The central nervous system is protected from the influence of the external environment by a blood-brain barrier. This cellular layer uses two properties to promote neuroprotection: a tight diffusion barrier and a complex array of transcellular transporters. Even though both properties are essential for proper humoral/CNS separation, little is known about their functional integration and regulation. In the Drosophila BBB tissue layer, the SPG, strong diffusion barrier properties had been previously identified, but the nature of its xenobiotic barrier had not been established. In this paper we characterize both physiologic aspects of the adult animal barrier and describe a novel system for the study of brain-specific small-molecule transport physiology. We combine in vivo physiologic assays for drug barrier function, and forward genetics to identify Mdr65 as an essential BBB transporter. Mdr65 loss of function leads to increased accumulation of ABC transporter substrates in the brain and increased sensitivity to cytotoxic xenobiotics. These studies suggest functional parallels between Mdr65 and the human MDR1/Pgp transporter, and together show strong evolutionary conservation of cell structure and chemoprotective mechanism in vertebrate and invertebrate CNS/humoral interfaces.
Chemical protection of the brain is a complex process involving many overlapping physiologic systems and thousands of genes (Abbott, 2005
; Sarkadi et al., 2006
; Zlokovic, 2008
). While recent advances in genomic and proteomic profiling of BBB components promise to provide a detailed description of the molecular players in BBB physiology, understanding how these components act in concert in a given environment remains a difficult problem for vertebrate systems to solve (Calabria and Shusta, 2006
). Integrative physiology is a discipline that promotes the use of appropriate model organisms to test physiologic function of particular genes and interacting genetic systems (Dow, 2007
). A powerful ambition of this approach is to find an experimental system advantageous for interpretation of gene function in different functional contexts (i.e. in the whole animal and/or cell-autonomous/tissue-based circumstances) (Yang et al., 2007
). We chose to focus our work in Drosophila
where a glial-dependent blood-brain barrier chemically insulates an open circulatory system from the retina, central brain and peripheral nerves (Carlson et al., 2000
; Stork et al., 2008
Previously, we discovered and began to characterize the BBB specific function of a Dm
orphan GPCR, Moody, that localizes with junctional complex components and likely controls diffusion barrier tightness through signals to the actin cytoskeleton (Bainton et al., 2005
; Schwabe et al., 2005
). Moody’s functional association with cellular junctions demonstrated for the first time the existence of hierarchichal control systems designed to direct specific aspects of BBB physiology. Interestingly, varying degrees of hypomorphic mutants in Moody demonstrate a range of phenotypes from subtle behavioral changes (i.e. drug responses) to outright disruption of the diffusion barrier in null animals. As GPCRs transmit information from external cellular stimuli as varied as photons and hormones, Moody’s discovery suggested the possibility that chemoprotective sensors may localize to and provide critical moment to moment evaluation and adjustment of barrier performance. However to pursue the control systems of chemical protection physiology, additional molecular and cellular components of the Dm
CNS xenoprotective interface had to be established ().
ABC transporters control localized pharmacokinetic penetration of drugs and are highly homologous between species thus it was logical to pursue their role at the Dm
BBB. Unfortunately, direct sequence comparisons of human MDR1/Pgp or Mrp1 to Dm
ABC B and C gene family members, respectively, did not yield any obvious candidates for specific chemoprotective genes (Dean and Annilo, 2005
). To unravel neuro-chemical protective function, we performed a reverse-genetic, physiologically-based screen that takes advantage of large collections of pre-existing mutants in many Dm
genes and identified PMdr65,
a loss of function allele of an ABC B transporter (). To confirm the PMdr65
mutant’s functional relevance to the CNS, we devised additional quantitative and in vivo
drug partition assays that address transporter-specific neuroprotective processes ( and ) and localized Mdr65 expression and function to the apical interface of the Dm
BBB (). Furthermore human MDR1/Pgp expressed at the Dm
BBB could similarly rescue drug transport, thus MDR1/Pgp can function cell autonomously to protect a CNS interstitial space. These data show that at least one ABC transporter in Dm
performs BBB specific duties similar to vertebrate MDR1/Pgp (Schinkel, 1999
) and suggests that the unique demands of CNS chemoprotection may select for transporters that are tuned to neural barrier requirements, even though very large evolutionary distances obscure the functional relationship of specific genes.
Coincident localization of the diffusion and xenobitotic transport barriers () demonstrated that the Dm
BBB combines vertebrate-like drug exclusion mechanisms to maintain a chemical barrier for the brain. This is an ideal setting to test the inter-relationship of chemical protection components at the cellular, organ and organismal levels (Strange, 2007
). For example in vertebrates MDR1/Pgp overexpression specifically promotes chemotheraptic drug resistance in cancer cells (Gottesman et al., 2002
). Here the quantity of an individual transporter at the cell membrane can alter the localized pharmacokinetics of a toxin by reducing partition into cells through increased efflux. However, at the BBB, the same gene functions in a complex cellular environment where xenobiotic protection has the potential to be dependent on not only the content of a single transporter, but also cell-type specific expression, spatial localization in a polarized cellular interface, additional transporters and other localized pharmacokinetic processes like diffusion barriers and metabolic enzymes (Sarkadi et al., 2006
; Zlokovic, 2008
). A strong drug barrier like the BBB can only function appropriately when all of these properties are manifested and correctly integrated. In this paper we show that increased quantities of BBB-specific Mdr65 induces CNS specific super-protection to cytotoxic substrates (). This is dependent on transporter localization, since several biologic tags to Mdr65 that prevent apical membrane association abrogate super-protection and rescue of xenobiotic sensitivity in Mdr65 nulls while exhibiting otherwise normal expression (data not shown). Thus a single overexpressed transporter can protect both an individual cell and, if properly localized, an entire viscous space like the CNS from drugs or chemicals. These data support the prevailing paradigm in vertebrate ABC transporter biology that end organ sensitivity must be matched with transporter type, quantity and discrete localization to promote xenobiotic efflux across cellular interfaces (Sarkadi et al., 2006
A great advantage of the Drosophila
model system is that cell autonomous gain or loss of function can be tested with anatomically directed genetic reagents, an approach that remains a technical challenge in vertebrates. Interestingly, selective, cell-type specific reduction of Mdr65 in the BBB produces a qualitatively similar xenobiotic phenotype to Mdr65 loss of function animals (about 1.7:1, mutant to WT ), thus much of Mdr65’s chemoprotective phenotype is targeted to the SPG. However this RNAi induced chemosensitive phenotype is not as strong as Mdr65 loss of function (2.4:1 mutant to WT ), suggesting additional roles for Mdr65 in whole animal small molecule pharmacokinetics. In fact, anatomically specific gene expression profiling demonstrates heightened levels of Mdr65 at other chemoprotective interfaces such as the gut and malphigian tubules (http://www.flyatlas.org/
). Thus, like vertebrate MDR1/Pgp, Mdr65 could play a role in broader xenobiotic/drug physiology, suggesting further evolutionary conservation between the way vertebrates and invertebrates organize and regulate chemical protection biology.
Finding innovative solutions to the drug delivery problems presented by the BBB – and indeed by all biological barriers – is likely to require an integrated understanding of the physiological mechanisms that allow barriers to maintain a balance between metabolic homeostasis and chemical protection. A genetic system like Dm offers the opportunity to employ inducible gene reduction systems and thus gain insight into acute responses to drug efflux loss of function, a condition similar to selectively localizing high levels of a transport inhibitor. Ultimately, these methods may be more gainfully applied to multiple, simultaneous gene reductions at the BBB interface. Such epistasis experiments targeting multiple localized small-molecule partition components will be a powerful method to uncover subtle interactions between localized pharmacokinetic control systems. With these tools in hand, future work will focus on systematic characterization of transport interfaces, through genomics or proteomics, and analysis of barrier responsiveness to various neurologic insults including cytotoxic drugs, hypoxia and metabolic stress.
Recent analyses of vertebrate barrier components point to a large number of biological pathways that may be involved in controlling and integrating the various aspects of barrier function (Enerson and Drewes, 2006
; Zlokovic, 2008
) (Ben Barres, personal communication). However, establishing the relevance of potential gene candidates is difficult without model systems that allow rapid analysis of proposed pathway function (Dow, 2007
; Yang et al., 2007
). We present a framework for using reverse genetics to build an integrated model of BBB physiology and to discover and test regulatory hypotheses of CNS chemoprotection. In addition this system has shown that simple genetic screens for breakdown of BBB function can identify new and unrecognized genes, like Moody, that are part of the regulatory hierarchy of neuroprotection hinting at modulatable control systems in vertebrate chemoprotection. Indeed, ongoing forward genetic screens have also identified mutants in several Dm
genes that are highly homologous to relevant vertebrate BBB genes involved in signaling, stress sensing and establishing and maintaining the structure of the BBB (data not shown). Thus BBB specific genes and processes found in model organisms, particularly Drosophila,
could lead to novel insights into the organization and cellular separation of the multiple protective BBB physiologies. These considerations, we believe, make our model system remarkably useful in terms of understanding how ancient and resilient organisms, such as the fruit fly, protect their CNS. Lastly, this approach may promote the identification of common, conserved regulatory pathways that contribute to chemical protection biology and BBB physiology across species.