Mutations in the Small GTPase Gene RAB39B Are Responsible for X-linked Mental Retardation Associated with Autism, Epilepsy, and Macrocephaly

Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5′ splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities.     

Rapid Decision-Making with Side-Specific Perceptual Discrimination in Ants

Background

Timely decision making is crucial for survival and reproduction. Organisms often face a speed-accuracy trade-off, as fully informed, accurate decisions require time-consuming gathering and treatment of information. Optimal strategies for decision-making should therefore vary depending on the context. In mammals, there is mounting evidence that multiple systems of perceptual discrimination based on different neural circuits emphasize either fast responses or accurate treatment of stimuli depending on the context.

Methodology/Principal Findings

We used the ant Camponotus aethiops to test the prediction that fast information processing achieved through direct neural pathways should be favored in situations where quick reactions are adaptive. Social insects discriminate readily between harmless group-members and dangerous strangers using easily accessible cuticular hydrocarbons as nestmate recognition cues. We show that i) tethered ants display rapid aggressive reactions upon presentation of non-nestmate odor (120 to 160 ms); ii) ants'' aggressiveness towards non-nestmates can be specifically reduced by exposure to non-nestmate odor only, showing that social interactions are not required to alter responses towards non-nestmates; iii) decision-making by ants does not require information transfer between brain hemispheres, but relies on side-specific decision rules.

Conclusions/Significance

Our results strongly suggest that first-order olfactory processing centers (up to the antennal lobes) are likely to play a key role in ant nestmate recognition. We hypothesize that the coarse level of discrimination achieved in the antennal lobes early in odor processing provides enough information to determine appropriate behavioral responses towards non-nestmates. This asks for a reappraisal of the mechanisms underlying social recognition in insects.     

Identification of the FANCI protein, a monoubiquitinated FANCD2 paralog required for DNA repair

Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.     

Soluble TNF-like cytokine (TL1A) production by immune complexes stimulated monocytes in rheumatoid arthritis

TNF-like cytokine (TL1A) is a newly identified member of the TNF superfamily of ligands that is important for T cell costimulation and Th1 polarization. However, despite increasing information about its functions, very little is known about expression of TL1A in normal or pathological states. In this study, we report that mononuclear phagocytes appear to be a major source of TL1A in rheumatoid arthritis (RA), as revealed by their strong TL1A expression in either synovial fluids or synovial tissue of rheumatoid factor (RF)-seropositive RA patients, but not RF-/RA patients. Accordingly, in vitro experiments revealed that human monocytes express and release significant amounts of soluble TL1A when stimulated with insoluble immune complexes (IC), polyethylene glycol precipitates from the serum of RF+/RA patients, or with insoluble ICs purified from RA synovial fluids. Monocyte-derived soluble TL1A was biologically active as determined by its capacity to induce apoptosis of the human erythroleukemic cell line TF-1, as well as to cooperate with IL-12 and IL-18 in inducing the production of IFN-gamma by CD4(+) T cells. Because RA is a chronic inflammatory disease with autoimmune etiology, in which ICs, autoantibodies (including RF), and various cytokines contribute to its pathology, our data suggest that TL1A could be involved in its pathogenesis and contribute to the severity of RA disease that is typical of RF+/RA patients.     

Guanosine inhibits CD40 receptor expression and function induced by cytokines and beta amyloid in mouse microglia cells

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-gamma (10 ng/ml) plus TNF-alpha (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta(1-42); 500 nM). Culture pretreatment with guanosine (10-300 microM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-gamma/TNF-alpha challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-gamma-induced phosphorylation on Ser(727) and translocation of STAT-1alpha to the nucleus as well as TNF-alpha-/Abeta-induced IkappaBalpha and NF-kappaB p65/RelA subunit phosphorylation, thus inhibiting NF-kappaB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-gamma/TNF-alpha-induced CD40 expression and function as well as on STAT-1alpha or NF-kappaB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer's disease.     

African trypanosomiasis: naturally occurring regulatory T cells favor trypanotolerance by limiting pathology associated with sustained type 1 inflammation

Tolerance to African trypanosomes requires the production of IFN-gamma in the early stage of infection that triggers the development of classically activated macrophages controlling parasite growth. However, once the first peak of parasitemia has been controlled, down-regulation of the type 1 immune response has been described. In this study, we have evaluated whether regulatory T cells (Tregs) contribute to the limitation of the immune response occurring during Trypanosoma congolense infection and hereby influence the outcome of the disease in trypanotolerant C57BL/6 host. Our data show that Foxp3+ Tregs originating from the naturally occurring Treg pool expanded in the spleen and the liver of infected mice. These cells produced IL-10 and limited the production of IFN-gamma by CD4+ and CD8+ effector T cells. Tregs also down-regulated classical activation of macrophages resulting in reduced TNF-alpha production. The Treg-mediated suppression of the type 1 inflammatory immune response did not hamper parasite clearance, but was beneficial for the host survival by limiting the tissue damages, including liver injury. Collectively, these data suggest a cardinal role for naturally occurring Tregs in the development of a trypanotolerant phenotype during African trypanosomiasis.     

Localization of synaptic proteins involved in neurosecretion in different membrane microdomains

A number of proteins and signalling molecules modulate voltage-gated calcium channel activity and neurosecretion. As recent findings have indicated the presence of Ca(v)2.1 (P/Q-type) channels and soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptors (SNAREs) in the cholesterol-enriched microdomains of neuroendocrine and neuronal cells, we investigated whether molecules known to modulate neurosecretion, such as the heterotrimeric G proteins and neuronal calcium sensor-1 (NCS-1), are also localized in these microdomains. After immuno-isolation, flotation gradients from Triton X-100-treated synaptosomal membranes revealed the presence of different detergent-resistant membranes (DRMs) containing proteins of the exocytic machinery (Ca(v)2.1 channels and SNAREs) or NCS-1; both DRM subtypes contained aliquots of heterotrimeric G protein subunits and phosphatidylinositol-4,5-bisphosphate. In line with the biochemical data, confocal imaging of immunolabelled membrane sheets revealed the localization of SNARE proteins and NCS-1 in different dot-like structures. This distribution was largely impaired by treatment with methyl-beta-cyclodextrin, thus suggesting the localization of all three proteins in cholesterol-dependent domains. Finally, bradykinin (which is known to activate the NCS-1 pathway) caused a significant increase in NCS-1 in the DRMs. These findings suggest that different membrane microdomains are involved in the spatial organization of the complex molecular network that converges on calcium channels and the secretory machinery.     

Linker molecules between laminins and dystroglycan ameliorate laminin-alpha2-deficient muscular dystrophy at all disease stages

Mutations in laminin-alpha2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-alpha2 are dystroglycan and alpha7beta1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to alpha7beta1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302-307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326-332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy.     

Iron deficiency differently affects metabolic responses in soybean roots

Iron deficiency responses were investigated in roots of soybean, a Strategy I plant species. Soybean responds to iron deficiency by decreasing growth, both at the root and shoot level. Chlorotic symptoms in younger leaves were evident after a few days of iron deficiency, with chlorophyll content being dramatically decreased. Moreover, several important differences were found as compared with other species belonging to the same Strategy I. The main differences are (i) a lower capacity to acidify the hydroponic culture medium, that was also reflected by a lower H(+)-ATPase activity as determined in a plasma membrane-enriched fraction isolated from the roots; (ii) a drastically reduced activity of the phosphoenolpyruvate carboxylase enzyme; (iii) a decrease in both cytosolic and vacuolar pHs; (iv) an increase in the vacuolar phosphate concentration, and (v) an increased exudation of organic carbon, particularly citrate, phenolics, and amino acids. Apparently, in soybean roots, some of the responses to iron deficiency, such as the acidification of the rhizosphere and other related processes, do not occur or occur only at a lower degree. These results suggest that the biochemical mechanisms induced by this nutritional disorder are differently regulated in this plant. A possible role of inorganic phosphate in the balance of intracellular pHs is also discussed.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.