ARTICLE https://doi.org/10.1038/s41467-021-22751-7 OPEN Brain-specific lipoprotein receptors interact with astrocyte derived apolipoprotein and mediate neuron-glia lipid shuttling Jun Yin1, Emma Spillman1,5, Ethan S. Cheng1, Jacob Short 1, Yang Chen1, Jingce Lei1, Mary Gibbs1, Justin S. Rosenthal1, Chengyu Sheng 1,6, Yuki X. Chen2,3, Kelly Veerasammy2,3, Tenzin Choetso2,3, ✉ Rinat Abzalimov2, Bei Wang 4, Chun Han 4,YeHe2 & Quan Yuan 1 1234567890():,; Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, we perform combined genetic, transcriptome, and biochemical analyses in the developing Drosophila larval brain. Here we report, 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 den- drite 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, we elucidate 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. 1 Dendrite Morphogenesis and Plasticity Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA. 2 The City University of New York, Graduate Center-Advanced Science Research Center, New York, NY, USA. 3 The City College of New York, CUNY, New York, NY, USA. 4 Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA. 5Present address: Department of Neurosciences, University of California, San Diego, San Diego, CA, USA. 6Present address: Department of Pharmacology, ✉ School of Basic Medical Sciences, Nanjing Medical University, Nanjing, China. email: [email protected] NATURE COMMUNICATIONS | (2021) 12:2408 | https://doi.org/10.1038/s41467-021-22751-7 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22751-7 ipid trafficking and homeostasis are critical for the devel- In Drosophila, the Apolipoprotein B (ApoB) family lipoprotein Lopment and maintenance of the nervous system. These pro- apolipophorin (apoLpp) is a major hemolymph lipid carrier and cesses are mediated by a large set of molecular carriers has two closely related lipophorin receptors (LpRs), LpR1 and shuttling a diverse group of lipid cargos in and out of designated LpR2, both of which are homologs of mammalian LDLR family cell types and cellular compartments1,2. In the central nervous proteins33–35. Notably, Drosophila LpRs have multiple isoforms system (CNS), lipid homeostasis heavily relies on neuron-glia cross produced by alternative splicing and differential promoter talk3. Studies in mammalian systems have indicated that, besides usage33. In the fly imaginal disc and oocyte, long isoforms of direct anatomical interactions, glia also supply neurons with LpRs (LpR-long) interact with lipid transfer particles (LTP, metabolic substrates, antioxidants, and trophic factors through Apoltp) and mediate endocytosis-independent neutral lipid secretion4–6. Intriguingly, apolipoproteins are among the most uptake, while short isoforms of LpRs (LpR-short) neither bind to abundant secretory factors that are produced and released by LTP, nor mediate lipid uptake in these peripheral tissues33,34.In mammalian astrocytes6,7, a group of glial cells with complex contrast, our previous genetic studies revealed specific expression morphology and highly branched structures that are intimately of LpR-short in larval ventral lateral neurons (LNvs), a group of associated with synapses6,8, suggesting a critical role for glia- visual projection neurons, and its functions in supporting den- derived lipoprotein and their lipid cargos in synapse formation and drite development and synaptic transmission36. This observation function2,9. This notion is supported by studies in cultured is validated by a recent study performed in a cultured Drosophila mammalian CNS neurons, where glia-derived cholesterol and neuronal cell line, where the LpR-dependent lipid uptake was phospholipids are essential for synaptogenesis9–11.Inaddition, directly visualized using fluorescently labeled ApoLpp37. Is there a recent findings in the Drosophila system also indicate essential functional significance behind the isoform-specific expression of functions of glia in synapse formation and neurotransmission12–15, LpRs? How do short isoforms of LpRs mediate lipid uptake in although the link between neuron-glia lipid transport and synaptic neurons? These are the questions we aim to address. function has yet to be established. In this study, we focus on the LpR1 gene and uncover its Characterized by their high metabolic rate and elaborate isoform-specific expression in neurons and its functions in reg- morphology, neurons require a continuous lipid supply ulating brain lipid content. Through systematic genetic and bio- throughout their lifetime. How lipoproteins and their receptors chemical analyses, we identify Glial Lazarillo (GLaz), an mediate neuron-glia lipid shuttling to meet that demand has been astrocyte-derived secreted lipocalin and a homolog of human a long-standing question in the neurobiology field. Numerous APOD, as a binding partner for the brain-specific LpR1-short and studies over the past decades have demonstrated the critical reveal their cooperative functions in supporting dendrite mor- functions of CNS lipid trafficking in synapse development and phogenesis, synaptic transmission, and lipid homeostasis in the cognitive functions3,16,17. In the mammalian system, deficiencies developing larval brain. In adult Drosophila, GLaz/APOD is in either apolipoproteins or their receptors lead to both structural found in CNS glia and has been shown to regulate stress resis- and functional deficits in the brain. For example, Apolipoprotein tance and contribute to longevity38,39. Recent studies also E (ApoE) delivers cholesterol, amyloid‑β, and other hydrophobic demonstrated that GLaz mediates neuron to glia lipid transfer molecules to neurons through its interaction with Very Low- and facilitates neuronal lipid clearance27,28. By identifying GLaz’s Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E function in neural development and its direct interaction with Receptor 2 (ApoER2)16,18. While the Apolipoprotein E (ApoE) LpR1, we not only uncover a pair of molecular carriers mediating knockout mice display significantly reduced dendrite size and neuron-glia lipid shuttling in the Drosophila CNS but also present synapse number as well as impaired learning and memory19, both evidence supporting isoform-specific expression as a key VLDLR and ApoER2 knockout animals also show deficits in mechanism for regulating the tissue distribution and ligand cerebellar morphology and impaired contextual fear conditioning specificity of neuronal lipoprotein receptors. This in turn leads to and long-term potentiation20,21. Genetic studies of other lipid the stage- and cell-type-specific regulation of lipid uptake. transport proteins and receptors, including APOD, Niemann–Pick Type C (NPC), Low-Density Lipoprotein Recep- tor (LDLR), and Low-density lipoprotein Receptor-related Pro- Results tein 1 (LRP1), further support the importance of lipid trafficking Isoform-specific expression and activity-dependent regulation in the proper development and function of the nervous of LpR1 in LNv neurons. To determine how neuronal lipopro- system22–25. tein receptors mediate lipid uptake, based on findings from pre- Due to the diversity of lipid transport proteins and lipoprotein vious studies, we focus our genetic and functional analyses on the receptors, as well as the complexity of their tissue- and cell- Drosophila LpR1 gene, which expresses in the larval CNS and is specific distributions, cellular and molecular mechanisms required for LNvs’ dendrite development and synaptic underlying neuron-glia lipid shuttling have not been well char- functions36. Sequence analyses revealed that LpR1 shares con- acterized in vivo under physiological conditions3,26. Recent served functional domains with the mammalian LDLR family findings in Drosophila highlight the protective functions of proteins, including LDLR receptor type A modules (LA), EGF neuron-glia metabolic coupling in neurons experiencing oxidative modules, and ß-propellers (Fig. 1a). Multiple isoforms of LpR1 stress or enhanced activity, demonstrating how neurons transfer have been detected in Drosophila cDNAs with distinct tissue lipids into glia for detoxification and storage27–30. Similarly, distributions and properties33,34. They are generally categorized observations made in the mammalian system also provided evi- as either long (LpR1-long) or short isoforms (LpR1-short) (Sup- dence illustrating fatty acid (FA) transport into astrocytes medi- plementary Fig. 1 and Fig. 1b). Compared to the long isoforms, ated by ApoE and the importance of neuronal lipid clearance31,32. LpR1-short isoforms have a shorter N-terminal domain and one In contrast, much less