Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila

The vertebrate Apolipoprotein D (ApoD) is a lipocalin secreted from subsets of neurons and glia during neural development and aging . A strong correlation exists between ApoD overexpression and numerous nervous system pathologies as well as obesity, diabetes, and many forms of cancer . However, the exact relationship between the function of ApoD and the pathophysiology of these diseases is still unknown. We have generated loss-of-function Drosophila mutants for the Glial Lazarillo (GLaz) gene , a homolog of ApoD in the fruit fly, mainly expressed in subsets of adult glial cells. The absence of GLaz reduces the organism's resistance to oxidative stress and starvation and shortens male lifespan. The mutant flies exhibit a smaller body mass due to a lower amount of neutral lipids stored in the fat body. Apoptotic neural cell death increases in aged flies or upon paraquat treatment, which also impairs neural function as assessed by behavioral tests. The higher sensitivity to oxidative stress and starvation and the reduced fat storage revert to control levels when a GFP-GLaz fusion protein is expressed under the control of the GLaz natural promoter. Finally, GLaz mutants have a higher concentration of lipid peroxidation products, pointing to a lipid peroxidation protection or scavenging as the mechanism of action for this lipocalin. In agreement with Walker et al. (, in this issue of Current Biology), who analyze the effects of overexpressing GLaz, we conclude that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration.     

Molecular interactions of the neuronal GPI-anchored lipocalin Lazarillo

Lazarillo, a glycoprotein involved in axon growth and guidance in the grasshopper embryo, is the only member of the lipocalin family that is attached to the cell surface by a GPI anchor. Recently, the study of Lazarillo homologous genes in Drosophila and mouse has revealed new functions in the regulation of lifespan, stress resistance and neurodegeneration. Here we report an analysis of biochemical properties of Lazarillo to gain insight into the molecular basis of its physiological function. Recombinant forms of the grasshopper protein were expressed in two different systems to test: (1) potential binding of several hydrophobic ligands; (2) protein-protein homophilic interactions; and (3) whether interaction with the function-blocking mAb 10E6 interferes with ligand binding. We tested 10 candidate ligands (retinoic acid, heme, bilirubin, biliverdin, ecdysterone, juvenile hormone, farnesol, arachidonic acid, linoleic acid and palmitic acid), and monitored binding using electrophoretic mobility shift, absorbance spectrum, and fluorimetry assays. Our work indicates binding to heme and retinoic acid, resulting in increased electrophoretic mobility, as well as to fatty acids, resulting in multimerization. Retinoic acid and fatty acids binding were confirmed by fluorescence titration, and heme binding was confirmed with absorbance spectrum assays. We demonstrate that Lazarillo oligomerizes in solution and can form clusters in the plasma membrane when expressed and GPI-anchored to the cell surface, however it is unable to mediate cell-cell adhesion. Finally, by ligand-mAb competition experiments we show that ligand-binding alone cannot be the key factor for Lazarillo to perform its function during axonal growth in the grasshopper embryo. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Ontogeny of identified cells from the median domain in the embryonic brain of the grasshopper Schistocerca gregaria

In this paper, we propose an ontogeny for previously identified cells from the median domain in the midline of the embryonic brain of the grasshopper Schistocerca gregaria. The so-called lateral cells (LCs) are characteristically located laterally within the median domain at its border with the protocerebral hemispheres. The LC occurs singly and can be identified in the early embryo on the basis of their expression of the cell surface lipocalin Lazarillo. Using immunocytochemical, dye injection, electron microscopical and histological methods, we show that these LC are neurons and derive as postmitotic cells directly from the epithelium of the median domain. Further, they and the other identified cells of the median domain such as the protocerebral commissure pioneers (PCP), co-express the Mes-3 antigen, consistent with a derivation from the mesectodermal germ layer of the embryo. Subsequent to axogenesis, electron microscopy reveals that these Mes-3-expressing LC fasciculate with the co-expressing PCPs within the developing protocerebral commissure. We present a model for the origin of all these cells based on histological data and bromodeoxyuridine incorporation. The model suggests a delamination of cells from the mesectoderm followed by a migration to their ultimate sites within the median domain.     

High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo

In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome.     

YodA from Escherichia coli is a metal-binding,lipocalin-like protein

We have determined the crystal structure of YodA, an Escherichia coli protein of unknown function. YodA had been identified under conditions of cadmium stress, and we confirm that it binds metals such as cadmium and zinc. We have also found nickel bound in one of the crystal forms. YodA is composed of two domains: a main lipocalin/calycin-like domain and a helical domain. The principal metal-binding site lies on one side of the calycin domain, thus making YodA the first metal-binding lipocalin known. Our experiments suggest that YodA expression may be part of a more general stress response. From sequence analogy with the C-terminal domain of a metal-binding receptor of a member of bacterial ATP-binding cassette transporters, we propose a three-dimensional model for this receptor and suggest that YodA may have a receptor-type partner in E. coli.     

Urinary Lipocalin Protein in a Female Rodent with Correlation to Phases in the Estrous Cycle: An Experimental Study Accompanied by In Silico Analysis

Male urinary lipocalin family proteins, practically odorant-binding proteins but also could be pheromones by themselves, in rodents act as a shuttle for chemosignal communication and facilitate delivery of the signals for access to congeners. However, presence of this protein in urine of female rodents has not yet been reported. Therefore, the present investigation was carried out to find if lipocalin family protein is present in the urine of female house rat and, if so, to find whether its expression differs between the phases in the estrous cycle. The rat urinary protein was separated in single dimensional gel electrophoresis. A 14.5 kDa lipocalin protein appeared in the urine prominently during the estrus and metestrus phases compared to proestrus and diestrus phases. The expression of this protein in the urine was very low in ovariectomized rats. MALDI-TOF/MS analysis affirmed the 14.5 kDa protein as a lipocalin family protein. Analysis adopting bio-informatics tools further proved the protein as a lipocalin family member. Thus, this study for the first time demonstrated the presence of a lipocalin family protein in the urine of a female rodent and it was highly expressed during estrus phase. This lipocalin protein in female rat urine may facilitate a chemosignal function independently of a pheromone or in association with a specific pheromone.     

Information filtering by synchronous spikes in a neural population

Information about time-dependent sensory stimuli is encoded by the spike trains of neurons. Here we consider a population of uncoupled but noisy neurons (each subject to some intrinsic noise) that are driven by a common broadband signal. We ask specifically how much information is encoded in the synchronous activity of the population and how this information transfer is distributed with respect to frequency bands. In order to obtain some insight into the mechanism of information filtering effects found previously in the literature, we develop a mathematical framework to calculate the coherence of the synchronous output with the common stimulus for populations of simple neuron models. Within this frame, the synchronous activity is treated as the product of filtered versions of the spike trains of a subset of neurons. We compare our results for the simple cases of (1) a Poisson neuron with a rate modulation and (2) an LIF neuron with intrinsic white current noise and a current stimulus. For the Poisson neuron, formulas are particularly simple but show only a low-pass behavior of the coherence of synchronous activity. For the LIF model, in contrast, the coherence function of the synchronous activity shows a clear peak at high frequencies, comparable to recent experimental findings. We uncover the mechanism for this shift in the maximum of the coherence and discuss some biological implications of our findings.     

Molecular cloning of a novel lipocalin-1 interacting human cell membrane receptor using phage display

Human lipocalin-1 (Lcn-1, also called tear lipocalin), a member of the lipocalin structural superfamily, is produced by a number of glands and tissues and is known to bind an unusually large array of hydrophobic ligands. Apart from its specific function in stabilizing the lipid film of human tear fluid, it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. To characterize proteins involved in the reception, detoxification, or degradation of these ligands, a cDNA phage-display library from human pituitary gland was constructed and screened for proteins interacting with Lcn-1. Using this method an Lcn-1 interacting phage was isolated that expressed a novel human protein. Molecular cloning and analysis of the entire cDNA indicated that it encodes a 55-kDa protein, lipocalin-1 interacting membrane receptor (LIMR), with nine putative transmembrane domains. The cell membrane location of this protein was confirmed by immunocytochemistry and Western blot analysis of membrane fractions of human NT2 cells. Independent biochemical investigations using a recombinant N-terminal fragment of LIMR also demonstrated a specific interaction with Lcn-1 in vitro. Based on these data, we suggest LIMR to be a receptor of Lcn-1 ligands. These findings constitute the first report of cloning of a lipocalin interacting, plasma membrane-located receptor, in general. In addition, a sequence comparison supports the biological relevance of this novel membrane protein, because genes with significant nucleotide sequence similarity are present in Takifugu rubripes, Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Bos taurus, and Sus scrofa. According to data derived from the human genome sequencing project, the LIMR-encoding gene has to be mapped on human chromosome 12, and its intron/exon organization could be established. The entire LIMR-encoding gene consists of about 13.7 kilobases in length and contains 16 introns with a length between 91 and 3438 base pairs.     

PIEZO2 ion channels in proprioception

PIEZO2 is a stretch-gated ion channel that is expressed at high levels in somatosensory neurons. Humans with rare mutations in the PIEZO2 gene have profound mechanosensory deficits that include a loss of the sense of proprioception. These striking phenotypes match those seen in conditional knockout mouse models demonstrating the highly conserved function for this gene. Here, we review the ramifications of loss of PIEZO2 function on normal daily activities and what studies like these have revealed about proprioception at the molecular and cellular level. Additionally, we highlight recent work that has uncovered the surprising functional and molecular diversity of proprioceptors. Together, these findings pioneer a path toward determining how the detection of mechanosensory input from muscles and tendons is used to control posture and refine motor performance.     

Neutrophil Gelatinase-associated Lipocalin in Normal and Neoplastic Human Tissues. Cell Type-specific Pattern of Expression

Neutrophil gelatinase-associated lipocalin (NGAL) has recently been identified in myeloperoxidase-negative neutrophil granules. Members of the lipocalin family are thought to bind and transport small lipophilic molecules such as retinoids and roles in cell regulation have been proposed. Recently, NGAL has also been demonstrated in the colonic mucosa in certain pathologic conditions.The aim of this study was to examine the distribution of NGAL in normal and neoplastic tissues by immunohistochemistry. Interestingly, NGAL was found in a variety of normal and pathological human tissues. A cell type-specific pattern of expression was seen in bronchus, stomach, small intestine, pancreas, kidney, prostate gland, and thymus. The comparative analysis of the putative rat homologue neu-related lipocalin showed a very similar pattern of expression with the exception of pancreas and kidney. Neoplastic human tissues showed a very heterogeneous expression of NGAL protein. High NGAL levels were found in adenocarcinomas of lung, colon and pancreas. In contrast, renal cell carcinomas of various subtypes and prostate cancers contained low NGAL levels. Lymphomas and thymic tumours were negative for NGAL immuno-labeling. Knowledge about the location of NGAL in normal cells and in disease states provides the first clues towards understanding its biological function.     

Specificity and randomness: structure-function relationships in neural circuits

A fundamental but unsolved problem in neuroscience is how connections between neurons might underlie information processing in central circuits. Building wiring diagrams of neural networks may accelerate our understanding of how they compute. But even if we had wiring diagrams, it is critical to know what neurons in a circuit are doing: their physiology. In both the retina and cerebral cortex, a great deal is known about topographic specificity, such as lamination and cell-type specificity of connections. Little, however, is known about connections as they relate to function. Here, we review how advances in functional imaging and electron microscopy have recently allowed the examination of relationships between sensory physiology and synaptic connections in cortical and retinal circuits.     

Structure and sequence relationships in the lipocalins and related proteins.

The lipocalins and fatty acid-binding proteins (FABPs) are two recently identified protein families that both function by binding small hydrophobic molecules. We have sought to clarify relationships within and between these two groups through an analysis of both structure and sequence. Within a similar overall folding pattern, we find large parts of the lipocalin and FABP structures to be quantitatively equivalent. The three largest structurally conserved regions within the lipocalin common core correspond to characteristic sequence motifs that we have used to determine the constitution of this family using an iterative sequence analysis procedure. This afforded a new interpretation of the family, which highlighted the difficulties of determining a comprehensive and coherent classification of the lipocalins. The first of the three conserved sequence motifs is also common to the FABPs and corresponds to a conserved structural element characteristic of both families. Similarities of structure and sequence within the two families suggests that they form part of a larger "structural superfamily"; we have christened this overall group the calycins to reflect the cup-shaped structure of its members.     

Apolipoprotein D expression absence in degenerating neurons of human central nervous system

Apolipoprotein D (apo D), a lipocalin transporter of small hydrophobic molecules could play an important role in several neurodegenerative diseases. However, its role in those diseases remains unclear. There has been reported increments of apo D in relation with different neuropathologic diseases. Recently, we reported the absence of apo D in neurons of substantia nigra which can contribute to the lability of neurons to oxidative damage. In order to determine the relationship between apo D expression and neuronal death, we studied the expression of apo D in various regions of human brains from patients without any neurological or psychological disorders, in relation with the neuronal damage revealed by Fluoro-Jade B staining. The absence of expression for apo D in injured neurons and the negative staining for Fluoro-Jade B of neurons that express apo D was observed in all sections studied. These findings are in accordance with the role possibly played by apo D in the neuroprotection of the nervous system.     

The hector G-protein coupled receptor is required in a subset of fruitless neurons for male courtship behavior

Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.