High-throughput functional analysis of autism genes in zebrafish identifies convergence in dopaminergic and neuroimmune pathways.

Advancing from gene discovery in autism spectrum disorders (ASDs) to the identification of biologically relevant mechanisms remains a central challenge. Here, we perform parallel in vivo functional analysis of 10 ASD genes at the behavioral, structural, and circuit levels in zebrafish mutants, revealing both unique and overlapping effects of gene loss of function. Whole-brain mapping identifies the forebrain and cerebellum as the most significant contributors to brain size differences, while regions involved in sensory-motor control, particularly dopaminergic regions, are associated with altered baseline brain activity. Finally, we show a global increase in microglia resulting from ASD gene loss of function in select mutants, implicating neuroimmune dysfunction as a key pathway relevant to ASD biology.

Expression of dlx genes in the normal and regenerating brain of adult zebrafish.

Dysfunctions in the GABAergic system lead to various pathological conditions and impaired inhibitory function is one of the causes behind neuropathies characterized by neuronal hyper excitability. The Dlx homeobox genes are involved in the development of nervous system, neural crest, branchial arches and developing appendages. Dlx genes also take part in neuronal migration and differentiation during development, more precisely, in the migration and differentiation of GABAergic neurons. Functional analysis of dlx genes has mainly been carried out in developing zebrafish embryos and larvae, however information regarding the expression and roles of these genes in the adult zebrafish brain is still lacking. The extensive neurogenesis that takes place in the adult zebrafish brain, makes them a good model for the visualization of mechanisms involving dlx genes during adulthood in physiological conditions and during regeneration of the nervous system. We have identified the adult brain regions where transcripts of dlx1a, dlx2a, dlx5a and dlx6a genes are normally found and have confirmed that within telencephalic domains, there is high overlapping expression of the four dlx paralogs with a marker for GABAergic neurons. Co-localization analyses carried with the Tg(dlx6a-1.4kbdlx5a/dlx6a:GFP) reporter line have also shown that in some areas of the diencephalon, cells expressing the dlx5a/6a bigene may have a neural stem cell identity. Furthermore, investigations in a response to stab wound lesions, have demonstrated a possible participation of the dlx5a/6a bigene, most likely of dlx5a, during regeneration of the adult zebrafish brain. These observations suggest a possible participation of dlx-expressing cells during brain regeneration in adult zebrafish and also provide information on the role of dlx genes under normal physiological conditions in adults.

Adding MASP1 to the lectin pathway-Leprosy association puzzle: Hints from gene polymorphisms and protein levels.

BACKGROUND: Deposition of complement factors on Mycobacterium leprae may enhance phagocytosis. Such deposition may occur through the lectin pathway of complement. Three proteins of the lectin pathway are produced from the gene MASP1: Mannan-binding lectin-associated serine protease 1 (MASP-1) and MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44). Despite their obvious importance, the roles played by these proteins have never been investigated in leprosy disease. METHODOLOGY: We haplotyped five MASP1 polymorphisms by multiplex sequence-specific PCR (intronic rs7609662*G>A and rs13064994*C>T, exon 12 3'-untranslated rs72549262*C>G, rs1109452*C>T and rs850314*G>A) and measured MASP-1, MASP-3 and MAp44 serum levels in 196 leprosy patients (60%, lepromatous) and 193 controls. PRINCIPAL FINDINGS: Lower MASP-3 and MAp44 levels were observed in patients, compared with controls (P = 0.0002 and P<0.0001, respectively) and in lepromatous, compared with non-lepromatous patients (P = 0.008 and P = 0.002, respectively). Higher MASP-3 levels were present in controls carrying variants/haplotypes associated with leprosy resistance (rs13064994*T, rs1109452_rs850314*CG within GT_CCG and rs850314*A: OR = 0.5-0.6, Pcorr = 0.01-0.04). Controls with rs1109452*T, included in susceptibility haplotypes (GT_GTG/GT_CTG: OR = 2.0, Pcorr = 0.03), had higher MASP-1 and lower MASP-3 levels (P≤0.009). Those with GC_CCG, presented increasing susceptibility (OR = 1.7, Pcorr = 0.006) and higher MAp44 levels (P = 0.015). MASP-3 expression decreased in patients, compared with controls carrying rs1109452_rs850314*CA or CG (P≤0.02), which may rely on exon 12 CpG methylation and/or miR-2861/miR-3181 mRNA binding. CONCLUSION: Polymorphisms regulating MASP-3/MAp44 availability in serum modulate leprosy susceptibility, underlining the importance of lectin pathway regulation against pathogens that exploit phagocytosis to parasitize host macrophages.

Hepatitis B Virus Infection Among Leprosy Patients: A Case for Polymorphisms Compromising Activation of the Lectin Pathway and Complement Receptors.

Thousands of leprosy patients not only suffer from physical deformities, but also either have or have had hepatitis B virus (HBV) coinfection. Polymorphisms of the complement system modulate susceptibility to leprosy, but genetic susceptibility to past or present HBV infection is unknown. We used sequencing and multiplex sequence-specific PCR to genotype 72 polymorphisms of seven genes (MBL2, FCN1, FCN2, FCN3, MASP1, MASP2, C3) encoding components of the lectin pathway, and two genes encoding complement receptors (CR1, VSIG4) in 190 patients, of which 74 were positive for HBsAg and/or anti-HBc (HBV+, 93.2% with a resolved infection) and 116 lepromatous patients, and 408 HBV-blood donors. In addition, we tested for levels of proteins of the lectin pathway. We found no difference between serum concentrations of mannan-binding lectin (MBL), MBL-associated serine proteins (MASP-1, MASP-2, MASP-3, MAp44), ficolin-3 (FCN-3), soluble complement receptor 1 (sCR1) and MBL mediated C4 activation, measured by ELISA or TRIFMA in up to 167 HBV+ and HBV- patients. Haplotypes lowering protein levels or encoding dysfunctional proteins increased susceptibility to HBV infection: MBL2*LYQC (OR = 3.4, p = 0.02), MASP1*AC_CC (OR = 4.0, p = 0.015) and MASP2*1C2-l (OR = 5.4, p = 0.03). Conversely, FCN1*3C2 haplotype, associated with higher gene expression, was protective (OR = 0.56, P = 0.033). Other haplotypes associated with HBV susceptibility were: MASP2*2B1-i (OR = 19.25, P = 0.003), CR1*3A (OR = 2.65, P = 0.011) and VSIG4*TGGRCG (OR = 12.55, P = 0.014). Some polymorphisms in ficolin genes associated with lower protein levels increased susceptibility to leprosy/HBV infection: FCN*1 (OR = 1.66, P = 0.029), FCN2*GGGCAC (OR = 6.73, P = 0.008), and FCN3*del_del_C (OR = 12.54, P = 0.037), and to lepromatous disease/HBV infection: FCN2*TA (OR = 2.5, P = 0.009), whereas FCN2*MAG was associated with increased FCN-2 expression and resistance against coinfection (OR = 0.29, P = 0.026). These associations were independent of demographic factors and did not increase susceptibility to leprosy per se, except MASP2*1C2-l. Associations for FCN2, FCN3, MASP1, MASP2, and VSIG4 variants were also independent of each other. In conclusion, polymorphisms compromising activation of the lectin pathway of complement increase susceptibility to HBV infection, with ficolin polymorphisms playing a major role in modulating the susceptibility among leprosy patients.

Mannose-Binding Lectin Does Not Act as a Biomarker for the Progression of Preinvasive Lesions of Invasive Cervical Cancer.

OBJECTIVE: To evaluate serum concentrations of mannose-binding lectin (MBL) in women presenting with different human papillomavirus (HPV)-associated cervical lesions. SUBJECTS AND METHODS: A total of 364 women, who underwent screening for cervical cancer or treatment at the Erasto Gaertner Cancer Hospital (HEG), Curitiba, Brazil, were enrolled in the study. Based on the latest cervical colposcopy-guided biopsy results, the women were divided into 4 groups: cervical intraepithelial neoplasia CIN-I (n = 54), CIN-II (n = 72), CIN-III (n = 145), and invasive cancer (n = 93). A time-resolved immunofluorometric assay was used to measure the MBL concentrations in serum. The statistical analysis was done using GraphPad Prism 6.0. Comparisons were performed by Kruskal-Wallis and Mann-Whitney tests and analyzed by χ2 test; continuous variables are presented as medians and categorical variables as frequencies. RESULTS: The median MBL concentrations in decreasing order were as follows: invasive cancer: 1,452 ng/mL, CIN-I: 1,324 ng/mL, CIN-II: 1,104 ng/mL, and CIN-III 1,098 ng/mL. However, no statistical significance was found among the 4 groups with HPV-associated lesions (p = 0.11). Equally, the MBL levels did not show a significant association between the age of the patients and the severity of the cervical lesions (p = 0.68). No statistical significance was found in the median values of MBL or in the status of MBL deficient (<100 ng/mL) and high producers (>1,000 ng/mL) among the women in each group (p = 0.77). CONCLUSION: In this study, there was no statistically significant difference in MBL serum levels among the groups with CIN. Hence MBL serum concentration appeared not to have influenced the progression of HPV-related preinvasive cervical lesions into invasive cancer.

Pyridoxine-Dependent Epilepsy in Zebrafish Caused by Aldh7a1 Deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare disease characterized by mutations in the lysine degradation gene ALDH7A1 leading to recurrent neonatal seizures, which are uniquely alleviated by high doses of pyridoxine or pyridoxal 5'-phosphate (vitamin B6 vitamers). Despite treatment, neurodevelopmental disabilities are still observed in most PDE patients underlining the need for adjunct therapies. Over 60 years after the initial description of PDE, we report the first animal model for this disease: an aldh7a1-null zebrafish (Danio rerio) displaying deficient lysine metabolism and spontaneous and recurrent seizures in the larval stage (10 days postfertilization). Epileptiform electrographic activity was observed uniquely in mutants as a series of population bursts in tectal recordings. Remarkably, as is the case in human PDE, the seizures show an almost immediate sensitivity to pyridoxine and pyridoxal 5'-phosphate, with a resulting extension of the life span. Lysine supplementation aggravates the phenotype, inducing earlier seizure onset and death. By using mass spectrometry techniques, we further explored the metabolic effect of aldh7a1 knockout. Impaired lysine degradation with accumulation of PDE biomarkers, B6 deficiency, and low γ-aminobutyric acid levels were observed in the aldh7a1-/- larvae, which may play a significant role in the seizure phenotype and PDE pathogenesis. This novel model provides valuable insights into PDE pathophysiology; further research may offer new opportunities for drug discovery to control seizure activity and improve neurodevelopmental outcomes for PDE.

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain.

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons.