Sorting nexin 3 mutation impairs development and neuronal function in Caenorhabditis elegans.

The sorting nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signaling and has been associated with several disorders, namely Alzheimer's disease and Down's syndrome. Despite the growing association of SNXs with neurodegeneration, not much is known about their function in the nervous system. The aim of this work was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, to dissect the role of distinct SNXs, particularly in the nervous system. By screening the C. elegans SNXs deletion mutants for morphological, developmental and behavioral alterations, we show here that snx-3 gene mutation leads to an array of developmental defects, such as delayed hatching, decreased brood size and life span and reduced body length. Additionally, ∆snx-3 worms present increased susceptibility to osmotic, thermo and oxidative stress and distinct behavioral deficits, namely, a chemotaxis defect which is independent of the described snx-3 role in Wnt secretion. ∆snx-3 animals also display abnormal GABAergic neuronal architecture and wiring and altered AIY interneuron structure. Pan-neuronal expression of C. elegans snx-3 cDNA in the ∆snx-3 mutant is able to rescue its locomotion defects, as well as its chemotaxis toward isoamyl alcohol. Altogether, the present work provides the first in vivo evidence of the SNX-3 role in the nervous system.

SNX27: A trans-species cognitive modulator with implications for anxiety and stress susceptibility.

Sorting Nexin 27 (SNX27) is a brain-enriched endosome-associated cargo adaptor that shapes excitatory control, being relevant for cognitive and reward processing, and for several neurological conditions. Despite this, SNX27's role in the nervous system remains poorly explored. To further understand SNX27 function, we performed an extensive behavioral characterization comprising motor, cognitive and emotional dimensions of SNX27+/- mice. Furthermore, attending on the recently described association between SNX27 function and cellular stress signaling mechanisms in vitro, we explored SNX27-stress interplay using a Caenorhabditis elegans Δsnx-27 mutant and wild-type (WT) rodents after stress exposure. SNX27+/- mice, as C. elegans Δsnx-27 mutants, present cognitive impairments, highlighting a conserved role for SNX27 in cognitive modulation across species. Interestingly, SNX27 downmodulation leads to anxiety-like behavior in mice evaluated in the Elevated Plus Maze (EPM). This anxious phenotype is associated with increased dendritic complexity of the bed nucleus of the stria terminalis (BNST) neurons, and increased complexity of the basolateral amygdala (BLA) pyramidal neurons. These findings highlight the still unknown role of SNX27 in anxiety regulation. Moreover, we uncovered a direct link between SNX27 dysfunction and stress susceptibility in C. elegans and found that stress-exposed rodents display decreased SNX27 levels in stress-susceptible brain regions. Altogether, we provided new insights on SNX27's relevance in anxiety-related behaviors and neuronal structure in stress-associated brain regions.

Age-Related Sexual Dimorphism on the Longitudinal Progression of Blood Immune Cells in BALB/cByJ Mice.

The study of immune system aging is of relevance, considering its myriad of interactions and role in protecting and maintaining body homeostasis. While mouse models have been extensively used to study immune system aging, little is known on how the main immune populations progress over time and what is the impact of sex. To contribute to filling this gap, male and female BALB/cByJ mice were longitudinally evaluated, from 3 to 18 months old, for the main blood populations, assessed by flow cytometry. Using linear mixed-effect models, we observed that the percentages of neutrophils, monocytes, eosinophils, and total natural killer (NK) cells increase with aging, while those of B cells, T cells (including CD4+ and CD8+ subsets), and Ly6C+ NK cells decrease. Males present higher percentages of neutrophils and classical monocytes Ly6Chigh over time, while females present higher percentages of total T cells, both CD4+ and CD8+, eosinophils, and NK cells. Males and females display similar percentages of B cells, even though with opposite accelerated progressions over time. This study revealed that mouse models recapitulate what is observed in humans during aging: an overall proportional decrease in the adaptive and an increase in the innate immune cells. Additionally, it uncovers an age-related sexual dimorphism in the proportion of immune cells in circulation, further strengthening the need to explore the impact of sex when addressing immune system aging using mouse models.

WDR60-mediated dynein-2 loading into cilia powers retrograde IFT and transition zone crossing.

The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homologue, WDR-60, and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing retrograde motor availability at the ciliary tip. Consistent with this, we show that fewer dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT, and the NPHP module as a roadblock to dynein-2 passage through the transition zone.

Functional specialization and differential regulation of short-chain carboxylic acid transporters in the pathogen Candida albicans.

The major fungal pathogen Candida albicans has the metabolic flexibility to assimilate a wide range of nutrients in its human host. Previous studies have suggested that C. albicans can encounter glucose-poor microenvironments during infection and that the ability to use alternative non-fermentable carbon sources contributes to its virulence. JEN1 encodes a monocarboxylate transporter in C. albicans and we show that its paralogue, JEN2, encodes a novel dicarboxylate plasma membrane transporter, subjected to glucose repression. A strain deleted in both genes lost the ability to transport lactic, malic and succinic acids by a mediated mechanism and it displayed a growth defect on these substrates. Although no significant morphogenetic or virulence defects were found in the double mutant strain, both JEN1 and JEN2 were strongly induced during infection. Jen1-GFP (green fluorescent protein) and Jen2-GFP were upregulated following the phagocytosis of C. albicans cells by neutrophils and macrophages, displaying similar behaviour to an Icl1-GFP fusion. In the murine model of systemic candidiasis approximately 20-25% of C. albicans cells infecting the kidney expressed Jen1-GFP and Jen2-GFP. Our data suggest that Jen1 and Jen2 are expressed in glucose-poor niches within the host, and that these short-chain carboxylic acid transporters may be important in the early stages of infection.

Sorting Out Sorting Nexins Functions in the Nervous System in Health and Disease.

Endocytosis is a fundamental process that controls protein/lipid composition of the plasma membrane, thereby shaping cellular metabolism, sensing, adhesion, signaling, and nutrient uptake. Endocytosis is essential for the cell to adapt to its surrounding environment, and a tight regulation of the endocytic mechanisms is required to maintain cell function and survival. This is particularly significant in the central nervous system (CNS), where composition of neuronal cell surface is crucial for synaptic functioning. In fact, distinct pathologies of the CNS are tightly linked to abnormal endolysosomal function, and several genome wide association analysis (GWAS) and biochemical studies have identified intracellular trafficking regulators as genetic risk factors for such pathologies. The sorting nexins (SNXs) are a family of proteins involved in protein trafficking regulation and signaling. SNXs dysregulation occurs in patients with Alzheimer's disease (AD), Down's syndrome (DS), schizophrenia, ataxia and epilepsy, among others, establishing clear roles for this protein family in pathology. Interestingly, restoration of SNXs levels has been shown to trigger synaptic plasticity recovery in a DS mouse model. This review encompasses an historical and evolutionary overview of SNXs protein family, focusing on its organization, phyla conservation, and evolution throughout the development of the nervous system during speciation. We will also survey SNXs molecular interactions and highlight how defects on SNXs underlie distinct pathologies of the CNS. Ultimately, we discuss possible strategies of intervention, surveying how our knowledge about the fundamental processes regulated by SNXs can be applied to the identification of novel therapeutic avenues for SNXs-related disorders.

Plasmids for in vivo construction of integrative Candida albicans vectors in Saccharomyces cerevisiae.

A general system has been devised for the in vivo construction of Candida albicans integrative vectors in Saccharomyces cerevisiae. The system is especially useful for the integration of genes in C. albicans that cannot be propagated in Escherichia coli, possibly because of their toxic effects. The ligation of S. cerevisiae 2 µ sequences to a C. albicans integrative vector permits in vivo maintenance and gap repair cloning within S. cerevisiae. After the vector assembly, it can be purified from S. cerevisiae or amplified by PCR and then used for transformation of C. albicans. The S. cerevisiae 2 µ sequence is completely removed by linearization prior to C. albicans transformation, such that no unwanted DNA is transferred in the final construct. The system was successfully used to clone and reintegrate the C. albicans JEN2 gene, which encodes a membrane protein that is apparently toxic to E. coli. Three popular C. albicans integrative vectors, CIp10, CIp20 and CIp30, are now available in versions that permit gap repair in S. cerevisiae.

SNX31: a novel sorting nexin associated with the uroplakin-degrading multivesicular bodies in terminally differentiated urothelial cells.

Uroplakins (UP), a group of integral membrane proteins, are major urothelial differentiation products that form 2D crystals of 16-nm particles (urothelial plaques) covering the apical surface of mammalian bladder urothelium. They contribute to the urothelial barrier function and, one of them, UPIa, serves as the receptor for uropathogenic Escherichia coli. It is therefore important to understand the mechanism by which these surface-associated uroplakins are degraded. While it is known that endocytosed uroplakin plaques are targeted to and line the multivesicular bodies (MVBs), it is unclear how these rigid-looking plaques can go to the highly curved membranes of intraluminal vesicles (ILVs). From a cDNA subtraction library, we identified a highly urothelium-specific sorting nexin, SNX31. SNX31 is expressed, like uroplakins, in terminally differentiated urothelial umbrella cells where it is predominantly associated with MVBs. Apical membrane proteins including uroplakins that are surface biotin-tagged are endocytosed and targeted to the SNX31-positive MVBs. EM localization demonstrated that SNX31 and uroplakins are both associated not only with the limiting membranes of MVBs containing uroplakin plaques, but also with ILVs. SNX31 can bind, on one hand, the PtdIns3P-enriched lipids via its N-terminal PX-domain, and, on the other hand, it binds uroplakins as demonstrated by co-immunoprecipitation and proximity ligation assay, and by its reduced membrane association in uroplakin II-deficient urothelium. The fact that in urothelial umbrella cells MVBs are the only major intracellular organelles enriched in both PtdIns3P and uroplakins may explain SNX31's MVB-specificity in these cells. However, in MDCK and other cultured cells transfected SNX31 can bind to early endosomes possibly via lipids. These data support a model in which SNX31 mediates the endocytic degradation of uroplakins by disassembling/collapsing the MVB-associated uroplakin plaques, thus enabling the uroplakin-containing (but 'softened') membranes to bud and form the ILVs for lysosomal degradation and/or exosome formation.

Role of the DHH1 gene in the regulation of monocarboxylic acids transporters expression in Saccharomyces cerevisiae.

Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170:301-306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects.

A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis.

Endocytosis regulates the plasma membrane protein landscape in response to environmental cues. In yeast, the endocytosis of transporters depends on their ubiquitylation by the Nedd4-like ubiquitin ligase Rsp5, but how extracellular signals trigger this ubiquitylation is unknown. Various carbon source transporters are known to be ubiquitylated and endocytosed when glucose-starved cells are exposed to glucose. We show that this required the conserved arrestin-related protein Rod1/Art4, which was activated in response to glucose addition. Indeed, Rod1 was a direct target of the glucose signaling pathway composed of the AMPK homologue Snf1 and the PP1 phosphatase Glc7/Reg1. Glucose promoted Rod1 dephosphorylation and its subsequent release from a phospho-dependent interaction with 14-3-3 proteins. Consequently, this allowed Rod1 ubiquitylation by Rsp5, which was a prerequisite for transporter endocytosis. This paper therefore demonstrates that the arrestin-related protein Rod1 relays glucose signaling to transporter endocytosis and provides the first molecular insights into the nutrient-induced activation of an arrestin-related protein through a switch in post-translational modifications.

Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of lysine 63-linked ubiquitin chains.

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys(63) linked chain(s), and Jen1-Lys(338) is one of the target residues. Ubiquitin-Lys(63)-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys(63)-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.