurobiol Aging 19 , 401 – 405 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 48. Engidawork , E. et al. β-Amyloid precursor protein, ETS-2 and collagen alpha 1 (VI) chain precursor, encoded on chromosome 21, are not overexpressed in fetal Down syndrome: further evidence against gene dosage effect . in Protein Expression in Down Syndrome Brain 335–346 (Springer Vienna, Vienna, 2001) . doi: 10.1007/978-3-7091-6262-0_28 . OpenUrl CrossRef 49. Cheon , M. S. , Dierssen , M. , Kim , S. H. & Lubec , G . Protein expression of BACE1, BACE2 and APP in Down syndrome brains . Amino Acids 35 , 339 – 343 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ Engidawork , E. & Lubec , G . Molecular changes in fetal Down syndrome brain . Journal of Neurochemistry vol. 84 895 – 904 Preprint at doi: 10.1046/j.1471-4159.2003.01614.x ( 2003 ). OpenUrl CrossRef PubMed Web of Science 51. ↵ Lu , J. et al. S100B and APP promote a gliocentric shift and impaired neurogenesis in down syndrome neural progenitors . PLoS One 6 , ( 2011 ). 52. ↵ Lu , H. E. et al. Modeling neurogenesis impairment in down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells . Exp Cell Res 319 , 498 – 505 ( 2013 ). OpenUrl CrossRef PubMed 53. ↵ Freude , K. K. , Penjwini , M. , Davis , J. L. , LaFerla , F. M. & Blurton-Jones , M . Soluble Amyloid Precursor Protein Induces Rapid Neural Differentiation of Human Embryonic Stem Cells . Journal of Biological Chemistry 286 , 24264 – 24274 ( 2011 ). OpenUrl Abstract / FREE Full Text 54. ↵ Heo , C. et al. Effects of the monomeric, oligomeric, and fibrillar Aβ42 peptides on the proliferation and differentiation of adult neural stem cells from subventricular zone . J Neurochem 102 , 493 – 500 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 55. ↵ Esposito , G. et al. Genomic and functional profiling of human Down syndrome neural progenitors implicates S100B and aquaporin 4 in cell injury . Hum Mol Genet 17 , 440 – 457 ( 2008 ). OpenUrl CrossRef PubMed Web of Science 56. ↵ Sobol , M. et al. Transcriptome and Proteome Profiling of Neural Induced Pluripotent Stem Cells from Individuals with Down Syndrome Disclose Dynamic Dysregulations of Key Pathways and Cellular Functions . Mol Neurobiol 56 , 7113 – 7127 ( 2019 ). OpenUrl CrossRef PubMed 57. ↵ Patel , N. et al. MicroRNAs can regulate human APP levels . Mol Neurodegener 3 , ( 2008 ). 58. Shi , W. L. et al. Integrated miRNA and mRNA expression profiling in fetal hippocampus with Down syndrome . J Biomed Sci 23 , ( 2016 ). 59. ↵ Brás , A. , Rodrigues , A. S. , Gomes , B. & Rueff , J. Down syndrome and microRNAs . Biomedical Reports vol. 8 11 – 16 Preprint at doi: 10.3892/br.2017.1019 ( 2018 ). OpenUrl CrossRef PubMed 60. ↵ Lemere , C. A. et al. Sequence of Deposition of Heterogeneous Amyloid β-Peptides and APO E in Down Syndrome: Implications for Initial Events in Amyloid Plaque Formation . Neurobiol Dis 3 , 16 – 32 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 61. Leverenz , J. B. & Raskind , M. A . Early Amyloid Deposition in the Medial Temporal Lobe of Young Down Syndrome Patients: A Regional Quantitative Analysis . Exp Neurol 150 , 296 – 304 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 62. Davidson , Y. S. , Robinson , A. , Prasher , V. P. & Mann , D. M. A . The age of onset and evolution of Braak tangle stage and Thal amyloid pathology of Alzheimer’s disease in individuals with Down syndrome . Acta Neuropathol Commun 6 , 56 ( 2018 ). 63. ↵ Mufson , E. J. , Miguel , J. C. & Perez , S. E. Alzheimer’s neuropathology in Down syndrome: From gestation to old age . in The Neurobiology of Aging and Alzheimer Disease in Down Syndrome 11 – 44 (Elsevier, 2022 ). doi: 10.1016/B978-0-12-818845-3.00011-6 . OpenUrl CrossRef 64. ↵ Gyure , K. A. , Durham , R. , Stewart , W. F. , Smialek , J. E. & Troncoso , J. C . Intraneuronal Aβ-Amyloid Precedes Development of Amyloid Plaques in Down Syndrome . Arch Pathol Lab Med 125 , 489 – 492 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 65. Serrano-Pozo , A. , Frosch , M. P. , Masliah , E. & Hyman , B. T . Neuropathological alterations in Alzheimer disease . Cold Spring Harb Perspect Med 1 , ( 2011 ). 66. ↵ Perez , S. E. et al. Frontal cortex and striatal cellular and molecular pathobiology in individuals with Down syndrome with and without dementia . Acta Neuropathol 137 , 413 – 436 ( 2019 ). OpenUrl CrossRef PubMed 67. ↵ Teller , J. K. et al. Presence of soluble amyloid β–peptide precedes amyloid plaque formation in Down’s syndrome . Nat Med 2 , 93 – 95 ( 1996 ). OpenUrl CrossRef PubMed Web of Science 68. ↵ Gonzalez , C. et al. Modeling amyloid beta and tau pathology in human cerebral organoids . Mol Psychiatry 23 , 2363 – 2374 ( 2018 ). OpenUrl CrossRef PubMed 69. ↵ Zhao , H. H. & Haddad , G. G . Alzheimer’s disease like neuropathology in Down syndrome cortical organoids . Front Cell Neurosci 16 , ( 2022 ). 70. ↵ Tosh , J. L. et al. Genetic dissection of down syndrome-associated alterations in APP/amyloid-β biology using mouse models . Sci Rep 11 , ( 2021 ). 71. ↵ Gleeson , J. G. et al. doublecortin, a Brain-Specific Gene Mutated in Human X-Linked Lissencephaly and Double Cortex Syndrome, Encodes a Putative Signaling Protein . Cell 92 , 63 – 72 ( 1998 ). OpenUrl CrossRef PubMed Web of Science 72. Gleeson , J. G. , Lin , P. T. , Flanagan , L. A. & Walsh , C. A . Doublecortin Is a Microtubule-Associated Protein and Is Expressed Widely by Migrating Neurons . Neuron 23 , 257 – 271 ( 1999 ). OpenUrl CrossRef PubMed Web of Science 73. Tischfield , M. A. et al. Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance . Cell 140 , 74 – 87 ( 2010 ). OpenUrl CrossRef PubMed Web of Science 74. ↵ Qu , C. et al. Direct binding of TUBB3 with DCC couples netrin-1 signaling to intracellular microtubule dynamics in axon outgrowth and guidance . J Cell Sci 126 , 3070 – 3081 ( 2013 ). OpenUrl Abstract / FREE Full Text 75. ↵ Wu , C. I. et al. APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer’s pathology in trisomy 21 neurons . Mol Psychiatry 27 , 1970 – 1989 ( 2022 ). OpenUrl PubMed 76. ↵ Stuart , T. et al. Comprehensive Integration of Single-Cell Data . Cell 177 , 1888 – 1902 .e21 ( 2019 ). OpenUrl CrossRef PubMed 77. ↵ Kanton , S. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development . Nature 574 , 418 – 422 ( 2019 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 27, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. 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Share Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids Karen Rakowiecki , Deepika Patel , Luis Aponte Cofresi , Orly Lazarov bioRxiv 2025.07.26.666926; doi: https://doi.org/10.1101/2025.07.26.666926 Share This Article: Copy Citation Tools Amyloid precursor protein mediates deficits in corticogenesis in Down syndrome cortical organoids Karen Rakowiecki , Deepika Patel , Luis Aponte Cofresi , Orly Lazarov bioRxiv 2025.07.26.666926; doi: https://doi.org/10.1101/2025.07.26.666926 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7636) Biochemistry (17704) Bioengineering (13897) Bioinformatics (41963) Biophysics (21460) Cancer Biology (18598) Cell Biology (25525) Clinical Trials (138) Developmental Biology (13383) Ecology (19908) Epidemiology (2067) Evolutionary Biology (24325) Genetics (15613) Genomics (22512) Immunology (17738) Microbiology (40422) Molecular Biology (17190) Neuroscience (88634) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4825) Physiology (7645) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)