The Drosophila blood-brain barrier as interface between neurons and hemolymph

MOD-03345; No of Pages 6 Mechanisms of Development xxx (2015) xxx–xxx

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The Drosophila blood-brain barrier as interface between neurons and hemolymph Stefanie Schirmeier 1, Christian Klämbt ⁎ Institut für Neuro- und Verhaltensbiologie, Badestr. 9, 48149 Münster, Germany

a r t i c l e

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Article history: Received 4 December 2014 Received in revised form 1 June 2015 Accepted 16 June 2015 Available online xxxx Keywords: Drosophila Blood–brain barrier Glial cells Nutrient transport Metabolic signaling Neuroblast proliferation

a b s t r a c t The blood–brain barrier is an evolutionary ancient structure that provides direct support and protection of the nervous system. In all systems, it establishes a tight diffusion barrier that hinders uncontrolled paracellular diffusion into the nervous system. In invertebrates, the blood–brain barrier separates the nervous system from the hemolymph. Thus, the barrier-forming cells need to actively import ions and nutrients into the nervous system. In addition, metabolic or environmental signals from the external world have to be transmitted across the barrier into the nervous system. The first blood–brain barrier that formed during evolution was most likely based on glial cells. Invertebrates as well as primitive vertebrates still have a purely glial-based blood–brain barrier. Here we review the development and function of the barrier forming glial cells at the example of Drosophila. © 2015 Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction In the past, research on the Drosophila model has disclosed many fundamentally important developmental processes. Not only the molecular and genetic code underlying axis determination is similar in flies and mammals but also paths to normal eye or heart development, metabolic control and finally disease progression obey evolutionary conserved mechanisms (Gonzalez, 2013; Nüsslein-Volhard and Wieschaus, 1980; Padmanabha and Baker, 2014; Qian and Bodmer, 2012; Rajan and Perrimon, 2013). In addition, work on Drosophila and other invertebrate models has provided an enormous advance in our understanding of neuroscience. For example, the molecular strategies organisms use to define neuronal cell types appear evolutionary conserved. In all species analyzed so far, proneural genes promote neural development and all proneural genes known encode related bHLH transcription factors (Bertrand et al., 2002). Even the specification of distinct neuronal cell fates appears to require the activity of conserved transcription factors (Thor and Thomas, 2002). We are not surprised by the fact that all organisms rely on the very same set of main neurotransmitters. Likewise, synaptic function and the mechanisms underlying neuronal conductance are organized in very similar ways across the animal kingdom. Given the many conserved processes operating during neural development, it appears likely that more specific functional elements such as the blood–

⁎ Corresponding author. Tel.: +49 251 832 1122. E-mail address: [email protected] (C. Klämbt). 1 née Limmer.

brain barrier are evolutionary conserved as well. Thus, the study of the Drosophila blood–brain barrier may have more general implications. How and when did the blood–brain barrier appear during evolution of the metazoa? Obviously, neurons had to be formed first. In the simplest multicellular organisms with an epithelial organization, such as sponge- or cnidaria-like animals, special cells that were able to sense the environment emerged (Fig. 1). These cells were likely those that evolved into the first sensory neurons (Bullock and Horridge, 1965; Hartline, 2011). As more of these sensory neurons specialized, they gained a requirement of a direct support as well as protection by other cells. This is for example seen in photoreceptor neurons which always form together with supporting pigment cells and molecular data indicate that this pairwise appearance of sensory and support cell has emerged only once during evolution (Arendt, 2003; Gehring and Ikeo, 1999; Nilsson, 2004). Neuronal support cells then may have evolved into the glial lineage. However, although neuronal assistance may have evolved as the first glial cell task — a second, equally important glial function must have developed concomitantly. Since nurturing and protection by isolation are intimately coupled, glial cells formed an increasingly tight barrier allowing establishment of a constant ion milieu in the brain. Thus, we propose that early on glial cells were separated into the supportive glial cells within the CNS and an outer glial cell layer forming the barrier to the remaining body (Fig. 1). Again, first signs of such supporting cells can be found in Cnidaria (Holtmann and Thurm, 2001a,b) although true glial cells cannot be seen in these animals (Hartline, 2011). Glial cells, however, are present in simple Acoela which originate before the split of protostomia and deuterostomia (Bailly et al., 2014; Bery et al., 2010; Hartline, 2011). Even in simple nematodes such as Caenorhabditis elegans, whose nervous system consists of only 302 neurons, glial cells forming a sheath

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Please cite this article as: Schirmeier, S., Klämbt, C., The Drosophila blood-brain barrier as interface between neurons and hemolymph, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.06.002

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Fig. 1. Evolution of the blood–brain barrier. (A) Epithelia establish the outer surface of the animal. ec: epithelial cell, n: nucleus. (B) Within epithelia, sensory neurons developed that are accompanied by support cells. (C) Epithelial support cells evolved into glial cells. (D) Some neurons were transferred into the interior of the animal, where they were covered by a glial blood–brain barrier.

around the cephalic sensory neurons extend processes around the nerve ring that may serve as simple blood–brain barrier like structure (Oikonomou and Shaham, 2011; Stout et al., 2014). A glial barrier around the nervous system is found throughout protostomia as well as in primitive deuterostomia. However, the blood–brain barrier had to change dramatically as vascularization developed. While the invertebrate nervous system floats in the hemolymph and thus just needs a tight barrier sheath, vertebrates developed a closed circulatory system. This implied that in the vascularized brain, all capillaries had to be “tight” in order to guarantee the insulation of the different compartments (body versus brain). Interestingly, in primitive vertebrates, such as in elasmobranch fish (sharks, skates, and rays), but also in some bony fish (sturgeon), the blood–brain barrier is still established by glial cells. These perivascular astrocytes form highly interdigitating lamellae without forming any tight junctions (Bundgaard and Abbott, 2008; Fig. 2). The barrier appears to be established by increasing the diffusion path between the perivascular glia. In mammals, the blood–brain barrier is formed by endothelial cells which – in the brain – are induced to form tight junctions by pericytes (Armulik et al., 2010; Daneman et al., 2010; Fig. 2). Astrocytes develop after the blood–brain barrier is established but their end-feet cover the entire endothelial/pericyte surface, where they are able to modulate blood–brain barrier properties (Abbott et al., 2006; Janzer and Raff, 1987; Mathiisen et al., 2010). In insects such as Drosophila, the blood–brain barrier is established by the perineurial and the subperineurial glial cells

(Carlson et al., 2000; Stork et al., 2008; Figs. 2, 3). The subperineurial glial cells form so-called septate junctions which prevent paracellular diffusion just as the tight junctions in the mammalian endothelial blood–brain barrier. Interestingly, homologous proteins such as Claudin 5 are involved in the tightness of the barrier in flies as well as in mammals (Nitta, 2003; Stork et al., 2008) again pointing towards the evolutionary conservation of this structure. In all animals, the blood–brain barrier separates the microenvironment around neurons and their processes from the remaining body fluids. This interface is also called neuro-vascular unit to highlight the intricate interaction of the two systems. The establishment of a barrier and the concomitant separation of brain and body compartments had immediate advantages for neuronal functionality, but also some disadvantages. On the one hand, the ion concentration in the brain can be kept at constant levels, which obviously helps to establish the sophisticated neuronal cross-talk that relies on minute changes in membrane potential. On the other hand, the separation of the nervous system from the remaining body called for the development of highly efficient and selective transport systems for metabolites and periphery derived signals. Thus, during evolution the formation of barrier functions coincides with the establishment of specific transport mechanisms. Based on evolutionary arguments, it can be anticipated that many of the relevant transport and relay mechanisms operating in the mammalian nervous system are already in place in the invertebrate blood–brain barrier. Here, we review the current knowledge on the invertebrate “neuro-vascular unit” which is an interface that not only controls brain homeostasis but also actively directs development and function of the nervous system. 2. The blood–brain barrier and the development of the Drosophila nervous system In holometabolus insects, such as Drosophila, neurogenesis occurs in two phases. During embryogenesis the larval nervous system is generated by special stem cells called neuroblasts (Hartenstein and Wodarz, 2013). Their formation occurs in so-called proneural clusters specified by the balanced expression of proneural and neurogenic genes. To date, all embryonic neuroblast lineages have been identified and, from single cell tracing experiments, the trajectories of many of the neurons are known (Bossing et al., 1996; Jenett et al., 2012; Landgraf et al., 1997; Li et al., 2014; Rickert et al., 2011; Schmid et al., 1999; Schmidt et al., 1997; Urbach and Technau, 2003). Due to the advances in automated image analysis and 3D reconstruction programs, the establishment of a complete interaction map of the larval brain by serial electron microscopic analysis is in reach (Cardona et al., 2010; Sprecher et al., 2011). About 10% of all neural cells in Drosophila are glial cells. The glial cells or subsets of them can be labeled using many different molecular markers (Halter et al., 1995; Klämbt and Goodman, 1991; Stork et al., 2008, 2012; Xiong and Montell, 1995). The developmental origins of

Fig. 2. The blood–brain barrier in invertebrates and vertebrates. Schematic view of the blood–brain barrier in Drosophila, sepia, sturgeon and the mouse. In Drosophila, a glial blood–brain barrier is found. It is built by two glial cell layers, the perineurial glia (PG) and the subperineurial glia (SPG), which form septate junctions (SJ) to prevent paracellular diffusion (NL: neural lamella). In sepia, restricting junctions (RJ) are formed between perivascular glial cells (PG) in capillaries and venous vessels and between pericytes (PC) in arterial vessels to prevent paracellular diffusion (EC: endothelial cells, BM: basal membrane). In contrast, in the sturgeon no occluding junctions are formed, but the overlap of the glial cells (G) abutting the endothelial cells (EC) is strongly increased to elongate the diffusion path and thereby prevent uncontrolled diffusion (BM: basal membrane). In the mouse, capillary-forming endothelial cells (EC) establish tight junctions (TJ) to seal the blood–brain barrier (BM: basal membrane, PC: Pericyte, AG: astrocytic glia).

Please cite this article as: Schirmeier, S., Klämbt, C., The Drosophila blood-brain barrier as interface between neurons and hemolymph, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.06.002

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Fig. 3. Transport across the blood–brain barrier. Schematic view of the Drosophila hemolymph–nervous system interface. The blood–brain barrier is generated by perineurial glia (PG) and subperineurial glia (SPG). Septate junctions (SJ) between the SPG block paracellular diffusion into the nervous system. The cortex glia (CG) intermingles with neuronal cell bodies (in the embryo) and wraps around neuroblasts (NB) and clusters of neurons (N) (during postembryonic stages). Astrocyte-like glia (AG) are in contact with synapses. All glial cells are interconnected with gap junctions (GJ). Different transport routes across the blood–brain barrier are indicated.

the diverse glial cell types and their division patterns have been described as well (Awasaki et al., 2008; Beckervordersandforth et al., 2008; Hilchen et al., 2008; Stork et al., 2008). There are six morphologically different glial cell classes in Drosophila: the perineurial and the subperineurial glial cells constitute the blood–brain barrier. They reside at the outer surface of the nervous system and establish a tight paracellular diffusion barrier. Unlike the endothelial barrier found in mammals, Drosophila subperineurial glial cells establish pleated septate junctions (Figs. 2,3). Below the blood–brain barrier glial cell layer are the cortex glial cells that wrap all neuronal cell bodies. The ensheathing glial cells form a separate barrier around the neuropil, which harbors synapses, dendrites and axons. The neuropil is invaded by astrocytelike glia which perform similar functions as their mammalian counterparts (Awasaki et al., 2008; Stacey et al., 2010; Stork et al., 2014). In the peripheral nervous system, wrapping glial cells ensheath axons (Stork et al., 2008). Every single glial cell type can be targeted by specific expression of the yeast transcription factor Gal4 that activates gene expression downstream of a so-called UAS element (Duffy, 2002). Thus, a genetic cross of a Gal4 driver strain with an UAS responder construct results in the specific expression of the UAS coupled gene. An additional temporal control can be established by adding the expression of Gal80, a temperature sensitive inhibitor of Gal4 (McGuire et al., 2004). This, combined with the wealth of genetic variants, puts Drosophila into a prime position to analyze blood–brain barrier functions. After a period of quiescence during late embryonic and early (first instar) larval stages, the proliferation of neuroblasts needs to be reactivated at the second larval instar. The reactivation of neuroblast proliferation requires a nutritional checkpoint since the availability of dietary amino acids is crucial for the re-initiation of proliferation (Britton and Edgar, 1998). The fat body, the functional equivalent of the mammalian liver and adipose tissue, senses the availability of amino acids via the cationic amino acid transporter Slimfast (Colombani et al., 2003). Via Target of rapamycin (TOR) signaling, the fat body controls neuroblast proliferation (Britton and Edgar, 1998; Chell and Brand, 2010; Sousa-Nunes et al., 2011). Up to date, the signal that is secreted by the fat body is unknown. The way it acts, however,

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has been elucidated recently. The blood–brain barrier forming subperineurial glial cells sense the signal and subsequently initiate secretion of insulin-like peptides (Fig. 3). In turn, these hormones activate the insulin/IGF pathway in neuroblasts triggering their proliferation (Chell and Brand, 2010; Sousa-Nunes et al., 2011; Spéder and Brand, 2014; Limmer and Klämbt, 2014). Eight different insulin-like peptides are known in Drosophila (Drosophila insulin like peptide (Dilp) 1–8) (Kannan and Fridell, 2013). The expression of Dilp6 and Dilp2 in the subperineurial glial cells strictly depends on the availability of amino acids (Chell and Brand, 2010). Upon amino acid deprivation Dilp6 and Dilp2 are not expressed and neuroblasts do not leave quiescence. This phenotype can be rescued by forced expression of either of the two insulin-like proteins in the subperineurial glial cells, which leads to an exit of quiescence even under dietary restriction (Chell and Brand, 2010; Sousa-Nunes et al., 2011). Furthermore, inhibition of glial Dilp secretion, by blocking vesicular trafficking through expression of a dominant-negative, temperature-sensitive mutant dynamin, prevents reactivation of neuroblast proliferation under normal feeding conditions without influencing the overall body growth (Chell and Brand, 2010). Dilp signals from other classical Dilp-sources, like the IPCs (insulin producing cells) or the fat body, do not seem to be able to compensate for loss of Dilp secretion by the glial cells, since overexpression of Dilps in the fat body or the IPCs does not rescue a starvation induced block of NSC reactivation, while forced expression in glial cells or neurons does (Sousa-Nunes et al., 2011). Insulin signaling may be more complex and cells of the blood–brain barrier may exert a more active role in modulating this signaling cascade in glia or neuroblasts. Recently it has been reported that glial cells of the blood–brain barrier secrete a decoy insulin receptor (SDR) to negatively regulate insulin signaling (Okamoto et al., 2013). Although SDR is predominantly secreted into the hemolymph, there may be a function in the CNS as well. Reactivation of neuroblast proliferation via insulin signaling occurs at the same time throughout the entire brain (Spéder and Brand, 2014). How can such a synchronous reaction be achieved? Glial cells in all organisms are characterized by intensive cell coupling via gap junctions (Dahl and Muller, 2014; Holcroft et al., 2013; Nualart-Marti et al., 2013; Parys et al., 2010; Theis and Giaume, 2012; Tress et al., 2012). All different glial cells of Drosophila form extensive gap junctions (Holcroft et al., 2013) and these are essential to synchronize insulin signaling via Ca2+ oscillations and thereby coordinate NSC reactivation throughout the brain (Spéder and Brand, 2014). Gap junctions are built by transmembrane proteins that form dense, almost crystalline arrays of hemi-channels. In invertebrates, those proteins are innexins, while in vertebrates gap junctions are formed by connexins. The Drosophila genome encodes 8 innexins, most of which are expressed in the nervous system (Holcroft et al., 2013). Mutations in the innexin-encoding gene (ogre) have been associated with defects in neuroblast proliferation already 30 years ago (Lipshitz and Kankel, 1985). In addition, RNAi based knockdown of innexin2 and ogre results in reduced growth of the adult brain (Holcroft et al., 2013). Panglial knockdown of both innexins, ogre and inx2, leads to a decrease in dilp6 expression in the subperineurial glial cells (Spéder and Brand, 2014). Interestingly, innexins do not just seem to influence Dilp6 expression, but also its secretion into the brain tissue and Dilp6 expressed in the subperineurial glia is not secreted in ogre mutants (Spéder and Brand, 2014). The subperineurial glial cells of feeding larvae exhibit synchronized Ca2+ waves before the reactivation of neuroblasts. In ogre mutants the synchrony of those calcium waves is lost (Spéder and Brand, 2014). In addition, Ca2 + waves in the larval subperineurial glial cells are lost under amino acid deprivation — even though the gap junctional coupling is still present. When Ca2 + influx into the subperineurial glial cells was blocked by hyperpolarization, both Dilp6 expression and secretion were blocked. Furthermore, scavenging of intracellular Ca2 + by overexpression of Calmodulin led to the same effect. Together these data demonstrate that neuroblast reactivation is regulated by

Please cite this article as: Schirmeier, S., Klämbt, C., The Drosophila blood-brain barrier as interface between neurons and hemolymph, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.06.002

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the nutritional state of the animal that is measured in the fat body and conveyed to the neuroblasts via a synchronous Ca2+ mediated secretion of insulin-like peptides by the subperineurial glia of the Drosophila blood–brain barrier. Nevertheless, the question of how innexins work to synchronize insulin release remains open. One can imagine two scenarios: first, the subperineurial glial cells could be coupled by functional gap junctions to enable a direct spread of the Ca2+ wave. Such coupling can be found in the endothelial cells forming the mammalian blood– brain barrier, but remains to be demonstrated in Drosophila (De Bock et al., 2012; Leybaert and Sanderson, 2012). Second, Innexins, as vertebrate connexins, could form hemichannels, which facilitate Ca2+ entry or the secretion of the second messenger ATP. ATP in turn may stimulate purinergic receptor dependent Dilp secretion in neighboring cells. Connexin43 and Pannexin1 hemichannels have been shown to mediate ATP release from glial cells in vertebrates (Iglesias et al., 2009; Kang et al., 2008). In Drosophila, P2X-like receptors are lacking (Fountain, 2013) and currently not much information is available on G proteincoupled adenosine receptors such as AdoR (Dolezelova et al., 2007). In addition to the insulin-related signaling cascade, additional glial proteins may operate to orchestrate neuroblast proliferation. For example, glial cells express the secreted protein Anachronism, which controls proliferation of the neural stem cells by a still elusive mechanism (Ebens et al., 1993; Miller et al., 2009). However, proteins homologous to Anachronism are not known in vertebrates. Moreover, miRNA signaling is shown to operate in glial cells to control the formation of neuroblasts in the optic lobes (Morante et al., 2013). 3. Physiological functions of the blood–brain barrier Probably, the most basic function of the blood–brain barrier is to mediate neuronal nutrient supply (Fig. 3). In the mammalian nervous system this is manifested by the lactate shuttle operating in astrocytes and the support of long axons by oligodendrocytes (Fünfschilling et al., 2012; Pellerin and Magistretti, 2012). In Drosophila, many evolutionary conserved metabolite transporters are encoded in the genome and many of them are expressed in the glial cells of the blood–brain barrier (DeSalvo et al., 2014; Featherstone, 2011). However, their function in organizing the transport of metabolites across the blood–brain barrier has not yet been thoroughly analyzed. Disruption of the integrity of the blood–brain barrier clearly affects behavior and in fact the analysis of behavioral traits led to the identification of the Drosophila gene moody, which regulates some aspects of blood–brain barrier function (Bainton et al., 2005; Schwabe et al., 2005). Moody mutants were found in a genetic screen due to their increased sensitivity to cocaine and nicotine (Bainton et al., 2005). Moody encodes a G protein-coupled receptor (GPCRs) and at least two protein isoforms are generated by differential splicing. The Moody proteins are continuously expressed by the subperineurial glial cells, where they are actively required to maintain the integrity of the blood–brain barrier. How the loss of Moody-mediated signaling results in a slight opening of the blood–brain barrier is not understood. It was recently shown that moody controls the dynamic formation of specialized actin-rich structures at the cell boundaries, which may contribute to septate junction formation (Hatan et al., 2011). Disruption of actin dynamics results in an abrogation of the blood–brain barrier suggesting that GPCR signaling controls actin dynamics, which has to be explored in the future. Additionally, disruption of other genes acting in the subperineurial glia may cause behavioral deficits. For example suppression of kinesin heavy chain (khc) or tubulinß3 function specifically in the glial cells of the blood–brain barrier resulted in impaired walking and flying abilities of adult flies (Schmidt et al., 2012). This phenotype is most likely explained by a disruption of directed vesicle transport towards the growing septate junctions in the subperineurial glial cells. In khc as well as rab21/rab30 knockdown animals localization of the septate junction protein Neurexin IV is disrupted resulting in a local impairment of the

blood–brain barrier. The concomitant increase of the potassium ion concentration in the nervous system in turn impedes normal neuronal function. The control of ion and neurotransmitter homeostasis is a general glial cell function and its disruption frequently affects normal behavior (Zwarts et al., 2014). However, the blood–brain barrier may also convey more specific signaling tasks. For example, it has been described that hemolymph proteins originating from the fat body modulate the mating behavior of Drosophila (Lazareva et al., 2007). Sex determination in Drosophila is regulated in a cell-autonomous manner and, thus, every cell has to individually determine its sex. Interestingly, the sex of the blood–brain barrier glial cells matters and their feminization reduces male courtship behavior in a moody dependent manner. Thus possibly, sex-specific, still elusive ligands of Moody are an important part of this regulation. 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Please cite this article as: Schirmeier, S., Klämbt, C., The Drosophila blood-brain barrier as interface between neurons and hemolymph, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.06.002

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Please cite this article as: Schirmeier, S., Klämbt, C., The Drosophila blood-brain barrier as interface between neurons and hemolymph, Mechanisms of Development (2015), http://dx.doi.org/10.1016/j.mod.2015.06.002