The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network.

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

Redundant functions of the SLC5A transporters Rumpel, Bumpel, and Kumpel in ensheathing glial cells.

Neuronal processing is energy demanding and relies on sugar metabolism. To nurture the Drosophila nervous system, the blood-brain barrier forming glial cells take up trehalose from the hemolymph and then distribute the metabolic products further to all neurons. This function is provided by glucose and lactate transporters of the solute carrier (SLC) 5A family. Here we identified three SLC5A genes that are specifically expressed in overlapping sets of CNS glial cells, rumpel, bumpel and kumpel. We generated mutants in all genes and all mutants are viable and fertile, lacking discernible phenotypes. Loss of rumpel causes subtle locomotor phenotypes and flies display increased daytime sleep. In addition, in bumpel kumpel double mutants, and to an even greater extent in rumpel bumpel kumpel triple mutants, oogenesis is disrupted at the onset of the vitollegenic phase. This indicates a partially redundant function between these genes. Rescue experiments exploring this effect indicate that oogenesis can be affected by CNS glial cells. Moreover, expression of heterologous mammalian SLC5A transporters, with known transport properties, suggest that Bumpel and/or Kumpel transport glucose or lactate. Overall, our results imply a redundancy in SLC5A nutrient sensing functions in Drosophila glial cells, affecting ovarian development and behavior.

The Drosophila NCAM homolog Fas2 signals independently of adhesion.

The development of tissues and organs requires close interaction of cells. To achieve this, cells express adhesion proteins such as the neural cell adhesion molecule (NCAM) or its Drosophila ortholog Fasciclin 2 (Fas2). Both are members of the Ig-domain superfamily of proteins that mediate homophilic adhesion. These proteins are expressed as isoforms differing in their membrane anchorage and their cytoplasmic domains. To study the function of single isoforms, we have conducted a comprehensive genetic analysis of Fas2 We reveal the expression pattern of all major Fas2 isoforms, two of which are GPI anchored. The remaining five isoforms carry transmembrane domains with variable cytoplasmic tails. We generated Fas2 mutants expressing only single isoforms. In contrast to the null mutation, which causes embryonic lethality, these mutants are viable, indicating redundancy among the different isoforms. Cell type-specific rescue experiments showed that glial-secreted Fas2 can rescue the Fas2 mutant phenotype to viability. This demonstrates that cytoplasmic Fas2 domains have no apparent essential functions and indicate that Fas2 has function(s) other than homophilic adhesion. In conclusion, our data suggest novel mechanistic aspects of a long-studied adhesion protein.

The Drosophila Blood-Brain Barrier Adapts to Cell Growth by Unfolding of Pre-existing Septate Junctions.

The blood-brain barrier is crucial for nervous system function. It is established early during development and stays intact during growth of the brain. In invertebrates, septate junctions are the occluding junctions of this barrier. Here, we used Drosophila to address how septate junctions grow during larval stages when brain size increases dramatically. We show that septate junctions are preassembled as long, highly folded strands during embryonic stages, connecting cell vertices. During subsequent cell growth, these corrugated strands are stretched out and stay intact during larval life with very little protein turnover. The G-protein coupled receptor Moody orchestrates the continuous organization of junctional strands in a process requiring F-actin. Consequently, in moody mutants, septate junction strands cannot properly stretch out during cell growth. To compensate for the loss of blood-brain barrier function, moody mutants form interdigitating cell-cell protrusions, resembling the evolutionary ancient barrier type found in primitive vertebrates or invertebrates such as cuttlefish.

Kinesin heavy chain function in Drosophila glial cells controls neuronal activity.

Kinesin heavy chain (Khc) is crucially required for axonal transport and khc mutants show axonal swellings and paralysis. Here, we demonstrate that in Drosophila khc is equally important in glial cells. Glial-specific downregulation of khc by RNA interference suppresses neuronal excitability and results in spastic flies. The specificity of the phenotype was verified by interspecies rescue experiments and further mutant analyses. Khc is mostly required in the subperineurial glia forming the blood-brain barrier. Following glial-specific knockdown, peripheral nerves are swollen with maldistributed mitochondria. To better understand khc function, we determined Khc-dependent Rab proteins in glia and present evidence that Neurexin IV, a well known blood-brain barrier constituent, is one of the relevant cargo proteins. Our work shows that the role of Khc for neuronal excitability must be considered in the light of its necessity for directed transport in glia.

Drosophila Neurexin IV stabilizes neuron-glia interactions at the CNS midline by binding to Wrapper.

Ensheathment of axons by glial membranes is a key feature of complex nervous systems ensuring the separation of single axons or axonal fascicles. Nevertheless, the molecules that mediate the recognition and specific adhesion of glial and axonal membranes are largely unknown. We use the Drosophila midline of the embryonic central nervous system as a model to investigate these neuron glia interactions. During development, the midline glial cells acquire close contact to commissural axons and eventually extend processes into the commissures to wrap individual axon fascicles. Here, we show that this wrapping of axons depends on the interaction of the neuronal transmembrane protein Neurexin IV with the glial Ig-domain protein Wrapper. Although Neurexin IV has been previously described to be an essential component of epithelial septate junctions (SJ), we show that its function in mediating glial wrapping at the CNS midline is independent of SJ formation. Moreover, differential splicing generates two different Neurexin IV isoforms. One mRNA is enriched in septate junction-forming tissues, whereas the other mRNA is expressed by neurons and recruited to the midline by Wrapper. Although both Neurexin IV isoforms are able to bind Wrapper, the neuronal isoform has a higher affinity for Wrapper. We conclude that Neurexin IV can mediate different adhesive cell-cell contacts depending on the isoforms expressed and the context of its interaction partners.

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila.

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Here we report that Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, is directing peripheral glial cell maturation. We show that crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, we identified some of the in vivo target genes that encode proteins localized in autocellular septate junctions. In conclusion, here we show that glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions.