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First published online 5 September 2007
doi: 10.1242/dev.004572

Development 134, 3733-3742 (2007)
Published by The Company of Biologists 2007
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Drosophila Niemann-Pick Type C-2 genes control sterol homeostasis and steroid biosynthesis: a model of human neurodegenerative disease

Xun Huang1,2, James T. Warren3, JoAnn Buchanan4, Lawrence I. Gilbert3 and Matthew P. Scott1,*

1 Departments of Developmental Biology, Genetics and Bioengineering, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA 94305-5439, USA.
2 Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
3 Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
4 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5439, USA.


Figure 1
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  		Fig. 1. The npc2 gene family in Drosophila melanogaster. (A) The gene structure and the phylogenetic tree of eight npc2-like genes. Two gene clusters contain five npc2-like genes (CG31410, CG12813 and CG3934; CG11314 and CG11315), which can be identified based upon protein sequence similarities and gene location. (B) Protein sequence alignment of Npc2 proteins. hNpc2, homo sapiens NPC2; CeNpc2, Caenorhabdtis elegans Npc2 (NCR-2); ScNpc2p, Saccharomyces cerevisiae Npc2. 1, 2, 3 indicate the positions of three intron positions described in the text. 4 marks the intron position of C. elegans npc2 (ncr-2). The asterisks denote the five key residues previously found to be important for Npc2 function.

 

Figure 2
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  		Fig. 2. Transcription patterns of npc2-like genes ascertained with in situ hybridization in Drosophila melanogaster. npc2a is deposited maternally (A) and is broadly expressed with a higher level of expression in many tissues, including the midgut (arrow in B), salivary gland (arrow in C) and ventral nerve cord (arrow in D). (E) npc2a in situ staining signal was not detected in the homozygous npc2a mutant embryos. (F) The salivary gland expression of npc2d. (G,H) npc2g was specifically expressed in head mesoderm (arrow in G) and fat body (arrow in H). (I) npc2b was specifically expressed in trachea (arrow) and hypopharynx (arrowhead). (J) npc2b in situ staining signal was not detected in the homozygous npc2b mutant. (K,L) npc2a and npc2b were expressed in larval brain hemispheres and ring gland (arrows), respectively.

 

Figure 3
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  		Fig. 3. npc2a mutants and their sterol accumulation phenotypes. (A) The gene structure of npc2a and the chromosome intervals deleted in three Drosophila npc2a alleles. (B-G) Filipin staining reveals the sterol distribution patterns in wild type (B,D,F) and npc2a mutants (C,E,G). B and C are filipin-stained wing imaginal discs from third-instar larvae: wild type (B) and npc2a mutant (C). The magnified views (B',C') show that in wild type, sterol is located mainly at cell-cell boundaries, whereas in npc2a mutants sterol accumulates in a punctate pattern that is not restricted to those boundaries. (D,E) Aberrant sterol accumulation was observed in a striped pattern in npc2a (E) but not wild-type eggs (D). (F,G) Filipin staining highlighted the lumen of the Malpighian tubules in wild type. In npc2a mutants, massive punctate accumulations of filipin staining were visible inside Malpighian tubules.

 

Figure 4
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  		Fig. 4. Ultrastructural defects in third-instar larval Malpighian tubules of npc2a mutants. (A) Wild-type Drosophila melanogaster;(B-D) npc2a mutants. Large multi-lamellar structures (arrows in B and C) and multivesicular bodies (arrow in D) are present in npc2a mutants but not wild-type tubules. The multi-lamellar structures were often clustered together to form large inclusions with or without electron-dense whorls within (arrowhead in C and arrow in B, respectively). M, mitochondria.

 

Figure 5
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  		Fig. 5. npc2a and npc2b act redundantly in regulating sterol homeostasis in Drosophila. Filipin staining patterns of third-instar larval tracheas (A) and brains (B) in different genetic backgrounds. (A) npc2a and npc2b act redundantly in regulating sterol homeostasis in trachea. A small number of filipin-stained particles of sterol accumulation (arrow) was found in npc2a animals. By contrast, there was no sterol accumulation phenotype in npc2b mutants or in wild-type animals (not shown). However, massive sterol accumulation (arrow) was found in npc1a animals as well as npc2a; npc2b double mutants. (B) In brains, the punctated filipin-stained pattern (arrows) was found in both npc2a single and npc2a; npc2b double mutants but not wild type or npc2b single mutants.

 

Figure 6
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  		Fig. 6. Sterol requirement and sterol content of npc2 mutants. (A) Rescue of mutant Drosophila melanogaster by food supplementation with 20E or other sterols. The particular developmental stage in which the mutants died is shown as a percentage of the total. The x-axis indicates the developmental stage and the y-axis is the percentage of lethal. Without dietary supplements, nearly all mutants died by the pupal stage. Supplementation with 20E caused substantial rescue, allowing survival of more than 80% of the mutant animals until adulthood. Similarly, cholesterol and its immediate precursor 7-dehydrocholesterol allowed about 80% of the mutant animals to survive to adulthood. (B) The total sterol content of third-instar larvae was not changed in npc2 mutants. Three samples were measured for each genotype and error bars represent standard deviation.

 

Figure 7
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  		Fig. 7. Neurodegeneration in npc2a; npc2b double mutant Drosophila. (A) Survival data for flies of different genotypes. All mutant designations refer to homozygous animals. KG05307 indicates the starting strain for generating npc2a mutants. The x-axis indicates the time in days and the y-axis shows the percentage of flies surviving. (B) Evidence for apoptotic cell death in the nervous system of mutant flies. Wild-type brains (upper left) had little or no TUNEL staining, so there was little normal cell death. A small number of cells were stained by TUNEL in npc2a mutants (upper right, arrow). Lower left and, magnified, lower right: npc2a; npc2b double mutants had far more frequent death of neurons (arrow) and tracheal cells (arrowhead). (C) The apoptotic cells (labeled by TUNEL) in npc2a; npc2b double mutants included neurons (labeled by anti-Elav, arrow in the merged panel) and non-neurons (arrowhead in the merged panel). (D) Synaptotagmin staining of wild-type and npc2a axon bundles. The accumulation of Synaptotagmin (arrow) within axon tracts was observed in a small number of axons in npc2a mutants but not wild type.

 

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© The Company of Biologists Ltd 2007