Mutations in SRPX2 (Sushi-Repeat Protein, X-linked 2) cause rolandic epilepsy with speech impairment (RESDX syndrome) or with altered development of the speech cortex (bilateral perisylvian polymicrogyria). The physiological roles of SRPX2 remain unknown to date. One way to infer the function of SRPX2 relies on the identification of the as yet unknown SRPX2 protein partners. Using a combination of interactome approaches including yeast two-hybrid screening, co-immunoprecipitation experiments, cell surface binding and surface plasmon resonance (SPR), we show that SRPX2 is a ligand for uPAR, the urokinase-type plasminogen activator (uPA) receptor. Previous studies have shown that uPAR^–/– knock-out mice exhibited enhanced susceptibility to epileptic seizures and had brain cortical anomalies consistent with altered neuronal migration and maturation, all features that are reminiscent to the phenotypes caused by SRPX2 mutations. SPR analysis indicated that the p.Y72S mutation associated with rolandic epilepsy and perisylvian polymicrogyria, led to a 5.8-fold gain-of-affinity of SRPX2 with uPAR. uPAR is a crucial component of the extracellular plasminogen proteolysis system; two more SRPX2 partners identified here, the cysteine protease cathepsin B (CTSB) and the metalloproteinase ADAMTS4, are also components of the extracellular proteolysis machinery and CTSB is a well-known activator of uPA. The identification of functionally related SRPX2 partners provides the first and exciting insights into the possible role of SRPX2 in the brain, and suggests that a network of SRPX2-interacting proteins classically involved in the proteolytic remodeling of the extracellular matrix and including uPAR participates in the functioning, in the development and in disorders of the speech cortex.

Loss-of-function mutations in progranulin (GRN) cause ubiquitin- and TAR DNA-binding protein 43 (TDP-43)-positive frontotemporal dementia (FTLD-U), a progressive neurodegenerative disease affecting ~10% of early-onset dementia patients. Here we expand the role of GRN in FTLD-U and demonstrate that a common genetic variant (rs5848), located in the 3'-untranslated region (UTR) of GRN in a binding-site for miR-659, is a major susceptibility factor for FTLD-U. In a series of pathologically confirmed FTLD-U patients without GRN mutations, we show that carriers homozygous for the T-allele of rs5848 have a 3.2-fold increased risk to develop FTLD-U compared with homozygous C-allele carriers (95% CI: 1.50–6.73). We further demonstrate that miR-659 can regulate GRN expression in vitro, with miR-659 binding more efficiently to the high risk T-allele of rs5848 resulting in augmented translational inhibition of GRN. A significant reduction in GRN protein was observed in homozygous T-allele carriers in vivo, through biochemical and immunohistochemical methods, mimicking the effect of heterozygous loss-of-function GRN mutations. In support of these findings, the neuropathology of homozygous rs5848 T-allele carriers frequently resembled the pathological FTLD-U subtype of GRN mutation carriers. We suggest that the expression of GRN is regulated by miRNAs and that common genetic variability in a miRNA binding-site can significantly increase the risk for FTLD-U. Translational regulation by miRNAs may represent a common mechanism underlying complex neurodegenerative disorders.

Axenfeld–Rieger syndrome (ARS) patients with PITX2 point mutations exhibit a wide range of clinical features including mild craniofacial dysmorphism and dental anomalies. Identifying new PITX2 targets and transcriptional mechanisms are important to understand the molecular basis of these anomalies. Chromatin immunoprecipitation assays demonstrate PITX2 binding to the FoxJ1 promoter and PITX2C transgenic mouse fibroblasts and PITX2-transfected cells have increased endogenous FoxJ1 expression. FoxJ1 is expressed at embryonic day 14.5 (E14.5) in early tooth germs, then down-regulated from E15.5–E17.5 and re-expressed in the inner enamel epithelium, oral epithelium, tongue epithelium, sub-mandibular salivary gland and hair follicles during E18.5 and neonate day 1. FoxJ1 and Pitx2 exhibit overlapping expression patterns in the dental and oral epithelium. PITX2 activates the FoxJ1 promoter and, Lef-1 and β-catenin interact with PITX2 to synergistically regulate the FoxJ1 promoter. FoxJ1 physically interacts with the PITX2 homeodomain to synergistically regulate FoxJ1, providing a positive feedback mechanism for FoxJ1 expression. Furthermore, FoxJ1, PITX2, Lef-1 and β-catenin act in concert to activate the FoxJ1 promoter. The PITX2 T68P ARS mutant protein physically interacts with FoxJ1; however, it cannot activate the FoxJ1 promoter. These data indicate a mechanism for the activity of the ARS mutant proteins in specific cell types and provides a basis for craniofacial/ tooth anomalies observed in these patients. These data reveal novel transcriptional mechanisms of FoxJ1 and demonstrate a new role of FoxJ1 in oro-facial morphogenesis.

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease, caused by mutations of at least nine different genes. Several extrarenal manifestations characterize this disorder, including cerebellar defects, situs inversus and retinitis pigmentosa. While the clinical manifestations vary significantly in NPHP, mutations of NPHP5 and NPHP6 are always associated with progressive blindness. This clinical finding suggests that the gene products, nephrocystin-5 and nephrocystin-6, participate in overlapping signaling pathways to maintain photoreceptor homeostasis. To analyze the genetic interaction between these two proteins in more detail, we studied zebrafish embryos after depletion of NPHP5 and NPHP6. Knockdown of zebrafish zNPHP5 and zNPHP6 produced similar phenotypes, and synergistic effects were observed after the combined knockdown of zNPHP5 and zNPHP6. The N-terminal domain of nephrocystin-6-bound nephrocystin-5, and mapping studies delineated the interacting site from amino acid 696 to 896 of NPHP6. In Xenopus laevis, knockdown of NPHP5 caused substantial neural tube closure defects. This phenotype was copied by expression of the nephrocystin-5-binding fragment of nephrocystin-6, and rescued by co-expression of nephrocystin-5, supporting a physical interaction between both gene products in vivo. Since the N- and C-terminal fragments of nephrocystin-6 engage in the formation of homo- and heteromeric protein complexes, conformational changes seem to regulate the interaction of nephrocystin-6 with its binding partners.

Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. To elucidate the pathological mechanisms leading from the mutated GNE to the HIBM phenotype, we attempted to identify and characterize early occurring downstream events by analyzing the genomic expression patterns of muscle specimens from 10 HIBM patients carrying the M712T Persian Jewish founder mutation and presenting mild histological changes, compared with 10 healthy matched control individuals, using GeneChip expression microarrays. When analyzing the expression profile data sets by the intersection of three statistic methods (Student’s t-test, TNoM and Info score), we found that the HIBM-specific transcriptome consists of 374 differentially expressed genes. The specificity of the HIBM transcriptome was assessed by the minimal transcript overlap found between HIBM and the transcriptome of nine additional muscle disorders including adult onset limb girdle myopathies, inflammatory myopathies and early onset conditions. A strikingly high proportion (18.6%) of the overall differentially expressed mRNAs of known function were found to encode for proteins implicated in various mitochondrial processes, revealing mitochondria pathways dysregulation. Mitochondrial morphological analysis by video-rate confocal microscopy showed a high degree of mitochondrial branching in cells of HIBM patients. The subtle involvement of mitochondrial processes identified in HIBM reveals an unexpected facet of HIBM pathophysiology which could at least partially explain the slow evolution of this disorder and give new insights in the disease mechanism.

Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp^2H) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp^2H embryos, demonstrating a gene–environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.