The ubiquitin system: a regulatory hub for intellectual disability and autism spectrum disorder

Intellectual disability (ID) and autism spectrum disorder (ASD) are two of the most common neurodevelopmental disorders. Both disorders are extremely heterogenous, and only ~ 40% of reported cases have so far been attributed to genetic mutations. Of the many cellular processes that are affected, the ubiquitin system (UbS) is of particular relevance in that it can rapidly regulate multiple signaling cascades simultaneously. The UbS is a post-translational modification process that revolves around the covalent attachment of a ubiquitin moiety to a substrate, thereby influencing different elements of protein biology, including trafficking, signal transduction, and degradation. Importantly, the UbS has been implicated in regulating multiple pathophysiological pathways related to ASD and ID. This review will discuss how the UbS acts as major signaling hub in the pathogenesis of ASD and ID, raising the prospect of treating broader patient cohorts by targeting the UbS as a common point of convergence of various mutations

Heterozygosity for Nuclear Factor One X in mice models features of Malan syndrome

Background: Nuclear Factor One X (NFIX) haploinsufficiency in humans results in Malan syndrome, a disorder characterized by overgrowth, macrocephaly and intellectual disability. Although clinical assessments have determined the underlying symptomology of Malan syndrome, the fundamental mechanisms contributing to the enlarged head circumference and intellectual disability in these patients remains undefined. Methods: Here, we used Nfix heterozygous mice as a model to investigate these aspects of Malan syndrome. Volumetric magnetic resonance imaging (MRI) was used to calculate the volumes of 20 brain sub regions. Diffusion tensor MRI was used to perform tractography-based analyses of the corpus callosum, hippocampal commissure, and anterior commissure, as well as structural connectome mapping of the whole brain. Immunohistochemistry examined the neocortical cellular populations. Two behavioral assays were performed, including the active place avoidance task to assess spatial navigation and learning and memory function, and the 3-chambered sociability task to examine social behaviour. Findings: Adult Nfix mice exhibit significantly increased brain volume (megalencephaly) compared to wildtypes, with the cerebral cortex showing the highest increase. Moreover, all three forebrain commissures, in particular the anterior commissure, revealed significantly reduced fractional anisotropy, axial and radial diffusivity, and tract density intensity. Structural connectome analyses revealed aberrant connectivity between many crucial brain regions. Finally, Nfix mice exhibit behavioral deficits that model intellectual disability. Interpretation: Collectively, these data provide a significant conceptual advance in our understanding of Malan syndrome by suggesting that megalencephaly underlies the enlarged head size of these patients, and that disrupted cortical connectivity may contribute to the intellectual disability these patients exhibit. Fund: Australian Research Council (ARC) Discovery Project Grants, ARC Fellowship, NYSTEM and Australian Postgraduate Fellowships

The p75 neurotrophin receptor is required for the survival of neuronal progenitors and normal formation of the basal forebrain, striatum, thalamus and neocortex

During development, the p75 neurotrophin receptor (p75) is widely expressed in the nervous system where it regulates neuronal differentiation, migration and axonal outgrowth. p75 also mediates the survival and death of newly born neurons, with functional outcomes being dependent on both timing and cellular context. Here we show that knockout of p75 from embryonic day 10 (E10) in neural progenitors using a conditional Nestin-Cre; p75 floxed mouse causes increased apoptosis of progenitor cells. By E14.5, the number of Tbr2-positive progenitor cells was significantly reduced and the rate of neurogenesis was halved. Furthermore, in adult knockout mice, there were fewer cortical pyramidal neurons, interneurons, cholinergic basal forebrain neurons, and striatal neurons, corresponding to a relative reduction in volume of these structures. Thalamic midline fusion during early postnatal development was also impaired in Nestin-Cre p75 floxed mice, indicating a novel role of p75 in the formation of this structure. The phenotype of this strain demonstrates that p75 regulates multiple aspects of brain development, including cortical progenitor cell survival, and that expression during early neurogenesis is required for appropriate formation of telencephalic structures

Usp9X Controls Ankyrin-Repeat Domain Protein Homeostasis during Dendritic Spine Development

Variants in the ANK3 gene encoding ankyrin-G are\ua0associated with neurodevelopmental disorders, including intellectual disability, autism, schizophrenia, and bipolar disorder. However, no upstream regulators of ankyrin-G at synapses are known. Here, we\ua0show that ankyrin-G interacts with Usp9X, a neurodevelopmental-disorder-associated deubiquitinase (DUB). Usp9X phosphorylation enhances their interaction, decreases ankyrin-G polyubiquitination, and stabilizes ankyrin-G to maintain dendritic spine development. In forebrain-specific Usp9X knockout mice (Usp9X), ankyrin-G as well as multiple ankyrin-repeat domain (ANKRD)-containing proteins are transiently reduced at 2 but recovered at 12\ua0weeks postnatally. However, reduced cortical spine density in knockouts persists into adulthood. Usp9X mice display increase of ankyrin-G ubiquitination and aggregation and hyperactivity. USP9X mutations in patients with intellectual disability and autism ablate its catalytic activity or ankyrin-G interaction. Our data reveal a DUB-dependent mechanism of ANKRD protein homeostasis, the impairment of which only transiently\ua0affects ANKRD protein levels but leads to\ua0persistent neuronal, behavioral, and clinical abnormalities

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-β signaling pathways in cancer

Abstract Background Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis. Results Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. Conclusions Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers

Elp2 mutations perturb the epitranscriptome and lead to a complex neurodevelopmental phenotype

Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.</p

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling

Background: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. Methods: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Results: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Conclusions: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function

Cell-Centric View of Apoptosis and Apoptotic Cell Death-Inducing Antitumoral Strategies

Programmed cell death and especially apoptotic cell death, occurs under physiological conditions and is also desirable under pathological circumstances. However, the more we learn about cellular signaling cascades, the less plausible it becomes to find restricted and well-limited signaling pathways. In this context, an extensive description of pathway-connections is necessary in order to point out the main regulatory molecules as well as to select the most appropriate therapeutic targets. On the other hand, irregularities in programmed cell death pathways often lead to tumor development and cancer-related mortality is projected to continue increasing despite the effort to develop more active and selective antitumoral compounds. In fact, tumor cell plasticity represents a major challenge in chemotherapy and improvement on anticancer therapies seems to rely on appropriate drug combinations. An overview of the current status regarding apoptotic pathways as well as available chemotherapeutic compounds provides a new perspective of possible future anticancer strategies

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling.

BackgroundThe X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.MethodsWe used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology.ResultsTwelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory.ConclusionsOur data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function

Partial Loss of USP9X Function Leads to a Male Neurodevelopmental and Behavioral Disorder Converging on Transforming Growth Factor β Signaling

The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor β signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor β signaling and hippocampal function