Lipoprotein particles cross the blood-brain barrier in Drosophila

Abstract

The blood-brain barrier (BBB) regulates passage of nutrients and signaling molecules from the circulation into the brain. Whether lipoproteins cross the BBB in vivo has been controversial, and no clear requirement for circulating lipoproteins in brain development has been shown. We address these issues in Drosophila, which has an functionally conserved BBB, and lipoproteins that resemble those of vertebrates. We show that the Drosophila lipoprotein lipophorin exists in two isoforms. Both isoforms cross the BBB, but accumulate on distinct subsets of cells within the brain. In addition to acting as a lipid carrier, lipophorin carries both sterol-linked and GPI-linked proteins into the circulation and transports them across the BBB. Finally, lipophorin promotes neuroblast proliferation by a mechanism that does not depend on delivery of dietary lipids. Transport of lipophorin and its cargo across the BBB represents a novel mechanism by which peripherally synthesized proteins might enter the brain and influence its development. Furthermore, lipid-linkage may be an efficient method to transport therapeutic molecules across the BBB.

Figure 1.

Lpp particles cross the larval BBB. A, Scheme depicts the larval CNS at the level of the commissure: neuropil (magenta), optic anlage (yellow), cell bodies (gray) and the BBB (red). B, Motifs within apolipophorin. Lipid-binding pocket (LBP) (yellow oval), Furin cleavage site (FcS) (blue line), von Willebrand factor D domain (vWD) homology region (brown oval). Arrows indicate the position of peptides used to generate anti-ApoLII (magenta) and anti-ApoLI (black). C, Western blots of larval hemolymph iodixanol density gradients probed with anti-ApoLII and anti-ApoLI. Arrowheads indicate ApoLII (magenta), ApoLI (black) and Apo^FL (red). Fraction densities in g/ml. D–I, Confocal section at the level of the commissures from wild-type (D, F, H) and Lpp^RNAi (E, G, I) CNS stained with anti-Lpp (D, E; F–I; green), anti-HRP (F, G; magenta), anti-Dpn (H, I; red) and DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride) (H, I; blue). J–O, Wild-type larval brains incubated at 0°C (J, L, N) or 23°C (K, M, O) with labeled Lpp (L, M; N, O, in green) and labeled IgG (J, K; N, O; magenta). Ten micrometer projections at the level of the commissure.

Figure 2.

^HAApoL-tev^Myc and ^HAApoLII localize differently within the CNS. A, Replacement of the apolipophorin furin cleavage site (blue) with TEV-protease cleavage sequences (red). HA (green) and Myc (gray) tags are shown. Other motifs as in Figure 1B. B, Western blots of hemolymph iodixanol density gradients from larvae expressing either ^HAApoL^Myc or ^HAApoL-tev^Myc. Blots are probed with anti-HA (green arrowheads) and anti-Myc (gray arrowheads). Size heterogeneity of ^HAApoL-tev^Myc detected with both N-terminal HA and C-terminal Myc tags suggests alternative splicing may generate different ApoL^FL isoforms. Fraction densities in g/ml. C–H, Ten micrometer projections of second instar brains from larvae expressing either ^HAApoL^Myc (C, C′, D), ^HAApoL-tev^Myc (E, E′, F), or ^HAApoL-tev^Myc + TevP^secreted (G, G′, H) in the fat body. Brains are stained with anti-HA (C, E, G; C′–H; green), anti-HRP (C′, E′, G′; magenta) and anti-Elav (D, F, H; red). Staining was observed only in the optic anlage for three of three brains expressing ^HAApoL^Myc. Both neuronal and optic anlage staining was observed for five of six brains from animals expressing ^HAApoL-tev^Myc + TevP^secreted. Optic anlage staining was never observed in five of five brains from animals expressing either ^HAApoL-tev^Myc alone or + TevP^cytoplasmic.

Figure 3.

Lpp loss of function blocks neuroblast proliferation. A–D, Confocal sections of second instar brains from wild-type (A, B) or Lpp^RNAi (C, D) larvae, stained with anti-HRP (A, C; magenta), anti-Ci¹⁵⁵ (A, C; green), anti-HRP (B, D; gray), anti-Dpn (B, D; green) or anti-HistoH3^P (B, D; red). Ci-155 labels the optic anlage, Dpn marks neuroblasts, and phospho-histone H3 indicates mitotic cells. E, Quantification of phospho-histone H3-positive cells within second instar brains. Green bar, Wild-type larvae fed normal food (n = 5, 83.4 ± 21.5); brown bar, wild-type larvae fed lipid-depleted food (n = 5, 31.2 ± 5.2); red bar, Lpp^RNAi larvae fed normal food (n = 6, 3.5 ± 1.0). Asterisks indicate significant differences from wild-type larvae fed normal food (p < 0.001). Error bars are SDs. F, Neuropil/CNS volume ratio of wild-type larvae raised on normal food (green bar) or lipid-depleted food (brown bar) and Lpp^RNAi larvae raised on normal food (red bar). Asterisks indicate significant differences from wild-type larvae fed normally (p < 0.0001). Error bars are SDs. G–I, Projections of whole brains stained with FilipinIII from wild-type larvae raised on normal food (G) or lipid-depleted food (H) and Lpp^RNAi larvae raised on normal food (I).

Figure 4.

^HAmCherry^GPI crosses the BBB on Lpp. A, Iodixanol density gradients of hemolymph from third instar wild-type (rows 1, 2, 3, 5) and Lpp^RNAi larvae (rows 4, 6) expressing ^HAmCherry^sec, ^HAmCherry^sterol or ^HAmCherry^GPI in the fat body. Western blots are probed with anti-ApoLII (first row) or anti-HA (rows 2–6). Fraction densities are shown in g/ml. B–D′, Forty micrometer projection at the level of the commissures from wild-type (B, C′) and Lpp^RNAi (D, D′) second instar brains expressing ^HAmCherry^sec (B, B′), ^HAmCherry^sterol (C, C′), or ^HAmCherry^GPI (D, E′) in the fat body stained with anti-HA (B′, C′, D′; B–D; green) and anti-HRP (B–D, magenta). ^HAmCherry^sec is not found within the CNS (0 ^HAmCherry-positive neurons in 10 brains examined). ^HAmCherry^GPI accumulates in the CNS (>50 ^HAmCherry-positive neurons in all 5 brains examined). ^HAmCherry^sterol accumulates weakly in the CNS (5, 13, and 22 ^HAmCherry-positive neurons in 3 brains examined).