Spatial mapping of juxtacrine axo-glial interactions identifies novel molecules in peripheral myelination.

Cell-cell interactions promote juxtacrine signals in specific subcellular domains, which are difficult to capture in the complexity of the nervous system. For example, contact between axons and Schwann cells triggers signals required for radial sorting and myelination. Failure in this interaction causes dysmyelination and axonal degeneration. Despite its importance, few molecules at the axo-glial surface are known. To identify novel molecules in axo-glial interactions, we modified the 'pseudopodia' sub-fractionation system and isolated the projections that glia extend when they receive juxtacrine signals from axons. By proteomics we identified the signalling networks present at the glial-leading edge, and novel proteins, including members of the Prohibitin family. Glial-specific deletion of Prohibitin-2 in mice impairs axo-glial interactions and myelination. We thus validate a novel method to model morphogenesis and juxtacrine signalling, provide insights into the molecular organization of the axo-glial contact, and identify a novel class of molecules in myelination.

Early markers of Fabry disease revealed by proteomics.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of the lysosomal hydrolase α-galactosidase A (α-GalA) that leads to the intra-lysosomal accumulation of globotriaosylceramide (Gb3) in various organ systems. As a consequence, a multisystems disorder develops, culminating in stroke, progressive renal and cardiac dysfunction. Enzyme replacement therapy (ERT) offers a specific treatment for patients affected by FD, though the monitoring of treatment is hindered by a lack of surrogate markers of response. Remarkably, due to the high heterogeneity of the Fabry phenotype, both diagnostic testing and treatment decisions are more challenging in females than in males; thus, reliable biomarkers for Fabry disease are needed, particularly for female patients. Here, we use a proteomic approach for the identification of disease-associated markers that can be used for the early diagnosis of FD as well as for monitoring the effectiveness of ERT. Our data show that the urinary proteome of Fabry naïve patients is different from that of normal subjects. In addition, biological pathways mainly affected by FD are related to immune response, inflammation, and energetic metabolism. In particular, the up-regulation of uromodulin, prostaglandin H2 d-isomerase and prosaposin in the urine of FD patients was demonstrated; these proteins might be involved in kidney damage at the tubular level, inflammation and immune response. Furthermore, comparing the expression of these proteins in Fabry patients before and after ERT treatment, a decrease of their concentration was observed, thus demonstrating the correlation between the identified markers and the effectiveness of the pharmacological treatment.

SIK1 localizes with nephrin in glomerular podocytes and its polymorphism predicts kidney injury.

Mutant α-adducin and endogenous ouabain levels exert a causal role in hypertension by affecting renal Na-K ATPase. In addition, mutant ß-adducin is involved in glomerular damage through nephrin down-regulation. Recently, the salt-inducible kinase 1 (SIK1) has been shown to exert a permissive role on mutant α-adducin effects on renal Na-K ATPase activity involved in blood pressure (BP) regulation and a SIK1 rs3746951 polymorphism has been associated with changes in vascular Na-K ATPase activity and BP. Here, we addressed the role of SIK1 on nephrin and glomerular functional modifications induced by mutant ß-adducin and ouabain, by using congenic substrains of the Milan rats expressing either mutant α- or ß-adducin, alone or in combination, ouabain hypertensive rats (OHR) and hypertensive patients. SIK1 co-localized and co-immunoprecipitated with nephrin from glomerular podocytes and associated with caveolar nephrin signaling. In cultured podocytes, nephrin-gene silencing decreased SIK1 expression. In mutant ß-adducin congenic rats and in OHR, the podocyte damage was associated with decreased nephrin and SIK1 expression. Conversely, when the effects of ß-adducin on podocytes were blocked by the presence of mutant α-adducin, nephrin and SIK1 expressions were restored. Ouabain effects were also reproduced in cultured podocytes. In hypertensive patients, nephrinuria, but not albuminuria, was higher in carriers of mutant SIK1 rs3746951 than in wild-type, implying a more direct effect of SIK1 on glomerular damage. These results demonstrate that, through nephrin, SIK1 is involved in the glomerular effects of mutant adducin and ouabain and a direct effect of SIK1 is also likely to occur in humans.

Molecular characterization of a recombinant replication protein (Rep) from the Antarctic bacterium Psychrobacter sp. TA144.

The Antarctic Gram-negative bacterium Psychrobacter sp. TA144 contains two small cryptic plasmids, called pTAUp and pTADw. pTAUp encodes a replication enzyme (PsyRep) whose activity is responsible for plasmid replication via the rolling circle replication pathway. Several attempts to produce the wild-type biologically active PsyRep in Escherichia coli failed, possibly due to auto-regulation of the protein population. However, the serendipitous occurrence of a frameshift mutation during the preparation of an expression vector resulted in the over-production of a recombinant protein, changed in its last 14 amino acid residues (PsyRep*), that precipitates in insoluble form. The purification of PsyRep* inclusion bodies and the successful refolding of the cold adapted enzyme allowed us to carry out its functional characterization. The mutated protein still displays a double stranded DNA nicking activity, while the change at the C-terminus impairs the enzyme specificity for the pTAUp cognate Ori+ sequence.

Molecular mechanisms regulating the subcellular localization of p95-APP1 between the endosomal recycling compartment and sites of actin organization at the cell surface.

Cell migration requires coordination between adhesion, actin organization and membrane traffic. Rac and ARF6 have been shown to cooperate for the organization of actin at the cell surface. Recently, the GIT family of ARF-GAPs has been identified, which includes proteins that can functionally interact with both ARF and Rac GTPases. The p95-APP1 protein is a member of this family, isolated as part of a multi-molecular complex interacting with GTP-Rac. Our previous work has indicated that this protein may be part of the machinery redirecting membrane recycling towards sites of protrusion during locomotion. By analyzing the distribution and the effects of truncated forms of p95-APP1, we show here that the lack of the ARF-GAP domain of p95-APP1 dramatically shifts its localization to large vesicles. The use of several markers of the endocytic pathway has revealed that the truncated p95-APP1 localizes specifically to a Rab11-, transferrin receptor-positive compartment. Other markers are excluded from the p95-APP1-positive vesicles, while known components of the multi-molecular complex colocalize with truncated p95-APP1 in this compartment. Coexpression of a constitutively active form of Rac induces the redistribution of the truncated constructs and of the associated PIX, PAK, and paxillin to peripheral sites of Rac-mediated actin organization, and the disassembly of the large Rab11-positive vesicles. Together, the data presented indicate that p95-APP1 is part of a complex that shuttles between the plasma membrane and the endocytic recycling compartment, and suggest that the dynamic redistribution of the p95-APP1-containing complex is mediated both by the ARF-GAP domain, and by the recruitment of the complex at the cell surface at sites of Rac activation.