Molecular Mechanisms Involved in Altered Differentiation of Neural Progenitors in Fragile X Syndrome

Fragile X syndrome (FXS) is the most common cause of genetically acquired intellectual disability and is strongly associated with autism spectrum disorders. FXS is an X-linked neurodevelopmental disorder, with an incidence of approximately 1 in 5000 males and 1 in 8000 females. It is primarily caused by a trinucleotide repeat expansion in the Fragile X mental retardation 1 (FMR1) gene leading to epigenetic silencing and loss of FMR1 protein (FMRP). Studies using Fmr1-knockout (Fmr1-KO) mice, modelling FXS, revealed that alterations in glutamatergic signaling play a central role in the aberrances of developmental processes in FXS brain. Tissue plasminogen activator (tPA) is a serine protease that potentiates signaling mediated by glutamate receptors. This thesis explored the effects of tPA and glutamate receptor signaling during early differentiation of FXS neural progenitor cells (NPCs). The differentiation of human and mouse FXS NPCs was characterized using calcium imaging, live cell imaging and immunostaining. Expression of tPA was increased in NPCs and brain of Fmr1-KO mice. The increased tPA was involved in altered neuronal migration and activity-dependent changes in FMRP-deficient mouse NPCs. NPCs were functionally characterized based on their responses to activation of type 1 metabotropic and ionotropic glutamate receptors. Increased differentiation of subpopulations of glutamate-responsive cells was observed in FXS NPCs. Treatment with 2-methyl-6-(phenylethynyl)-pyridine (MPEP), rescued abnormal differentiation of glutamate-responsive cells in both human and mouse FXS NPCs. In addition, MPEP treatment corrected morphological defects and migration of Fmr1-KO cells. Finally, an increased differentiation was evident for cells expressing calcium-permeable alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in FXS NPCs and reduced GluA2 expression resulted in increased calcium permeability of AMPA receptors. In summary, this study provides insight into the molecular mechanisms involved in early aberrant differentiation of FXS neuronal cells and will pave the way to develop new therapeutic approaches and biomarkers for FXS.N/

Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.Peer reviewe

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein

The absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Ca-v) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Ca-v channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Ca-v channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-gamma 1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Ca-v channels in FXS neural progenitors.Peer reviewe

Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system

Increased Calcium Influx through L-type Calcium Channels in Human and Mouse Neural Progenitors Lacking Fragile X Mental Retardation Protein

Fragiili-X-oireyhtymä (FXS) on yleisin periytyvän kehitysvammaisuuden aiheuttaja ja tunnettu geneettinen autismin kirjon häiriöiden syy. FXS taustalla on X-kromosomissa sijaitsevan FMR1-geenin CGG-toistojaksoalueen monistumisen aiheuttama metylaatio ja geenin inaktivoituminen, johtaen FMR1-proteiinin (FMRP) puutokseen. FMRP liittyy normaaliin aivojen kehitykseen ja aivosolujen välisten yhteyksien muodostumiseen. Kalsiumsignaalit säätelevät hermosolujen kehitykseen liittyviä prosesseja kuten kypsymistä, erilaistumista ja synapsien muodostumista, ja kalsiumkanavien säätelyn ongelmat on yhdistetty autismin kirjon häiriöihin. Tämän tutkimuksen tavoitteena oli selvittää L-tyypin kalsiumkanavien myötävaikutus FXS hermosoluesiasteiden erilaistumisen häiriöihin. Osoitamme, että solun depolarisaatiosta aiheutuvat solunsisäiset kalsiumvasteet korostuvat FXS-potilaiden indusoiduista pluripotenteista kantasoluista (iPCS) ja FMR1-poistogeenisten hiirten aivokudoksesta tuotetuissa hermosolujen esiasteissa. Kalsiumin lisääntynyt sisäänvirtaus nifedipiiniherkkien jänniteriippuvaisten kalsiumkanavien (Cav) kautta myötävaikuttaa liiallisiin depolarisaation ja tyypin 1 metabotrooppisen glutamaattireseptorin (mGluR) aktivaation vasteisiin. FXS patofysiologiaan liittyy tunnetusti tyypin 1 mGluR poikkeava toiminta. L-tyypin/T-tyypin Cav-kanavien ekspression suhde on suurentunut FXS-esiasteissa ja korreloi esiasteiden erilaistumiseen glutamaattiherkiksi soluiksi. Aivoista peräisin olevan hermokasvutekijän (BDNF) vähentäminen FXS-hiirten esiasteissa vähentää Cav-kanavien ilmentymistä ja aktivaatiovasteita, mitkä liittyvät lisääntyneeseen fosfolipaasi C-γ1 -lokaation fosforylaatioon TrkB-reseptoreissa ja erilaistuvien esiastepopulaatioiden muutoksiin. Tuloksemme osoittavat, että lisääntyneellä kalsiumin sisäänvirtauksella L-tyypin Cav-kanavien kautta FXS-hermosolujen esiasteissa on kehityksellisiä vaikutuksia.The absence of FMR1 protein (FMRP) causes fragile X syndrome (FXS) and disturbed FMRP function is implicated in several forms of human psychopathology. We show that intracellular calcium responses to depolarization are augmented in neural progenitors derived from human induced pluripotent stem cells and mouse brain with FXS. Increased calcium influx via nifedipine-sensitive voltage-gated calcium (Cav) channels contributes to the exaggerated responses to depolarization and type 1 metabotropic glutamate receptor activation. The ratio of L-type/T-type Cav channel expression is increased in FXS progenitors and correlates with enhanced progenitor differentiation to glutamate-responsive cells. Genetic reduction of brain-derived neurotrophic factor in FXS mouse progenitors diminishes the expression of Cav channels and activity-dependent responses, which are associated with increased phosphorylation of the phospholipase C-γ1 site within TrkB receptors and changes of differentiating progenitor subpopulations. Our results show developmental effects of increased calcium influx via L-type Cav channels in FXS neural progenitors

Table1_Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.pdf

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.</p

Video5_Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.avi

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.</p

Video2_Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.avi

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.</p

Video4_Reduced LYNX1 expression in transcriptome of human iPSC-derived neural progenitors modeling fragile X syndrome.avi

Lack of FMR1 protein results in fragile X syndrome (FXS), which is the most common inherited intellectual disability syndrome and serves as an excellent model disease to study molecular mechanisms resulting in neuropsychiatric comorbidities. We compared the transcriptomes of human neural progenitors (NPCs) generated from patient-derived induced pluripotent stem cells (iPSCs) of three FXS and three control male donors. Altered expression of RAD51C, PPIL3, GUCY1A2, MYD88, TRAPPC4, LYNX1, and GTF2A1L in FXS NPCs suggested changes related to triplet repeat instability, RNA splicing, testes development, and pathways previously shown to be affected in FXS. LYNX1 is a cholinergic brake of tissue plasminogen activator (tPA)-dependent plasticity, and its reduced expression was consistent with augmented tPA-dependent radial glial process growth in NPCs derived from FXS iPSC lines. There was evidence of human iPSC line donor-dependent variation reflecting potentially phenotypic variation. NPCs derived from an FXS male with concomitant epilepsy expressed differently several epilepsy-related genes, including genes shown to cause the auditory epilepsy phenotype in the murine model of FXS. Functional enrichment analysis highlighted regulation of insulin-like growth factor pathway in NPCs modeling FXS with epilepsy. Our results demonstrated potential of human iPSCs in disease modeling for discovery and development of therapeutic interventions by showing early gene expression changes in FXS iPSC-derived NPCs consistent with the known pathophysiological changes in FXS and by revealing disturbed FXS progenitor growth linked to reduced expression of LYNX1, suggesting dysregulated cholinergic system.</p