InteractiveFly: GeneBrief

Glial Lazarillo: Biological Overview | References

Lipid shuttling between neurons and glia contributes to the development and function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, A combined genetic, transcriptome, and biochemical analyses was performed in the developing Drosophila larval brain. This study reports, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, this study elucidated molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment (Yin, 2021).

Lipid trafficking and homeostasis are critical for the development and maintenance of the nervous system. These processes are mediated by a large set of molecular carriers shuttling a diverse group of lipid cargos in and out of designated cell types and cellular compartments. In the central nervous system (CNS), lipid homeostasis heavily relies on neuron-glia cross talk. Studies in mammalian systems have indicated that, besides direct anatomical interactions, glia also supply neurons with metabolic substrates, antioxidants, and trophic factors through secretion. Intriguingly, apolipoproteins are among the most abundant secretory factors that are produced and released by mammalian astrocytes, a group of glial cells with complex morphology and highly branched structures that are intimately associated with synapses, suggesting a critical role for glia-derived lipoprotein and their lipid cargos in synapse formation and function. This notion is supported by studies in cultured mammalian CNS neurons, where glia-derived cholesterol and phospholipids are essential for synaptogenesis. In addition, recent findings in the Drosophila system also indicate essential functions of glia in synapse formation and neurotransmission, although the link between neuron-glia lipid transport and synaptic function has yet to be established (Yin, 2021).

Characterized by their high metabolic rate and elaborate morphology, neurons require a continuous lipid supply throughout their lifetime. How lipoproteins and their receptors mediate neuron-glia lipid shuttling to meet that demand has been a long-standing question in the neurobiology field. Numerous studies over the past decades have demonstrated the critical functions of CNS lipid trafficking in synapse development and cognitive functions. In the mammalian system, deficiencies in either apolipoproteins or their receptors lead to both structural and functional deficits in the brain. For example, Apolipoprotein E (ApoE) delivers cholesterol, amyloid-β, and other hydrophobic molecules to neurons through its interaction with Very Low-Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E Receptor 2 (ApoER2). While the Apolipoprotein E (ApoE) knockout mice display significantly reduced dendrite size and synapse number as well as impaired learning and memory, both VLDLR and ApoER2 knockout animals also show deficits in cerebellar morphology and impaired contextual fear conditioning and long-term potentiation. Genetic studies of other lipid transport proteins and receptors, including APOD, Niemann-Pick Type C (NPC), Low-Density Lipoprotein Receptor (LDLR), and Low-density lipoprotein Receptor-related Protein 1 (LRP1), further support the importance of lipid trafficking in the proper development and function of the nervous system (Yin, 2021).

Due to the diversity of lipid transport proteins and lipoprotein receptors, as well as the complexity of their tissue- and cell-specific distributions, cellular and molecular mechanisms underlying neuron-glia lipid shuttling have not been well characterized in vivo under physiological conditions. Recent findings in Drosophila highlight the protective functions of neuron-glia metabolic coupling in neurons experiencing oxidative stress or enhanced activity, demonstrating how neurons transfer lipids into glia for detoxification and storage. Similarly, observations made in the mammalian system also provided evidence illustrating fatty acid (FA) transport into astrocytes mediated by ApoE and the importance of neuronal lipid clearance. In contrast, much less is known about how neurons acquire lipid cargos from glia-derived lipoproteins under normal conditions, especially during development, when neurite outgrowth and synapse formation produce a high lipid demand. Attempts were made to fill this gap by determining the functional significance and regulatory mechanisms underlying neuronal lipid uptake using the Drosophila larval brain as a model system (Yin, 2021).

In Drosophila, the Apolipoprotein B (ApoB) family lipoprotein Apolipophorin (ApoLpp) is a major hemolymph lipid carrier and has two closely related lipophorin receptors (LpRs), LpR1 and LpR2, both of which are homologs of mammalian LDLR family proteins. Notably, Drosophila LpRs have multiple isoforms produced by alternative splicing and differential promoter usage. In the fly imaginal disc and oocyte, long isoforms of LpRs (LpR-long) interact with lipid transfer particles (LTP, Apoltp) and mediate endocytosis-independent neutral lipid uptake, while short isoforms of LpRs (LpR-short) neither bind to LTP, nor mediate lipid uptake in these peripheral tissues. In contrast, previous genetic studies revealed specific expression of LpR-short in larval ventral lateral neurons (LNvs), a group of visual projection neurons, and its functions in supporting dendrite development and synaptic transmission (Yin, 2018). This observation is validated by a recent study performed in a cultured Drosophila neuronal cell line, where the LpR-dependent lipid uptake was directly visualized using fluorescently labeled ApoLpp (Matsuo, 2019). Is there a functional significance behind the isoform-specific expression of LpRs? How do short isoforms of LpRs mediate lipid uptake in neurons? These are the questions this study aims to address (Yin, 2021).

This study focused on the LpR1 gene and uncover its isoform-specific expression in neurons and its functions in regulating brain lipid content. Through systematic genetic and biochemical analyses, Glial Lazarillo (GLaz), an astrocyte-derived secreted lipocalin and a homolog of human APOD, was identified as a binding partner for the brain-specific LpR1-short; their cooperative functions are revealed in supporting dendrite morphogenesis, synaptic transmission, and lipid homeostasis in the developing larval brain. In adult Drosophila, GLaz/APOD is found in CNS glia and has been shown to regulate stress resistance and contribute to longevity. Recent studies also demonstrated that GLaz mediates neuron to glia lipid transfer and facilitates neuronal lipid clearance. By identifying GLaz's function in neural development and its direct interaction with LpR1, this study not only uncovered a pair of molecular carriers mediating neuron-glia lipid shuttling in the Drosophila CNS but also presents evidence supporting isoform-specific expression as a key mechanism for regulating the tissue distribution and ligand specificity of neuronal lipoprotein receptors. This in turn leads to the stage- and cell-type-specific regulation of lipid uptake (Yin, 2021).

Lipid-mediated communication between glia and neurons is essential for brain lipid homeostasis and serves critical functions in neural development and synaptic function. Using the developing Drosophila larval brain as a model, this study investigated how neurons acquire their lipid supply from neighboring astrocytes and the regulatory mechanisms associated with the neuron-glia lipid trafficking. Genetic and RNA-seq analyses reveal that lipid uptake in fly neurons is mediated largely by short isoforms of the LpR1 receptor, which recruits a lipid complex through direct interactions with astrocyte-derived apolipoprotein GLaz/APOD. This study identifies specific molecular carriers mediating neuron-glia lipid shuttling and reveals the isoform-specific expression of lipoprotein receptors as a mechanism that determines cell-type-specific recruitment of distinct lipid cargos (Yin, 2021).

Exon mapping of cell-specific RNA-seq libraries revealed that only short isoforms of LpR1 are expressed in LNvs and are upregulated by chronic elevation of neuronal activity. Expanding upon those studies, additional tissue-specific RNA-seq datasets were examined and qFISH analyses was proformed, which strengthened the conclusion that the short-isoforms of LpR1 are CNS-specific and are predominately expressed in neurons, while the long-isoforms of LpR1 are mainly expressed in peripheral tissues and mediate endocytosis-independent lipid uptake. In addition, genetic and biochemical analyses further reveal the functional significance of isoform specificity, including its impact on the receptor's distribution, binding partners, and ability to support specific types of lipid trafficking. These distinctions highlight transcriptional regulation as a key mechanism controlling the cell-type-specific distribution of lipoprotein receptors, their lipid cargos, and uptake efficacy. Results obtained from this study clearly indicate that the molecular and functional diversity of lipoprotein receptors far exceeds current understandings, which are mostly based on DNA sequence analyses. Single-cell transcriptome analyses with sufficient resolution to identify isoform-specific splicing events could potentially reveal the capacity and specificity of the lipid uptake machinery within each individual cell type, helping arrive at a better understanding of the regulatory mechanisms underlying the cell-type-specific lipid recruitment (Yin, 2021).

Lipid shuttling between neurons and glia contributes to energy balance, neural protection, synapse development, and function, and potentially utilizes conserved molecular machinery that is shared among different organisms. Studies in Drosophila and murine neuron-glia coculture systems have demonstrated that neuronal activity alters the metabolic programs of both neurons and glia, leading to the transfer of neuronal lipids into glia for detoxification and storage in the form of lipid droplets. Notably, in both systems, vertebrate ApoE is able to function as the lipid carrier supporting neuronal lipid transfer and lipid droplet accumulation in glia (Yin, 2021).

On the other hand, in the developing larval CNS, this study found an increased lipid demand in neurons coping with chronically elevated input activity. This increase is likely met, at least partially, by enhancing neuronal lipid uptake through the activity-induced upregulation of LpR1 expression. These observations demonstrate a strong influence imposed by activity on neurons' capacity for lipid recruitment through its effects on lipoprotein receptors. Together with earlier studies, these findings suggest that both sides of neuron-glia lipid shuttling are regulated by neuronal activity, and the regulatory mechanisms controlling the expression level of lipoprotein receptors and their interactions with specific ligand molecules likely have functional significance in activity-dependent structural and functional plasticity in the nervous system (Yin, 2021).

Recent studies in adult Drosophila compound eyes have demonstrated that GLaz is involved in lipid transfer from neuron to glia, while the current studies illustrated the interaction between GLaz and LpR1 and a possible role for GLaz in delivering lipid to neurons. Therefore, GLaz appears to be involved in both sides of the lipid trafficking and potentially serves as a key molecular target for regulatory mechanisms controlling lipid homeostasis in the fly brain (Yin, 2021).

The basic structure, molecular composition, and developmental processes of a synapse are shared among vertebrate and invertebrate systems. While synaptogenesis in mammalian neurons relies on cholesterol production by glial cells and its delivery by ApoE-containing lipoprotein complexes, whether the cholesterol and/or ApoE-like lipid carriers are required for building synapses in Drosophila neurons is not known. Importantly, the Drosophila genome does not contain a homolog of ApoE. It is also reported that flies do not have the ability to synthesize cholesterol, and only obtain it through their diet to produce essential hormones. The contrast between these two systems suggests exciting possibilities for studying the lipids involved in synapse construction by understanding the differences and similarities between lipid recruitment in Drosophila vs. mammalian neurons (Yin, 2021).

This study demonstrates that the short isoforms of LpR1 recruit lipids and support dendrite morphogenesis through their interactions with the astrocyte-secreted lipoprotein GLaz, the homolog of human APOD. Given the strong dendrite development phenotype, as well as the reduced life span and stress resistance observed in GLaz loss-of-function mutants, GLaz's hypothesized lipid cargo is likely a critical contributor towards synapse development and neuronal functions in the fly CNS. Currently, only a limited number of putative lipid ligands have been identified for GLaz's homologs; grasshopper Lazarillo binds to retinoic acid and fatty acids, and human APOD binds to arachidonic acid (AA) and progesterone (PG). Whether these lipids or other types of hydrophobic ligands bind to Drosophila GLaz has not been studied. Similar to human APOD, this study observed dimeric and tetrameric GLaz in the larval brain extract. This suggests that, instead of only being able to accommodate a small hydrophobic ligand as a single unit, the GLaz protein could participate in different types of lipoprotein complexes and exhibit distinctive behaviors in vivo through its multimeric forms (Yin, 2021).

APOD's function in the CNS has long been underestimated, despite the fact that APOD is highly elevated during aging and neural injury. When examining recent human and mouse astrocyte RNA-seq data, it was found that, although ApoE is the highest expressing apolipoprotein in mouse astrocytes, the most abundant apolipoprotein expressed in human astrocytes is APOD, strongly suggesting its functional importance in the CNS. In both Drosophila and mammalian systems, GLaz/APOD are produced by astrocytes and have both anti-oxidation and anti-inflammatory protective functions. Given the similarities between GLaz's and APOD's functional and biochemical properties, these findings on the GLaz-LpR1 interaction in Drosophila may facilitate the identification of a mammalian lipoprotein receptor that interacts with APOD and provide new insights into its protective role in aging and neurodegenerative disorders (Yin, 2021).

Gut cytokines modulate olfaction through metabolic reprogramming of glia

Infection-induced aversion against enteropathogens is a conserved sickness behaviour that can promote host survival. The aetiology of this behaviour remains poorly understood, but studies in Drosophila have linked olfactory and gustatory perception to avoidance behaviours against toxic microorganisms. Whether and how enteric infections directly influence sensory perception to induce or modulate such behaviours remains unknown. This study shows that enteropathogen infection in Drosophila can modulate olfaction through metabolic reprogramming of ensheathing glia of the antennal lobe. Infection-induced unpaired cytokine expression in the intestine activates JAK-STAT signalling in ensheathing glia, inducing the expression of glial monocarboxylate transporters and the apolipoprotein glial lazarillo (GLaz), and affecting metabolic coupling of glia and neurons at the antennal lobe. This modulates olfactory discrimination, promotes the avoidance of bacteria-laced food and increases fly survival. Although transient in young flies, gut-induced metabolic reprogramming of ensheathing glia becomes constitutive in old flies owing to age-related intestinal inflammation, which contributes to an age-related decline in olfactory discrimination. These findings identify adaptive glial metabolic reprogramming by gut-derived cytokines as a mechanism that causes lasting changes in a sensory system in ageing flies (Cai, 2021).

Olfactory perception influences nutrition and promotes physiological and mental well-being. In flies, a dedicated olfactory circuit elicits avoidance behaviours towards geosmin-a volatile compound that is released by mould and some bacteria. Olfactory receptors also mediate an initial attraction to food that contains certain enteropathogens. After infection with these pathogens, however, an avoidance behaviour is triggered by immune receptors in the brain, gustatory bitter neurons, and the neuropeptide leukokinin. Whether changes in olfactory perception contribute to this behavioural switch from attraction to avoidance remains unclear (Cai, 2021).

In Drosophila, odorants are sensed by olfactory receptor neurons in the head, the antenna and the maxillary palp. Olfactory receptor neurons synapse into projection neurons at the antennal lobe (AL), where the signal is converted into a spatiotemporal code in 50 glomerular compartments. Projection neurons axons project to higher olfactory centres to instruct innate and learned behaviour. In this system, glia and neurons operate as a tightly coupled unit to maintain olfactory sensitivity (Cai, 2021).

In ageing flies, olfactory perception of both aversive and attractive odours declines, but the mechanism(s) of this decline remain unclear. Olfactory perception and other neurological processes also decline in ageing mammals, often influenced by gastrointestinal signals (Cai, 2021).

This study investigated the communication between the gut and the brain, and how it influences infection-induced avoidance behaviour, infection tolerance, and olfactory decline during ageing (Cai, 2021).

A modified capillary feeder (CAFE) assay was used to measure choice between food that did or did not contain Erwinia carotovora carotovora 15 (Ecc15), a non-lethal enteropathogen that causes intestinal inflammation. Consistent with recent reports, naive flies consumed more Ecc15-containing food than normal food. However, when orally infected with Ecc15 for 24 h before the feeding assay, flies developed a distinct aversion to food that contained Ecc15. To assess whether this involved changes in olfactory perception, the 'preference index' for attractive (such as putrescine) or aversive (such as 3-octanol) odours was determined in T-maze assays. Preference or aversion for attractive or aversive odours, respectively, declined after infection, which indicates that infection causes a non-selective decline in olfactory discrimination. This was transient, as olfactory discrimination recovered 5 days after infection, coincident with the clearance of bacteria and epithelial regeneration in the intestine. Olfactory discrimination was not influenced by starvation or exposure to heat-killed Ecc15. Consistent with their reported role in sensing pathogenic bacteria, the CO2 receptor Gr63a or the odorant receptor co-receptor Orco was required for the attraction to Ecc15 food: Orco1 and Gr63a1 mutants ingested less Ecc15 food under naive conditions, and when infected, failed to further reduce ingestion of Ecc15-containing food. Together, these observations suggest that after an initial odorant-mediated attraction, flies develop aversion to enteropathogens, through a concerted activation of gustatory and immune receptors and suppression of olfaction (Cai, 2021).

After oral infection with Ecc15, damaged intestinal enterocytes produce the inflammatory IL-6-like cytokines Unpaired 2 and 3 (Upd2 and Upd3) to stimulate intestinal stem-cell proliferation and epithelial regeneration. Proteins of the Upd family activate the JAK-STAT signalling pathway through the receptor Domeless (Dome) and the JAK homologue Hopscotch (Hop). Using the 2xSTAT::GFP reporter for JAK-STAT pathway activity, this study found upregulated GFP expression in the brain 4 h after oral Ecc15 infection, as well as after oral infection with the more lethal enteropathogen Pseudomonas entomophila (PE) that damages the gut epithelium. JAK-STAT activity was observed in a sparse population of cells of the brain that stained positive for the glial marker Repo. Subtype-specific Gal4 drivers revealed that among the five subtypes of Drosophila glia (astrocytes, ensheathing, perineural, subperineural and cortex glia), ensheathing glia (EG) were the main population that upregulate STAT activity in response to Ecc15 infection. This was confirmed using four different Gal4 drivers to label EG and by flow cytometry. Infection did not influence numbers and membranous processes (labelled using UAS::mCD4GFP) of EG at the AL, and glomerular compartmentalization in the AL and lobe size remained unaffected. JAK-STAT activation in EG was sufficient and required for infection-induced changes in olfactory discrimination, as overexpression of constitutively active Hop (hop^tuml) in EG reduced olfactory discrimination, whereas loss of Dome or STAT in all glia (repo::Gal4), or specifically in EG (GMR56F03::Gal4), rescued the decline of olfactory discrimination caused by Ecc15 infection. Overexpression of hop^tuml in EG also reduced ingestion of Ecc15-containing food and promoted survival of flies fed PE-containing food, whereas knockdown of Dome or STAT in EG increased ingestion of Ecc15-containing food in infected flies and increased mortality on PE-containing food. It is proposed that the corresponding changes in ingestion of PE-laced food contribute to reduced mortality, but it is possible that additional genetic background conditions influence mortality, as seen, for example, in Orco-mutant flies, which ingest less bacteria but show increased susceptibility to PE (Cai, 2021).

To test whether gut-derived Upd proteins directly contribute to the infection-induced activation of JAK-STAT signalling in EGs, intestinal enterocyte-specific perturbations were performed using Mex1::Gal4, an enterocyte driver with no expression in the brain. Indeed, JAK-STAT activation in glia at the AL could be triggered in naive flies or prevented in infected flies by overexpression or knockdown, respectively, of Upd2 and Upd3 in enterocytes. Consistently, enterocyte-derived Upd2 and Upd3 were sufficient and required for the modulation of olfactory discrimination caused by infection. Knockdown of Upd2 or Upd3 did not affect olfaction in naive flies, and perturbing these ligands in fatbody (cg::Gal4) or haemocytes (hml::Gal4), tissues that are sources for Upd proteins in other contexts, did not significantly affect STAT activity in glia at the AL (Cai, 2021).

Loss of olfactory sensitivity is an early sign of normal ageing and neurodegeneration. In ageing Drosophila, olfactory perception has been reported to deteriorate before vision, a decline that was possible to recapitulate in T-maze assays. Glomerular compartments in the AL became less organized and less distinct in geriatric (60-70-day-old) flies and AL size increased with age. This correlates with a reduction in the number of EG and of glial membranous processes, changes that are expected to affect AL structure, and thus probably contribute to the age-related decline in olfaction (Cai, 2021).

Ageing in Drosophila is accompanied by the development of intestinal inflammation, and is associated with the constitutive expression and release of Upd cytokines. Consistently, JAK-STAT activity in the AL of EG was increased in old flies, and knockdown of Dome or STAT by RNA interference (RNAi) in EG specifically or in all glia rescued the decline of olfactory discrimination in old flies. The loss of Dome in EG also rescued the age-related decline of EGs and restored the size of the AL. JAK-STAT activation in the EG of old flies is a consequence of intestinal Upd release, as knocking down Upd2 and Upd3 in enterocytes alleviated STAT activation in the AL, and prevented the age-related decline of olfactory discrimination (Cai, 2021).

This age-related decline of olfactory discrimination was independent of the microbiota, as germ-free old flies still exhibited reduced olfaction sensitivity, increased JAK-STAT signalling in the AL, decreased numbers of EG, loss of glial cellular processes, and an enlarged AL. These results are consistent with the observation that the age-related increase in Upd released from the gut is also independent of the microbiota (Cai, 2021).

To understand why EG but not other glia selectively respond to Upd ligands and activate JAK-STAT signalling during ageing or infection, single-cell RNA sequencing (scRNA-seq) was performed on purified glia from young and old flies. Either all glia (labelled using repo::Gal4) or EG selectively (labelled using GMR56F03::Gal4) were profiled using Smart-seq2. The expression of dome was significantly higher in EG than in other glia, consistent with the specific upregulation of socs36E, a known target of JAK-STAT signalling, in EG but not in other glia during ageing. These results are supported by a similar upregulation of Socs36E in EG of old flies observed in a previous scRNA-seq dataset (Cai, 2021).

Bulk RNA sequencing analysis on glia (repo::Gal4, UAS::tdTomato) purified from central brains of flies expressing a 10xSTAT::GFP reporter revealed that the transcriptomes of STAT::GFP⁺ glia from Ecc15 infected and uninfected flies were more similar to each other than to STAT::GFP^- glia of either condition, which indicates that JAK-STAT induction has a stronger influence on glial transcriptomes than other infection-related changes. Differentially expressed genes (866 genes using a cut-off of twofold change, P < 0.001, false discovery rate (FDR) < 0.01 and reads > 0.5) were significantly enriched in genes that encode proteins involved in lipid metabolism and carbohydrate transmembrane transport. These included the lipid binding protein Glial lazarillo (GLaz, a homologue of apolipoprotein D in mammals), which facilitates lipid transport from neurons to glia in flies; the lipid droplet surface binding proteins Lsd-1 and Lsd-2; the diacylglycerol O-acyltransferase Midway, which is a central regulator of triacylglycerol biosynthesis, and Coatomer, which is responsible for protein delivery to lipid droplets (LDs). This induction of lipid storage genes was coupled with induction of the monocarboxylate transporter (MCT) Outsiders (Out), and the MCT accessory protein Basigin (Bsg), sugar transporters (Tret1-1 and Tret1-2), and 17 enzymes involved in β-oxidation (Cai, 2021).

Glial MCTs promote lipid production in neurons and LD accumulation in glia by establishing a neuron and glia 'lactate shuttle'. To test a potential role for STAT signalling in influencing this shuttle at the AL, the accumulation of LDs was assessed at the AL in infected young flies using a combination of a neutral lipid probe (LipidTox, deep red) and a lipid peroxidation probe (C11-Bodipy, 581/591). A transient accumulation of LDs was observed 24 h after infection that decreased 4 days after infection, possibly owing to increased levels of β-oxidation. Overexpression of hop^tuml in the EG of young flies also promoted LD accumulation, whereas knocking down Dome or STAT rescued infection-induced accumulation. GLaz and Out were required for LD accumulation after infection, and overexpression of Upd2 and Upd3 in the gut induced LD accumulation at the AL, whereas knockdown of Upd2 or Upd3 alleviated LD accumulation in infected flies. Infection or JAK-STAT perturbation did not influence lipid peroxidation in LDs in young flies (Cai, 2021).

During neuronal stress, neurons can preferentially transfer fatty acids to glia, causing lipid accumulation and increasing fatty acid β-oxidation in glia. This study observed a significant induction of LDs specifically in EG at the AL in old flies, phenocopying hop^tuml overexpression. As fatty acid β-oxidation is a source of reactive oxygen species (ROS) that can result in lipid peroxidation, and lipid peroxidation in pigment cells (glia of the retina) promotes the demise of photoreceptors in the retina (whereas oxidative stress contributes to age-related dysfunction of cholinergic projection neurons within the olfactory circuit) it was reasoned that overall levels of ROS might increase in glia with age. Various genetically encoded ROS sensors were expressed in all glia (repo::Gal4) or in EG only (GMR56F03::Gal4) to measure levels of hydrogen peroxide (H2O2; measured by RoGFP2_Orp1) or the glutathione redox potential (measured by RoGFP2_Grx1) within the mitochondria or cytosol, respectively. Cytosolic levels of H2O2 were increased in the EG of old flies (both cytosolic and mitochondrial H2O2 levels were increased in all glia), whereas the cytosolic glutathione redox potential remained unchanged. In contrast to acute intestinal infection in young flies, lipids were peroxidated in LDs of old flies. Knocking down STAT specifically in EG, or knocking down Upd2 and Upd3 in gut enterocytes, inhibited LD accumulation and alleviated lipid peroxidation in old flies (Cai, 2021).

Olfactory discrimination was partially rescued in old and young infected flies after knockdown of GLaz and Out in EG. Knockdown of GLaz and Out also led to more Ecc15 food consumption, increased mortality after PE exposure, and reduced LD accumulation in the glia of old flies (Cai, 2021).

To confirm that infection or ageing-induced metabolic changes in EG affect neuron or glia metabolic coupling at the AL, the consequences of perturbing projection neurons directly were assessed using GH146::Gal4. Knocking down Out but not lactate dehydrogenase (Ldh) in projection neurons rescued olfactory discrimination of infected or aged flies, whereas food preference or mortality was not influenced. Overexpression of lipase 4 (Lip-4) in projection neurons, or knockdown of the neuronal lipid binding protein Neural lazarillo (NLaz), significantly improved olfactory discrimination in infected or old flies, and overexpression of Lip-4 increased Ecc15 food consumption and increased mortality after PE exposure (Cai, 2021).

This work suggests that gut-derived inflammatory cytokines modulate the metabolic coupling of glia and neurons in the brain of Drosophila to induce an adaptive temporary halt of olfactory discrimination after intestinal infection, but also contribute to age-related olfactory decline. It is proposed that gut-derived Upd2 and Upd3 reprogram lipid metabolism in EG, increasing lactate and lipid transport between glia and olfactory neurons, resulting in LD accumulation and upregulation of mitochondrial β-oxidation, potentially a source of increased ROS production. Chronic activation of this metabolic shift in old flies results in the accumulation of peroxidated lipids in EG, promoting their decay and contributing to the previously described functional decline of olfactory neurons. Detailed characterization of this metabolic reprogramming, and further exploration of the role of lipid synthesis in projection neurons for glial lipid accumulation and for olfactory discrimination are important avenues for further study (Cai, 2021).

These findings further determine the regulation of avoidance behaviour against enteropathogens in insects. In addition to gustatory bitter neurons and immune receptors in octopaminergic neurons, Upd proteins constitute a direct endocrine signal from the damaged intestinal epithelium in this complex but essential behaviour. It is proposed that Upd-mediated suppression of olfactory discrimination is required to prevent olfaction-mediated attraction to a food source after pathogenicity has been established and aversion is induced by gustatory neurons. It remains unclear, however, whether gustatory neurons are also affected by JAK-STAT signalling in EG. Whether similar mechanisms are conserved and control infection-induced loss of sensory perception in vertebrates including humans will be interesting to explore (Cai, 2021).

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Lipocalin Apolipoprotein D (ApoD) is an extracellular lipid binding protein endowed with antioxidant capacity. This study performed a comprehensive analysis of ApoD intracellular traffic and demonstrates its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. ApoD was shown to be endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins (see Glial Lazarillo). These results set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases (Pascua-Maestro, 2017).

Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz

Apolipoprotein D (ApoD), a member of the Lipocalin family, is the gene most up-regulated with age in the mammalian brain. Its expression strongly correlates with aging-associated neurodegenerative and metabolic diseases. Two homologues of ApoD expressed in the Drosophila brain, Glial Lazarillo (GLaz) and Neural Lazarillo (NLaz), are known to alter longevity in male flies. However, sex differences in the aging process have not been explored so far for these genes. This study demonstrates that NLaz alters lifespan in both sexes, but unexpectedly the lack of GLaz influences longevity in a sex-specific way, reducing longevity in males but not in females. While NLaz has metabolic functions similar to ApoD, the regulation of GLaz expression upon aging is the closest to ApoD in the aging brain. A multivariate analysis of physiological parameters relevant to lifespan modulation uncovers both common and specialized functions for the two Lipocalins, and reveals that changes in protein homeostasis account for the observed sex-specific patterns of longevity. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. It was also demonstrated that food composition is an important environmental parameter influencing stress resistance and reproductive phenotypes of both Lipocalin mutants. Since ApoD shares many properties with the common ancestor of invertebrate Lipocalins, this global comparison with both GLaz and NLaz will lead to understand the complex functions of ApoD in mammalian aging and neurodegeneration (Ruiz, 2011).

This work highlights that the two Drosophila Lipocalins expressed in the nervous system have both functional redundancies and specializations. The response to oxidative stress and accumulation of lipid peroxides are among their common functions, while the transcriptional and behavioral response to starvation, the pattern of daily locomotor activity, storage of fat along aging, fertility, and courtship behavior differentiate NLaz from GLaz mutants. This framework is guiding current research, as more details need to be elucidated. However, it can already be asked how many of these shared or unique functions are conserved in the mammalian homologues also expressed in the brain (Ruiz, 2011).

The basal position of ApoD in the phylogenetic tree of vertebrate Lipocalins suggests that ApoD shares many properties with the common ancestor of invertebrate Lipocalins. However, it has to be taken into account that neither GLaz nor NLaz is a true orthologue of ApoD. Molecular phylogenetic analyses strongly suggest that the Drosophila Lipocalins originated from an independent duplication event, taking place within the invertebrate lineage. Subsequently, the resulting genes have diverged both in their protein coding sequence and their regulatory sequences (Ruiz, 2011).

Since ApoD in the adult mammalian brain is expressed mainly in glial cells, one might be tempted to directly conclude that GLaz is the closest Drosophila homologue. Furthermore, the expression data reported in this study strongly suggest that GLaz regulation through aging is most similar to the robust increase of mammalian ApoD in the aged brain (de Magalhaes; Loerch, 2008). Interestingly, ApoD is most similar to GLaz in protein sequence, but to NLaz in the intron-exon structure of the gene. ApoD has been shown to be up-regulated by oxidative stress in astrocytes, and this induction is mediated through the JNK pathway (Bajo-Grañeras, Ganfornina and Sanchez, unpublished observations cited in Ruiz, 2011), comparable to the NLaz JNK-mediated induction by stress in Drosophila. Thus, if the Drosophila data is extrapolated to learn about the functions of ApoD in mammalian aging and neurodegeneration the global comparison with both GLaz and NLaz, as reported in this study, must be taken into account (Ruiz, 2011).

To understand the multigenic control of aging, it must be taken into account that a layer of complexity is added due to the fact that each gene has pleiotropic effects, and each one has differing degrees of specialization or redundancy with members of the same gene family. This fact represents a daunting complication for the task of predicting the actions of putative anti-aging or anti-neurodegeneration drugs. However, complexity should not prevent investigating until a comprehensive understanding of the aging process is available (Ruiz, 2011).

Altered lipid metabolism in a Drosophila model of Friedreich's ataxia

Friedreich's ataxia (FRDA) is the most common form of autosomal recessive ataxia caused by a deficit in the mitochondrial protein frataxin. Although demyelination is a common symptom in FRDA patients, no multicellular model has yet been developed to study the involvement of glial cells in FRDA. Using the recently established RNAi lines for targeted suppression of frataxin in Drosophila, it was possible to study the effects of general versus glial-specific frataxin downregulation. In particular, it was of interest to study the interplay between lowered frataxin content, lipid accumulation and peroxidation and the consequences of these effects on the sensitivity to oxidative stress and fly fitness. Interestingly, ubiquitous frataxin reduction leads to an increase in fatty acids catalyzing an enhancement of lipid peroxidation levels, elevating the intracellular toxic potential. Specific loss of frataxin in glial cells triggers a similar phenotype which can be visualized by accumulating lipid droplets in glial cells. This phenotype is associated with a reduced lifespan, an increased sensitivity to oxidative insult, neurodegenerative effects and a serious impairment of locomotor activity. These symptoms fit very well with the observation of an increase in intracellular toxicity by lipid peroxides. Interestingly, co-expression of a Drosophila apolipoprotein D ortholog (glial lazarillo) has a strong protective effect in frataxin models, mainly by controlling the level of lipid peroxidation. These results clearly support a strong involvement of glial cells and lipid peroxidation in the generation of FRDA-like symptoms (Navarro, 2010).

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. This study has 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. It is concluded that GLaz has a protective role in stress situations and that its absence reduces lifespan and accelerates neurodegeneration (Sanchez, 2006).

Increased Apolipoprotein D (ApoD) expression has been reported in various neurological disorders, including Alzheimer's disease, schizophrenia, and stroke, and in the aging brain . However, whether ApoD is toxic or a defense is unknown. In a screen to identify genes that protect Drosophila against acute oxidative stress, a fly homolog of ApoD, Glial Lazarillo (GLaz), was isolated. In independent transgenic lines, overexpression of GLaz resulted in increased resistance to hyperoxia (100% O(2)) as well as a 29% extension of lifespan under normoxia. These flies also displayed marked improvements in climbing and walking ability after sublethal exposure to hyperoxia. Overexpression of Glaz also increased resistance to starvation without altering lipid or protein content. To determine whether GLaz might be important in protection against reperfusion injury, the flies were subjected to hypoxia, followed by recovery under normoxia. Overexpression of GLaz was protective against behavioral deficits caused in normal flies by this ischemia/reperfusion paradigm. This id the first to manipulate the levels of an ApoD homolog in a model organism. These data suggest that human ApoD may play a protective role and thus may constitute a therapeutic target to counteract certain neurological diseases (Walker, 2006).

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

This study 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. It is proposed that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed (Sanchez, 2000).

Search PubMed for articles about Drosophila Glial Lazarillo

Cai, X. T., Li, H., Borch Jensen, M., Maksoud, E., Borneo, J., Liang, Y., Quake, S. R., Luo, L., Haghighi, P. and Jasper, H. (2021). Gut cytokines modulate olfaction through metabolic reprogramming of glia. Nature 596(7870): 97-102. PubMed ID: 34290404

Matsuo, N., Nagao, K., Suito, T., Juni, N., Kato, U., Hara, Y. and Umeda, M. (2019). Different mechanisms for selective transport of fatty acids using a single class of lipoprotein in Drosophila. J Lipid Res 60(7): 1199-1211. PubMed ID: 31085629

Muffat, J., Walker, D. W. and Benzer, S. (2008). Human ApoD, an apolipoprotein up-regulated in neurodegenerative diseases, extends lifespan and increases stress resistance in Drosophila. Proc Natl Acad Sci U S A 105(19): 7088-7093. PubMed ID: 18458334

Navarro, J. A., Ohmann, E., Sanchez, D., Botella, J. A., Liebisch, G., Molto, M. D., Ganfornina, M. D., Schmitz, G. and Schneuwly, S. (2010). Altered lipid metabolism in a Drosophila model of Friedreich's ataxia. Hum Mol Genet 19(14): 2828-2840. PubMed ID: 20460268

Pascua-Maestro, R., Diez-Hermano, S., Lillo, C., Ganfornina, M. D. and Sanchez, D. (2017). Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress. PLoS Genet 13(2): e1006603. PubMed ID: 28182653

Ruiz, M., Sanchez, D., Canal, I., Acebes, A. and Ganfornina, M. D. (2011). Sex-dependent modulation of longevity by two Drosophila homologues of human Apolipoprotein D, GLaz and NLaz. Exp Gerontol 46(7): 579-589. PubMed ID: 21376794

Sanchez, D., Ganfornina, M. D., Torres-Schumann, S., Speese, S. D., Lora, J. M. and Bastiani, M. J. (2000). Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system. Int J Dev Biol 44(4): 349-359. PubMed ID: 10949044

Sanchez, D., Lopez-Arias, B., Torroja, L., Canal, I., Wang, X., Bastiani, M. J. and Ganfornina, M. D. (2006). Loss of glial lazarillo, a homolog of apolipoprotein D, reduces lifespan and stress resistance in Drosophila. Curr Biol 16(7): 680-686. PubMed ID: 16581513

Walker, D. W., Muffat, J., Rundel, C. and Benzer, S. (2006). Overexpression of a Drosophila homolog of apolipoprotein D leads to increased stress resistance and extended lifespan. Curr Biol 16(7): 674-679. PubMed ID: 16581512

Yin, J., Gibbs, M., Long, C., Rosenthal, J., Kim, H. S., Kim, A., Sheng, C., Ding, P., Javed, U. and Yuan, Q. (2018). Transcriptional Regulation of Lipophorin Receptors Supports Neuronal Adaptation to Chronic Elevations of Activity. Cell Rep 25(5): 1181-1192. PubMed ID: 30380410

Yin, J., Spillman, E., Cheng, E. S., Short, J., Chen, Y., Lei, J., Gibbs, M., Rosenthal, J. S., Sheng, C., Chen, Y. X., Veerasammy, K., Choetso, T., Abzalimov, R., Wang, B., Han, C., He, Y. and Yuan, Q. (2021). Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling. Nat Commun 12(1): 2408. PubMed ID: 33893307

date revised: 10 April 2022

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