SUMMARY Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of reenacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift towards GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Frank-Ter Haar syndrome (FTHS), also known as Ter Haar syndrome, is an autosomal-recessive disorder characterized by skeletal, cardiovascular, and eye abnormalities, such as increased intraocular pressure, prominent eyes, and hypertelorism. We have conducted homozygosity mapping on patients representing 12 FTHS families. A locus on chromosome 5q35.1 was identified for which patients from nine families shared homozygosity. For one family, a homozygous deletion mapped exactly to the smallest region of overlapping homozygosity, which contains a single gene, SH3PXD2B. This gene encodes the TKS4 protein, a phox homology (PX) and Src homology 3 (SH3) domain-containing adaptor protein and Src substrate. This protein was recently shown to be involved in the formation of actin-rich membrane protrusions called podosomes or invadopodia, which coordinate pericellular proteolysis with cell migration. Mice lacking Tks4 also showed pronounced skeletal, eye, and cardiac abnormalities and phenocopied the majority of the defects associated with FTHS. These findings establish a role for TKS4 in FTHS and embryonic development. Mutation analysis revealed five different homozygous mutations in SH3PXD2B in seven FTHS families. No SH3PXD2B mutations were detected in six other FTHS families, demonstrating the genetic heterogeneity of this condition. Interestingly however, dermal fibroblasts from one of the individuals without an SH3PXD2B mutation nevertheless expressed lower levels of the TKS4 protein, suggesting a common mechanism underlying disease causation.

Congenital Hepatic Fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure, however the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1del4/del4) mouse, which is orthologous of CHF, we show that Pkhd1del4/del4 cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including CXCL1, CXCL10 and CXCL12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1del4/del4 cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local TGFβ1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts. Conclusion our results show that fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment and collagen deposition. These findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis and macrophage polarization over time.

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. INTRODUCTIONRett syndrome (RTT) is a progressive neurological disorder that affects 1 in 10 000 girls worldwide and represents one of the most common causes of mental retardation in females. RTT is characterized by an apparently normal development for the first 6-18 months of life, followed by regression with the onset of clinical signs including mental retardation, loss of speech, acquired microcephaly, growth retardation, autistic features, seizures, ataxia and hand stereotypies. 1 Beside the classic form, several RTT variants have been described including the Zappella variant, the congenital form, the 'forme fruste' and the early onset seizures variant. [2][3][4][5] In past years, mutations in three genes have been associated with classic and/or variant RTT: MECP2 and CDKL5, located on the X chromosome, and FOXG1, on chromosome 14. 1,6,7 About 80% of classic RTT cases are caused by mutations in MECP2 that encodes for a methyl-CpG-binding protein involved in the regulation of gene expression. 8,9 To investigate the molecular mechanisms leading from MECP2 mutations to RTT onset, different mouse models have been generated and extensively characterized. 10-13 These models allowed identification of specific alterations in glutamatergic neurons: 13 cells lacking Mecp2 have reduced synapse numbers and, accordingly, they show a reduced synaptic response. The opposite effects are elicited by Mecp2 over-expression....

In the liver, CFTR regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e cholangiocytes). CF-related liver disease (CFLD) is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver, and other epithelia. This study investigates the mechanisms linking CFTR to TLR4 activity. We found that CFTR is associated in a multi-protein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Src activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates TLR4, resulting in activation of NF-κB, and increased pro-inflammatory cytokines production in response to endotoxins. This Src/NF-κB-dependent inflammatory process attracts inflammatory cells, but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF-cholangiocytes to LPS, rescued the junctional defect in-vitro and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr-KO mice). Conclusion Our findings reveal a novel function of CFTR as regulator of TLR4 responses and cell polarity in biliary epithelial cells. This mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo and maybe a novel therapeutic target in CFLD and other inflammatory cholangiopathies.

Cystic fibrosis-associated liver disease (CFLD) is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of TLR4/NF-kB-dependent immune mechanisms plays a major role in the pathogenesis of CFLD, and may represent a therapeutic target. Nuclear receptors (NRs) are transcription factors that regulate several intracellular functions. Some NRs, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counter-regulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in Cftr-KO mice exposed to DSS, and in vitro in primary cholangiocytes isolated from wild type and from Cftr-KO mice exposed to LPS. We found that in CFTR-defective biliary epithelium, expression of PPAR-γ is increased, but does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) upregulates PPAR-γ-dependent genes, while inhibiting the activation of NF-kB and the secretion of proinflammatory cytokines (LIX, MCP-1, MIP-2, G-CSF, KC) in response to LPS. PPAR-γ agonists modulate NF-kB-dependent inflammation by upregulating IkBα, a negative regulator of NF-kB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-KO mice exposed to a DSS-induced portal endotoxemia. Conclusion These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes.

Few studies have been conducted to understand post-zygotic accumulation of mutations in cells of the healthy human body. We reprogrammed 32 skin fibroblast cells from families of donors into human induced pluripotent stem cell (hiPSC) lines. The clonal nature of hiPSC lines allows a high-resolution analysis of the genomes of the founder fibroblast cells without being confounded by the artifacts of single-cell whole-genome amplification. We estimate that on average a fibroblast cell in children has 1035 mostly benign mosaic SNVs. On average, 235 SNVs could be directly confirmed in the original fibroblast population by ultradeep sequencing, down to an allele frequency (AF) of 0.1%. More sensitive droplet digital PCR experiments confirmed more SNVs as mosaic with AF as low as 0.01%, suggesting that 1035 mosaic SNVs per fibroblast cell is the true average. Similar analyses in adults revealed no significant increase in the number of SNVs per cell, suggesting that a major fraction of mosaic SNVs in fibroblasts arises during development. Mosaic SNVs were distributed uniformly across the genome and were enriched in a mutational signature previously observed in cancers and in de novo variants and which, we hypothesize, is a hallmark of normal cell proliferation. Finally, AF distribution of mosaic SNVs had distinct narrow peaks, which could be a characteristic of clonal cell selection, clonal expansion, or both. These findings reveal a large degree of somatic mosaicism in healthy human tissues, link de novo and cancer mutations to somatic mosaicism, and couple somatic mosaicism with cell proliferation.

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dys-regulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306C) and 2 patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5 and MECP2 mutated cells. The only major change in gene expression common to MECP2-and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the delta family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA - glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is down-regulated in both MECP2 and CDKL5-mutated iPS cells and up-regulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.