Proteomic analysis of the developing mammalian brain links PCDH19 to the Wnt/β-catenin signalling pathway

Clustering Epilepsy (CE) is a neurological disorder caused by pathogenic variants of the Protocadherin 19 (PCDH19) gene. PCDH19 encodes a protein involved in cell adhesion and Estrogen Receptor α mediated-gene regulation. To gain further insights into the molecular role of PCDH19 in the brain, we investigated the PCDH19 interactome in the developing mouse hippocampus and cortex. Combined with a meta-analysis of all reported PCDH19 interacting proteins, our results show that PCDH19 interacts with proteins involved in actin, microtubule, and gene regulation. We report CAPZA1, αN-catenin and, importantly, β-catenin as novel PCDH19 interacting proteins. Furthermore, we show that PCDH19 is a regulator of β-catenin transcriptional activity, and that this pathway is disrupted in CE individuals. Overall, our results support the involvement of PCDH19 in the cytoskeletal network and point to signalling pathways where PCDH19 plays critical roles

De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function

The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth

HMG Advance Access published July 2, 2013Loss-of-function mutations in UPF3B result in variable clinical presentations including intellectual disability (ID, syndromic and non-syndromic), autism, childhood onset schizophrenia and attention deficit hyperactivity disorder. UPF3B is a core member of the nonsense-mediatedmRNAdecay (NMD) pathway that functions to rapidly degrade transcripts with premature termination codons (PTCs). Traditionally identified in thousands of human diseases, PTCs were recently also found to be part of 'normal' genetic variation in human populations. Furthermore, many human transcripts have naturally occurring regulatory features compatible with 'endogenous'PTCsstrongly suggesting roles ofNMDbeyondPTCmRNAcontrol. In this study,weinvestigated the role of Upf3b andNMD in neural cells.Weprovide evidence that suggests Upf3b-dependentNMD(Upf3b-NMD) is regulated at multiple levels during development including regulation of expression and sub-cellular localization of Upf3b. Furthermore, complementary expression of Upf3b, Upf3a and Stau1 stratify the developing dorsal telencephalon, suggesting that alternativeNMD,andthe related Staufen1-mediatedmRNAdecay (SMD) pathways are differentially employed. A loss of Upf3b-NMD in neural progenitor cells (NPCs) resulted in the expansion of cell numbers at the expense of their differentiation. In primary hippocampal neurons, loss of Upf3b-NMD resulted in subtle neurite growth effects. Our data suggest that the cellular consequences of loss of Upf3b-NMD can be explained in-part by changes in expression of key NMD-feature containing transcripts, which are commonly deregulated also in patients with UPF3B mutations. Our research identifies novel pathological mechanisms of UPF3B mutations and at least partly explains the clinical phenotype of UPF3B patients.Lachlan A. Jolly, Claire C. Homan, Reuben Jacob, Simon Barry and Jozef Gec

The deubiquitylating enzyme USP9X promotes the polarity and self-renewal of neural progenitor cells.

Neural Progenitor Cells (NPCs) are the primordial cells of central nervous system (CNS). Understanding how they are regulated benefits our knowledge of normal development, the pathology of neurological disorders, and of therapeutic designs. One gene identified as a putative regulator of stem cell (SC) populations, including NPCs, by virtue of displaying commonly elevated expression in representative SC populations is USP9X. During development, USP9X mRNA is found highly expressed in the ventricular zones of the developing murine CNS. On this basis the USP9X protein, a substrate specific deubiquitylating enzyme, was hypothesized to be highly expressed in NPCs, and to function in the control of NPC behavior. USP9X protein was found enriched within the apical end-feet structures of NPCs, where it partially co-localised with N-cadherin. This expression domain is common to highly conserved NPC polarity imparting complexes, and to fate determinant networks, which are functionally integrated together to control NPC fate. Thus USP9X protein expression was consistent with a putative regulatory role in NPC fate. To identify cellular processes regulated by USP9X in NPCs, the effect of USP9X over-expression was analysed in embryonic stem cell (ESC)-derived NPCs. ESC lines were generated housing transgenes encoding USP9X under the transcriptional control of the human Nestin 2nd intron, and differentiated into neurons via NPC intermediates. The nestin-USP9X transgene expression resulted in two phenomena. First, it produced a dramatically altered cellular architecture wherein the majority (over 80%) of NPCs were arranged into ‘rosette’ colonies. These NPCs expressed markers of Radial Glial cells, named radial progenitors (RPs) thereafter, and were highly polarised akin to their in-vivo counterparts. Second, USP9X over expression caused a five-fold percentage increase of RPs and neurons. BrdU labelling, as well as the examination of the RP:neuron ratio indicated that nestin-USP9X enhanced the self-renewal of RPs but did not block their subsequent differentiation to neurons and astrocytes. nestin-USP9X RPs reformed rosette colonies following passage as single cells whereas control cells did not, suggesting it aids the establishment of polarity. From these data it was proposed that USP9X-induced polarisation of NPCs, provides an environment conducive for self-renewal. The nestin-USP9X transgene was subsequently used to generate transgenic embryos. Initially, three founding embryos were analysed. In two of three nestin-USP9X embryos, thickening, convolution, and disorganised CNS tissues were observed. A further four nestin- USP9X transgenic embryos generated through the breeding of a transgenic line revealed relatively milder defects of thickened CNS tissues. These effects are speculated to result from expansion of the NPC population, but await further experimental investigation. Together this study identifies USP9X as a regulator of NPC function. In-vivo, USP9X was found highly enriched in the apical end-feet structures, common to molecular networks that regulate NPC fate, and that also have components known to be USP9X substrates. In ESC derived NPCs, USP9X over-expression promoted polarity and self-renewal, which was also speculated to occur in NPCs in-vivo upon limited analysis. This work affirms the intrinsic relationship between polarity and NPC fate decisions, which is here suggested to be coordinated by USP9X regulated pathways.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Sciences, 201

The FAM deubiquitylating enzyme localizes to multiple points of protein trafficking in epithelia, where it associates with E-cadherin and (Beta)-catenin

Ubiquitylation is a necessary step in the endocytosis and lysosomal trafficking of many plasma membrane proteins and can also influence protein trafficking in the biosynthetic pathway. Although a molecular understanding of ubiquitylation in these processes is beginning to emerge, very little is known about the role deubiquitylation may play. Fat Facets in mouse (FAM) is substrate-specific deubiquitylating enzyme highly expressed in epithelia where it interacts with its substrate, β-catenin. Here we show, in the polarized intestinal epithelial cell line T84, FAM localized to multiple points of protein trafficking. FAM interacted with β-catenin and E-cadherin in T84 cells but only in subconfluent cultures. FAM extensively colocalized with β-catenin in cytoplasmic puncta but not at sites of cell-cell contact as well as immunoprecipitating with β-catenin and E-cadherin from a higher molecular weight complex (~500 kDa). At confluence FAM neither colocalized with, nor immunoprecipitated, β-catenin or E-cadherin, which were predominantly in a larger molecular weight complex (~2 MDa) at the cell surface. Overexpression of FAM in MCF-7 epithelial cells resulted in increased β-catenin levels, which localized to the plasma membrane. Expression of E-cadherin in L-cell fibroblasts resulted in the relocalization of FAM from the Golgi to cytoplasmic puncta. These data strongly suggest that FAM associates with E-cadherin and β-catenin during trafficking to the plasma membrane

Loss of Usp9x disrupts cell adhesion, and components of the Wnt and Notch signaling pathways in neural progenitors

Development of neural progenitors depends upon the coordination of appropriate intrinsic responses to extrinsic signalling pathways. Here we show the deubiquitylating enzyme, Usp9x regulates components of both intrinsic and extrinsic fate determinants. Nestin-cre mediated ablation of Usp9x from embryonic neural progenitors in vivo resulted in a transient disruption of cell adhesion and apical-basal polarity and, an increased number and ectopic localisation of intermediate neural progenitors. In contrast to other adhesion and polarity proteins, levels of β-catenin protein, especially S33/S37/T41 phospho-β-catenin, were markedly increased in Usp9x embryonic cortices. Loss of Usp9x altered composition of the β-catenin destruction complex possibly impeding degradation of S33/S37/T41 phospho-β-catenin. Pathway analysis of transcriptomic data identified Wnt signalling as significantly affected in Usp9x embryonic brains. Depletion of Usp9x in cultured human neural progenitors resulted in Wnt-reporter activation. Usp9x also regulated components of the Notch signalling pathway. Usp9x co-localized and associated with both Itch and Numb in embryonic neocortices. Loss of Usp9x led to decreased Itch and Numb levels, and a concomitant increase in levels of the Notch intracellular domain as well as, increased expression of the Notch target gene Hes5. Therefore Usp9x modulates and potentially coordinates multiple fate determinants in neural progenitors

Loss of Usp9x disrupts cortical architecture, hippocampal development and TGFb-mediated axonogenesis

The deubiquitylating enzyme Usp9x is highly expressed in the developing mouse brain, and increased Usp9x expression enhances the self-renewal of neural progenitors in vitro. USP9X is a candidate gene for human neurodevelopmental disorders, including lissencephaly, epilepsy and X-linked intellectual disability. To determine if Usp9x is critical to mammalian brain development we conditionally deleted the gene from neural progenitors, and their subsequent progeny. Mating Usp9xloxP/loxP mice with mice expressing Cre recombinase from the Nestin promoter deleted Usp9x throughout the entire brain, and resulted in early postnatal lethality. Although the overall brain architecture was intact, loss of Usp9x disrupted the cellular organization of the ventricular and sub-ventricular zones, and cortical plate. Usp9x absence also led to dramatic reductions in axonal length, in vivo and in vitro, which could in part be explained by a failure in Tgf-β signaling. Deletion of Usp9x from the dorsal telencephalon only, by mating with Emx1-cre mice, was compatible with survival to adulthood but resulted in reduction or loss of the corpus callosum, a dramatic decrease in hippocampal size, and disorganization of the hippocampal CA3 region. This latter phenotypic aspect resembled that observed in Doublecortin knock-out mice, which is an Usp9x interacting protein. This study establishes that Usp9x is critical for several aspects of CNS development, and suggests that its regulation of Tgf-β signaling extends to neurons

Impaired neural differentiation of MPS IIIA patient induced pluripotent stem cell-derived neural progenitor cells

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes βIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.Rebecca J. Lehmann, Lachlan A. Jolly, Brett V. Johnson, Megan S. Lord, Ha Na Kim, Jennifer T. Saville, Maria Fuller, Sharon Byers, Ainslie L.K. Derrick-Robert

Loss of <i>Usp9x</i> reduces neuronal outgrowth.

<p>Embryonic hippocampal neurons were isolated, transfected with a plasmid encoding Enhanced Green Fluorescent Protein, and grown in-vitro for 3, 5 or 7 days. (a) Example immunofluorescent images of wildtype (Nes-<i>Usp9x^+/Y</i>) and null (Nes-<i>Usp9x^−/Y</i>) neurons resolved using GFP expression (Green) and co-stained with the axonal and dendritic specific antibodies, TAU1 (cyan) and MAP2 (red) respectively. (b–c) Morphometric analysis was employed to record mean primary axonal length (b) and number of neurite termini (c). *p<0.05 by student 2-tailed t-test.</p

Loss of <i>Usp9x</i> disrupts TGF-β signalling in hippocampal neurons.

<p>(a–b) TGF-β luciferase reporter assays conducted in either wildtype (Nes-<i>Usp9x^+/Y</i>) or null (Nes-<i>Usp9x^−/Y</i>) embryonic hippocampal neuronal cultures. Hippocampal neurons were isolated and transfected with both renilla control and pGL3-TGF-β luciferase reporter plasmids. (a) Cells were grown for 3 days before analysis using dual-luciferase reporter assays and data normalised relative to wildtype readings. (b) Luciferase reporter activity in response to increasing concentrations of TGF-β. Data normalised to controls in the absence of TGF-β. All luciferase data from 6 biological replicates (i.e. cultures isolated from 6 <i>Usp9x^+/y</i> and 6 <i>Usp9x^−/Y</i> embryos). (c) Response of established TGFβ target genes in presence or absence of <i>Usp9x</i>, analysed by RT-qPCR. Isolated hippocampal neurons grown for 2 days prior to the addition of 1 ng/ml TGF-β. (d–e). Morphological analysis of hippocampal neurons exposed to 1 ng/ml TGF-β in the presence or absence of Usp9x. (d) Comparison of mean primary axonal length. (e). Comparison of number of neurite termini.</p