Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.

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NEURONAL ceroid lipofuscinoses (NCLs) are a group of fatal genetic neurodegenerative diseases characterized by the progressive death of central nervous system (CNS) neurons and the accumulation of autofluorescent proteinacious storage material in cells (see COOPER 2003; HALTIA 2003; GOEBEL and WISNIEWSKI 2004; WISNIEWSKI 2005 for reviews). NCL subtypes differ mainly in their age of onset, their rate of progression, and the morphology of the storage material that accumulates in the cells of patients. The collective incidence of the various forms of NCL ranges from 0.1 to 7/100,000 (discussed in WISNIEWSKI 2005). There are nine NCLs, each caused by mutation in a specific gene (MOLE 2004). Six of these genes are known. Infantile neuronal ceroid lipofuscinosis (INCL) is caused by mutations in the CLN1 gene (now named PPT1), which encodes palmitoyl-protein thioesterase 1 (VESA et al. 1995). Late-infantile NCL (LINCL) results from mutations in the CLN2 gene, which encodes tripeptidyl peptidase 1 (TPP1) (SLEAT et al. 1997). Juvenile NCL and two variant late infantile forms of NCL (vLINCL) are caused by mutations in the CLN3, CLN5, and CLN6 genes, respectively, all of which encode transmembrane proteins with unknown functions (INTERNATIONAL BATTEN DISEASE CONSORTIUM 1995; SAVUKOSKI et al. 1998; GAO et al. 2002; WHEELER et al. 2002). Progressive epilepsy with mental retardation (EPMR), the least severe form of NCL, results from mutations in the CLN8 gene. Patients suffering from this disease can live out a normal life span, but suffer from seizures and progressive mental retardation (RANTA et al. 1999). The CLN4 and CLN7 genes associated with adult NCL and Turkish vLINCL, respectively, have not been identified. It has been demonstrated recently, however, that a subset of Turkish vLINCL cases are actually allelic variants of EPMR, while the genetic causes of the remaining cases of Turkish vLINCL remain unknown (RANTA et al. 2004). Recently, a novel form of NCL was characterized that has been classified as CLN9 (SCHULZ et al. 2004). The CLN9 gene has yet to be identified. All NCLs can be classified as lysosomal storage disorders (LSDs), because of the characteristic inclusions that accumulate in the cells of patients with disease (see images in WISNIEWSKI et al. 2001). In addition, a number of the known CLN gene products are proteins associated with the lysosome. PPT1 and TPP1 are both proteins with known lysosomal functions. Battenin, the gene product of CLN3, although its function is unknown, has been demonstrated to localize to the lysosomal membrane (EZAKI et al. 2003).

INCL is the most severe form of NCL and accounts for 25% of all NCL cases in the United States (discussed in HOFMANN et al. 2001). Children born with INCL initially appear normal; however, by age 1 they begin to show signs of psychomotor degeneration and progressive vision loss, resulting in complete blindness by age 2. Most children with INCL lose all higher brain functions by age 3 but survive until their early to mid teens (SANTAVUORI et al. 1973; see also discussion in WISNIEWSKI 2005). CNS neurons, mainly those in the cerebral cortex and cerebellum, are selectively killed in INCL, while cells from other tissues are spared. In addition to neurodegeneration, the cellular accumulation of autofluorescent storage material in the form of granular osmiophilic deposits (GRODs) is a hallmark of INCL (HALTIA et al. 1973). GROD accumulation occurs in most, if not all, tissues, including neurons (MITCHISON et al. 1998). The primary protein components of INCL storage material are saposins A and D, proteins involved in the degradation of sphingolipids in the lysosome (TYYNELA et al. 1993). INCL is unique in this regard, since the primary protein component that accumulates in the storage material of all other NCLs is subunit C of mitochondrial ATPase.

INCL is caused by mutations in the PPT1 gene, which encodes the soluble lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1) (VESA et al. 1995). PPT1 cleaves the thioester bond that attaches long-chain fatty acids, predominately palmitate, to the cysteine residues of S-acylated proteins (CAMP and HOFMANN 1993; CAMP et al. 1994). It is clear that the loss of PPT1 activity is what ultimately causes the death of CNS neurons in INCL. What is not clear, however, is how loss of PPT1 activity causes cell death and why neurons are selectively sensitive. Many of the disease phenotypes characteristic of INCL clearly result from loss of PPT1's catabolic function in the lysosome, such as the accumulation of GRODs as well as lipid thioesters and the secondary dysregulation of other lysosomal enzymes (LU et al. 1996; PRASAD and PULLARKAT 1996). An attractive model for the underlying etiology of INCL is that disruption of normal lysosomal homeostasis causes the progressive accumulation of a toxic product or process that can ultimately kill cells and to which neurons are particularly sensitive (see discussions in GUPTA et al. 2001; LU et al. 2002). The fact that the products of at least four of the CLN genes are known or suspected to have lysosomal functions supports this hypothesis. Recent evidence suggests that the toxic process may be progressive dysfunction of the endoplasmic reticulum, leading to activation of the unfolded protein response and neuronal apoptosis (ZHANG et al. 2006). In addition to a catabolic role in the lysosome, however, there is evidence that PPT1 may have extralysosomal functions, specifically in neurons; loss of these functions could contribute to disease. PPT1 may localize to presynaptic termini of mammalian synapses, suggestive of a possible function in turnover or regulation of palmitoylated proteins needed for synaptic transmission (HEINONEN et al. 2000; LEHTOVIRTA et al. 2001; AHTIAINEN et al. 2003; see, however, VIRMANI et al. 2005). Consistent with a specific role in the nervous system, PPT1 expression is both temporally and spatially regulated during development of the mammalian CNS (ISOSOMPPI et al. 1999; SUOPANKI et al. 1999). There is also evidence that some fraction of PPT1 may localize to lipid rafts in the plasma membrane and is involved in regulating apoptosis via ceramide-mediated signaling pathways (DAWSON et al. 2002; GOSWAMI et al. 2005). While the extralysosomal localization of PPT1 is intriguing and could be related to the underlying etiology of INCL, more in vivo evidence is needed to demonstrate that PPT1 actually functions at these extralysosomal locations. Finally, it is clear that palmitoylated proteins are a substrate of PPT1 in vitro, and while they are also likely to be substrates in vivo, the identities of specific S-acylated proteins that are in vivo substrates of PPT1 are not known. This limits our understanding of both the normal in vivo functions of Ppt1 and the way(s) in which the loss of those functions leads to neurodegeneration in INCL.

Drosophila has been used successfully to investigate numerous human neurological diseases, including lysosomal storage disorders (BONINI and FORTINI 2003; DRISCOLL and GERSTBREIN 2003; BILEN and BONINI 2005; DERMAUT et al. 2005; HUANG et al. 2005; MYLLYKANGAS et al. 2005). These fly models can produce neurodegeneration phenotypes that recapitulate aspects of their respective human diseases and provide tractable model systems with which to investigate disease etiology and explore potential therapeutic modalities (MARSH and THOMPSON 2004; WOLFGANG et al. 2005). We previously characterized the Ppt1 ortholog in Drosophila as well as palmitoyl-protein thioesterase 1 enzyme activity as an initial step in assessing the suitability of using Drosophila as a model system for studying INCL (GLASER et al. 2003). Both the Ppt1 gene and Ppt1 enzyme activity are uniformly expressed among different tissues of the fly and throughout all stages of development in a pattern typical of housekeeping genes. A small deletion, Df(1)446-20, was generated as part of the analysis. Df(1)446-20 removes Ppt1 along with three neighboring genes. Flies homozygous for Df(1)446-20 and completely lacking Ppt1 enzyme activity are viable and fertile.

In this report, two new point-mutant alleles of Ppt1, along with Df(1)446-20, were used to characterize phenotypes caused by the loss of Ppt1. Ppt1-deficient flies accumulate autofluorescent storage material, a phenotype characteristic of all NCLs. The osmiophilic deposits have a highly laminar morphology more similar to deposits seen in Tay–Sachs disease than to the morphology of GRODs seen in INCL, and the deposits accumulate almost exclusively in the adult CNS. In addition, the life span of Ppt1-deficient flies is reduced by 30%, but unlike in human INCL, the life-span reduction is not the consequence of progressive neurodegeneration. Ppt1-deficient flies show no evidence of alterations in brain morphology or of apoptotic cell death of CNS neurons, even at extreme ages.

Drosophila strains and genetics:

Flies were maintained on cornmeal-brewer's yeast-glucose medium at 23° and 55% relative humidity. Creation of the Df(1)446-20 deficiency and the UAS:DmPpt1^8.1 cDNA transgene have been described previously (GLASER et al. 2003; KOREY and MACDONALD 2003). Flies containing the P{Act5C-Gal4}17bFO1 and P{GAL4-elav.L}3 transgenes were obtained from the Bloomington Stock Center. To demonstrate rescue of the aberrant storage phenotype by the Ppt1 cDNA transgene, Df(1)446-20; UAS:DmPpt^8.1/+; P{GAL4-elav.L}3/+ and Df(1)446-20; UAS:DmPpt^8.1/+; H²/+ flies were created by crossing Df(1)446-20/Y; CyO/+; P{GAL4-elav.L}3/H² males to Df(1)446-20; UAS:DmPpt^8.1/CyO females.

Electron microscopy:

Brains and other tissues of interest were dissected from flies of appropriate genotype and fixed in 6.5% glutaraldehyde in 0.1 M sodium phosphate buffer at pH 7.4 for 3–3.5 hr. The tissue was then washed 2 x 10 min in phosphate buffer and 2 x 10 min in 0.1 M sodium cacodylate buffer at pH 7.4, postfixed in 1% osmium in cacodylate buffer for 1 hr, washed 1 x 5 min in water, prestained in 2% uranyl acetate for 30 min, washed in water again for 5 min, dehydrated in an acetone series, and embedded in Epon 812/Araldite. Sections (0.08–0.25 µm thick) of each sample were stained with uranyl acetate and Reynold's lead and were observed using a Zeiss 910 transmission electron microscope. Two to 5 animals of each genotype were examined, and 40–50 cells were examined at random from each animal to identify abnormal ultrastructural deposits. The ages of adult flies used for analysis were 1–3 weeks or as indicated in each figure legend.

Isolation of point mutations in Ppt1:

Male w¹¹¹⁸ flies were mutagenized by feeding them 20 mM ethyl methanesulfonate in 1% sucrose using standard protocols (GRIGLIATTI 1986). Mutagenized males were mated en masse to virgin por¹⁵⁷⁵ os^s/FM6, w¹ females. Single */FM6 females were recovered, placed individually into vials, and allowed to mate with FM6/Y males. Progeny from crosses that produced viable */Y male progeny were identified, and a single */Y male was collected from each vial and assayed for Ppt1 enzyme activity using the fluorogenic substrate 4-methylumbelliferyl-6-thiopalmitoyl-ß-D-glucoside (4-MU-6S-palm-ß-glc). The enzyme assay was modified from our previous study (GLASER et al. 2003). Briefly, the head was dissected from each */Y fly and placed into the well of a 96-well plate. Each well contained 20 µl water plus 10 µl of substrate (0.375 mg/ml 4-MU-6S-palm-ß-glc, 0.2 M Na-phosphate/0.1 M citrate, pH 4.0, 15 mM DTT, 0.09% BSA, and 5 units/ml ß-glucosidase). The heads were then homogenized in the 4-MU-6S-palm-ß-glc substrate using a 96-well pestle. Plates were incubated at 30° for 2 hr after which 100 µl of stop buffer was added to each well, and the amount of fluorescence at 460 nm was measured. A total of 2200 viable mutagenized chromosomes were screened, and two independent strains were found that had no detectable Ppt1 enzyme activity. The Ppt1 gene in each strain was amplified from genomic DNA by PCR using Deep Vent Polymerase (New England Biolabs, Beverly, MA). Both strands of the gene from each strain were sequenced, and a single GC-to-AT transition mutation was found in each strain producing the S77F and A179T mutations of Ppt1.

RNA interference:

To construct a foldback RNAi construct of Ppt1, we designed two sets of PCR primers to amplify identical fragments encompassing the first 800 bp of the Ppt1 coding region. One fragment was 8 bp longer than the other to produce an unpaired region for the hairpin turn in the RNA. The first amplified fragment was flanked by EcoRI and BamHI restriction sites. The primers used were: 5'-CCGGAATTCCAATGATTTCTATCTGCTGT-3' and 5'-CGCGGATCCGTAAAGGGCTGGATGACTTT-3'. The second amplified fragment was flanked by XbaI and BamHI restriction sites. The primers used were: 5'-TGCTCTAGAGCAATGATTTCTATCTGCTGT-3' and 5'-CGCGGATCCTGCTCTCAGTAAAGGGCTGG-3'. The PCR fragments were amplified and cloned using the Zero Blunt TOPO TA kit (Invitrogen, San Diego). The two fragments were digested with XbaI/BamHI and EcoRI/BamHI, as appropriate, and ligated into a 1600-bp concatamer. The concatamer was then cut with XbaI and EcoRI and cloned into the pUAST transformation vector. The pUAST:Ppt1-RNAi construct was injected into Drosophila embryos, and transformants were obtained using standard methods (RUBIN and SPRADLING 1982). Two independent insertions of the pUAST:Ppt1-RNAi construct were recombined onto a single second chromosome to generate UAS:Ppt1-RNAi^8/7/CyO flies. UAS:Ppt1-RNAi^8/7/CyO females were crossed to y¹ w*; P{Act5C-GAL4}17bFO1/TM6B males to generate the UAS:Ppt1-RNAi^8/7/+; P{Act5C-GAL4}17bFO1/+ progeny used for EM analysis.

Analysis and quantitation of autofluorescence:

Tissues from animals of the appropriate genotype were dissected in saline and mounted directly in Vectashield (Vector Laboratories, Burlingame, CA). Tissues were examined and photographed on an Axioscope-2 Plus deconvolution microscope using a fluorescein filter set (Zeiss, Thornwood, NY). All images are flattened 2-µm Z-stacks collected using Openlab software (Improvision). Pixel intensities were distributed across a range of 0–127 arbitrary intensity units. To quantitate the amount of autofluorescence in the brain, the total number of pixels in a flattened Z-stack image of a whole brain was determined and parsed into 10-unit increments of increasing fluorescence intensity. All brain preparations used for quantitation were imaged using the same exposure time and intensity. In addition, the optic lobes were removed from brains used for quantitation to reduce variability caused by differences in the amount of optic lobe tissue recovered during dissection of the brain from the head capsule.

Viability curves:

Approximately 300 female flies of appropriate genotype were collected at 0–2 days of age and were distributed into vials with 30 females and 5 males per vial. The flies were maintained at 23° and were passaged twice per week. The number of dead females was counted at each passage. The data were analyzed using the Kaplan–Meier survival analysis package in StatView 5.0. P-values were calculated using the Mantel–Cox log rank statistic.

Loss of Ppt1 causes accumulation of aberrant storage material:

To determine whether aberrant storage material accumulates in flies lacking Ppt1, we dissected brains from Df(1)446-20 homozygous female flies and examined them by electron microscopy. Abnormal deposits were readily apparent (Figure 1). Unlike the irregular granular deposits (GRODs) seen in human INCL, the deposits in Ppt1-null flies were fairly homogenous in structure, being predominantly spherical in shape and composed of tens to hundreds of concentric layers of material, each layer being 4–12 nm in thickness, and often surrounding a granular core from which the laminar material seemed to emanate (Figure 1, A and B, see also Figure 3, B–D). The deposits are similar in structure to the membranous cytoplasmic bodies observed in human Tay–Sachs and Sandhoff diseases, as well as to deposits observed in flies mutant for the lysosomal sugar transporter benchwarmer and in flies mutant for dnpc1a, the fly homolog of human NPC1 that is mutated in Nieman–Pick type C disease (DERMAUT et al. 2005; HUANG et al. 2005; see later discussion). The laminar deposits were strongly osmiophilic and were found in the cytoplasm, typically near the nucleus, in 90% of the cell bodies in any given field of view. No deposits were detected in sections from the neuropil regions of the brain. Individual deposits averaged 0.5 µm in diameter in 5-day-old animals up to 2 µm in 35-day-old animals, indicating that the deposits grow in size through the accumulation of additional layers of storage material as the fly ages (compare Figure 1A and 1B). Deposits consisting of what appear to be aggregates of individual components became more common as the flies aged (Figure 1C). Storage material in the brains of Df(1)446-20 males formed deposits that were consistently larger and more often aggregated and that contained more prominent gaps and spaces between the layers of material, as compared to deposits in females (Figure 1D). Laminar deposits like those found in the brain were also found in the thoracic ganglion; more rarely, deposits were also detected that had a morphology reminiscent of the curvilinear and rectilinear profiles observed in the classic late infantile and variant late infantile forms of NCL (Figure 1E). There were no abnormalities in the morphology of other cellular organelles, and the laminar deposits did not appear to be derived from structurally abnormal mitochondria, as has been reported for Ppt1 mutants in Caenorhabditis elegans (arrowheads in Figure 1; PORTER et al. 2005).

View larger version (158K): In this window In a new window Download PPT slide   FIGURE 1.— Df(1)446-20 flies accumulate abnormal storage material. Tissues from Df(1)446-20 flies were analyzed by electron microscopy (EM). Osmiophilic laminar deposits were detected in the brains of 5-day (A) and 35-day (B and C) posteclosion females. Deposits in the brains of 14-day posteclosion males were larger and less compacted (D). Deposits reminiscent of curvilinear and rectilinear deposits were detected in 35-day adult thoracic ganglion (E). Rare deposits were detected in the brains of third-instar larvae (F), and less sharply defined deposits were also seen in cells of 35-day adult gut (G). Brains from wild-type flies lacked abnormal deposits, but they did have abundant vacuoles containing fine granular material (H). Enlargements of selected deposits are shown to the right in A, B, F, G, and H. Abnormal storage deposits are indicated by arrows. Mitochondria are indicated by arrowheads. Nuclei are indicated by the letter N. Bars, 1 µm.

 

View larger version (98K): In this window In a new window Download PPT slide   FIGURE 3.— Abnormal storage material accumulates in the brains of females with point mutations in Ppt1. (A) The locations of the S77F and A179T point mutations of Ppt1 are shown in the crystal structure of bovine PPT1 (BELLIZZI et al. 2000). The protein backbone is shown in yellow. Palmitate in the binding pocket of the enzyme is shown in red. The active site of the enzyme is located at the top of the palmitate, in the middle of the enzyme in the orientation displayed. Serine 77 and alanine 179 are shown in purple in spacing-filling mode, with alanine 179 located at the bottom of the binding pocket and serine 77 located in an indentation at the top of the molecule. Brains from female flies of genotypes Df(1)446-20/Ppt1S77F (B), Df(1)446-20/Ppt1A179T (C), and Ppt1S77F/Ppt1A179T (D) were examined by EM. Laminar deposits identical to those observed in Df(1)446-20 animals were found in all three trans-heterozygous genotypes.

 
Laminar deposits were also found in some tissues outside the adult CNS, including third-instar larval brain and adult gut epithelium (Figure 1, F and G). Compared to the adult brain, however, these tissues had deposits that were smaller, more diffuse, less osmiophilic, and much less abundant, particularly in larval brain. It is unclear whether the deposits seen in the larval brain are precursors of the deposits seen in adults. No abnormal storage material was found in muscle or fat body cells in the adult (data not shown). Finally, no abnormal deposits were detected in the brains of w¹¹¹⁸ or Oregon R control flies or Df(1)446-20 heterozygotes (Figure 1H and data not shown). Vesicles containing uniform granular contents were common in brain cells of young wild-type flies but were not detected in the Df (1)446-20 mutant cells (compare Figure 1H to Figure 1, A–G).

Loss of Ppt1 is necessary and sufficient for accumulation of abnormal storage material:

The Df(1)446-20 deletion removes three genes in addition to Ppt1 (GLASER et al. 2003), so it was necessary to demonstrate that the loss of the Ppt1gene was what specifically caused accumulation of the abnormal storage material detected by EM. Initial attempts to rescue the storage material phenotype in Dp(1)446-20 flies using a genomic clone from the Ppt1 locus were unsuccessful. Ppt1 expression was negligible no matter where the clone was inserted in the genome despite inclusion of significant amounts of flanking sequence (our unpublished observation). To circumvent this problem, we used Ppt1 cDNA clone UAS:DmPpt1^8.1 to express Ppt1 enzyme activity in Df(1)446-20 flies. UAS:DmPpt1^8.1 was created for previous studies and can be controlled using the GAL4/UAS system (BRAND and PERRIMON 1993; KOREY and MACDONALD 2003). Pan-neural expression of UAS:DmPpt1^8.1 was induced in Df(1)446-20 flies using an elav:GAL4 driver. The level of Ppt1 enzyme activity measured in extracts of whole heads from these flies was 71.8% of the level observed in heads from wild-type flies (data not shown). This means that enzyme levels specifically in the brain were about threefold higher than those of wild type. This is because the brain contributes only about one-quarter of the total Ppt1 enzyme activity that is measured in a whole-head extract, as estimated by comparing enzyme levels in extracts of dissected brains vs. those of whole heads in wild-type animals (our unpublished observation). This level of neuron-specific overexpression of Ppt1 did not produce any obvious phenotypes in young adult flies, but did reduce viability in animals older than 2–3 weeks of age (data not shown). This was not unexpected, since overexpression of Ppt1 is known to be deleterious (KOREY and MACDONALD 2003).

Brains from Df(1)446-20 flies expressing UAS:DmPpt1^8.1 were isolated and examined by EM. No laminar deposits or aberrant storage material of any type were detected, whereas laminar deposits were readily detected in brain cells in sibling controls (Figure 2). This result demonstrates that deletion of Ppt1 is necessary for the accumulation of aberrant storage material seen in Df(1)446-20 flies.

View larger version (103K): In this window In a new window Download PPT slide   FIGURE 2.— Neuron-specific expression of a Ppt1 cDNA rescues the abnormal storage material phenotype. Brains from female flies of genotypes Df(1)446-20; UAS:DmPpt18.1/+; P{GAL4-elav.L}3/+ (A) and from sibling control flies of genotype Df(1)446-20; UAS:DmPpt18.1/+; H2/+ (B) were analyzed by EM. No laminar deposits were detected in Df(1)446-20 flies expressing the Ppt1 cDNA, but deposits were readily detected in sibling controls lacking the GAL4 driver. Abnormal storage deposits are indicated by arrows. Nuclei are indicated by the letter N. Bars, 1 µm.

 
To demonstrate that mutation to Ppt1 is sufficient to cause accumulation of abnormal storage material, we generated point mutations in the Ppt1 gene using ethyl methanesulfonate mutagenesis and analyzed the mutants by EM. Individual male flies hemizygous for mutagenized X chromosomes were screened for Ppt1 enzyme activity using the fluorogenic substrate 4-methylumbelliferyl-6-thiopalmitoyl-ß-D-glucoside (see MATERIALS AND METHODS). Two mutant alleles of Ppt1 were recovered that produced no detectable enzyme activity capable of metabolizing the methylumbelliferyl substrate. The mutant alleles were then cloned from genomic DNA by PCR and sequenced. Each allele was found to contain a single point mutation that changes a conserved amino acid, specifically, serine 77 to phenylalanine (S77F) and alanine 179 to threonine (A179T). The analogous residues in human PPT1 are serine 69 and alanine 171. The A179T mutation is located within the distal end of the substrate-binding pocket (Figure 3A). The alanine side chain likely comes in contact with fatty acid substrates, suggesting that the alanine-to-threonine mutation could interfere with substrate binding. The S77F mutation is located at the bottom of an indentation on a side of the enzyme separate from the substrate-binding pocket and the active site residues (Figure 3A). The change of serine 77 to a bulky phenylalanine could reduce Ppt1 stability. In this regard, it is interesting to note that the side chain of serine 77 is immediately adjacent to a conserved vicinal disulfide bridge that may be important in Ppt1 structure. Finally, females were created that were trans-heterozygous for each pairwise combination of the Ppt1 point mutations and the Df(1)446-20 deletion, specifically, Df(1)446-20/Ppt1^S77F, Df(1)446-20/Ppt1^A179T, and Ppt1^S77F/Ppt1^A179T. Brains from these females were isolated and analyzed by EM for the presence of abnormal storage material. Laminar deposits identical in structure to those observed in Df(1)446-20 animals were found in all three genotypes (Figure 3, B–D). This result provides compelling genetic evidence that it is solely the loss of Ppt1 enzyme activity, and not background mutations, that causes the observed accumulation of abnormal storage material.

RNAi-mediated knockdown of Ppt1 expression was also used to demonstrate that reduction of Ppt1 enzyme activity causes accumulation of abnormal storage material. A Ppt1 RNAi transgene that consists of an inverted repeat of the first 800 bp of the Ppt1 cDNA separated by a short spacer was constructed and inserted into the Drosophila germline (see MATERIALS AND METHODS). The transgene can be controlled using the Gal4/UAS system. The strongest suppression of Ppt1 enzyme activity was observed when expression of the RNAi transgene was driven using the tissue-general Act5C:Gal4 driver. Levels of Ppt1 enzyme activity assayed in extracts of female heads varied from fly to fly, ranging from 0 to 10% of wild-type levels (data not shown). Brains were isolated from the heads of such Ppt1 knockdown flies and then assayed by EM for the presence of abnormal storage material. Laminar deposits like those seen in both Df(1)446-20 and Ppt1 point mutant flies were observed (Figure 4). In comparison to flies containing a mutation in the Ppt1 gene, the RNAi flies had deposits that were less abundant, being found in 30% of the cells in a given field of view instead of the 90% seen in Ppt1 null flies. Also, the deposits seen in RNAi flies most often had notably fewer laminar layers, although deposits with a larger number of layers were occasionally observed (Figure 4). This result further supports the conclusion that a deficiency of Ppt1 enzyme activity results in the accumulation of abnormal storage material in the form of laminar cytoplasmic deposits.

View larger version (129K): In this window In a new window Download PPT slide   FIGURE 4.— RNAi-mediated knockdown of Ppt1 causes accumulation of abnormal storage material. Brains from flies of genotype UAS:DmPpt1-RNAi8/7/+; P{Act5C-Gal4}17bFO1/+ were examined by EM. The majority of abnormal storage deposits had fewer laminar layers than did deposits in Ppt1 mutant flies (A), although deposits with a larger numbers of layers were also observed (B).

 

Loss of Ppt1 causes accumulation of CNS-specific autofluorescent inclusions:

Tissues were dissected from Df(1)446-20 homozygous female flies and were examined by fluorescence microscopy to determine whether abnormal storage material in Ppt1-mutant flies could be detected as autofluorescent inclusions. Adult Df(1)446-20 flies clearly accumulated autofluorescent inclusions in the CNS (Figure 5). All animals, including Drosophila, accumulate autofluorescent material called lipofuscin as a normal consequence of aging (PORTA 2002). The autofluorescent inclusions detected in Df(1)446-20 flies, however, were more abundant and larger in size than the lipofuscin that accumulated in age-matched wild-type control flies (Figure 5A). The autofluorescent inclusions, which fluoresced across a wide range of wavelengths, were found in the adult CNS, including the brain and ventral ganglia, but not in the larval CNS, or in any other adult tissue examined, or in flies heterozygous for the Df(1)446-20 deletion (Figure 5, A and B, and data not shown). Since the autofluorescent inclusions were observed in young adults but not in larvae, they must begin forming during metaphorphosis. To address the timing of their appearance, we quantified autofluorescent inclusions at various times during metamorphosis and in adults at different ages. By 36 hr after pupation, autofluorescent inclusions were detected in the pupal brain, but no difference was observed between Df(1)446-20 and wild-type flies (Figure 6). By 72 hr after pupation, however, autofluorescent inclusions in the Df(1)446-20 brains were both significantly more abundant and significantly brighter than the lipofuscin found in wild-type brains, and this difference persisted in adult flies (Figure 6).

View larger version (68K): In this window In a new window Download PPT slide   FIGURE 5.— Adult Df(1)446-20 homozygous flies accumulate autofluorescent inclusions in the CNS. (A) Flattened optical Z-stacks of Df(1)446-20 or w1118 brains from female third-instar larvae (3L), 1-day-old adult females (1 d), 29-day-old adult females (29 d), and 58-day-old adult females (58 d) were obtained by deconvolution light microscopy. (B) Autofluorescent inclusions were also detected in the thoracic ganglion of Df(1)446-20 flies, but not in non-CNS tissues, including fat body, gut, or ovary. The solidly white tissues in the fat body sample of Df(1)446-20 and in the ovary preparations are autofluorescent tissues, like the chorion of developing eggs, and differ from the punctate autofluorescent inclusions found in the CNS.

 

View larger version (18K): In this window In a new window Download PPT slide   FIGURE 6.— Autofluorescent inclusions form during metamorphosis and increase in number and intensity with age. Brains were isolated at various times postpupation (hours) and posteclosion (days), and flattened optical Z-stacks like those shown in Figure 5 were obtained. The amount of autofluorescence in each brain was quantitated by counting the number of pixels in the image in 10-unit increments of increasing pixel intensity. Results for brains from female Df(1)446-20 (shaded lines) and w1118 (solid lines) are shown. Each mean and standard deviation value was calculated from three to five independent samples.

 
To demonstrate that mutation to Ppt1 is sufficient to cause autofluorescent inclusions, we isolated brains from Df(1)446-20/Ppt1^S77F, Df(1)446-20/Ppt1^A179T, and Ppt1^S77F/Ppt1^A179T females and analyzed them for autofluorescence. Autofluorescent inclusions were substantially more abundant in all three trans-heterozygous genotypes than in wild-type controls, supporting the conclusion that loss of Ppt1 activity causes the accumulation of autofluorescent inclusions (Figure 7). Surprisingly, pan-neural expression of the Ppt1 cDNA did not rescue the autofluorescence phenotype, nor did tissue-general expression of the Ppt1 RNAi transgene phenocopy the autofluorescence. Autofluorescence was rescued, however, in Ppt1-deficient males containing Dp(1;Y)578, a Y chromosome containing a duplication of the 8A region of the X chromosome where Ppt1 is located (data not shown). The presence of autofluorescent inclusions in three different trans-heterozygous females, each having two independently derived Ppt1 mutations, combined with rescue by Dp(1;Y)578 provides strong genetic evidence that Ppt1 deficiency is the cause of the autofluorescence phenotype. The relationship between the autofluorescent inclusions and the laminar bodies seen by EM is unclear. While they could be the same deposits, it is also possible that they are different types of deposits independently produced by loss of Ppt1.

View larger version (74K): In this window In a new window Download PPT slide   FIGURE 7.— Mutation to Ppt1 is sufficient to cause autofluorescence. Brains from female flies of genotypes Df(1)446-20/Ppt1S77F (A), Df(1)446-20/Ppt1A179T (B), Ppt1S77F/Ppt1A179T (C), and w1118 (D) were examined by deconvolution light microscopy. Autofluorescent inclusions like those observed in Df(1)446-20 animals were readily detected in all three trans-heterozygous genotypes.

 

Ppt1-mutant flies have a reduced life span but no obvious neurodegeneration:

Ppt1-mutant flies are viable and fertile, but their life span is reduced significantly compared to that of w¹¹¹⁸ controls. The median life spans of Df(1)446-20/Ppt1^S77F, Df(1)446-20/Ppt1^A179T, and Ppt1^A179T/Ppt1^S77F females were reduced 42, 37, and 20%, respectively (Figure 8 and Table 1). Seeing a life span reduction in all three trans-heterozygous genotypes is consistent with mutation to the Ppt1 gene causing the observed reductions. Effects of genetic background, however, cannot be completely excluded, particularly given the modest effects. The greater reduction in deficiency heterozygotes compared to point mutant heterozygotes could indicate that the point mutants of Ppt1 are not null alleles, at least for Ppt1 functions related to reduction in life span. Alternatively, background mutations on the Df(1)446-20 chromosome could reduce life span dominantly beyond the reduction caused by loss of Ppt1; however, a reduction in life span in Df(1)446-20 heterozygotes has not been observed. The median life spans of male flies hemizygous for Df(1)446-20, Ppt1^S77F, or Ppt1^A179T were reduced 32, 29, and 21%, respectively, compared to those of w¹¹¹⁸ controls (P < 0.001; data not shown). While this result is consistent with the idea that loss of Ppt1 reduces viability in adult males, hemizygosity of the male X chromosome prevents us from drawing strong conclusions. Finally, we were unable to determine if pan-neural expression of the UAS:DmPpt1^8.1 cDNA can rescue the reduction in life span. elav-driven expression of the cDNA causes a reduction in viability on its own, likely due to overexpression of Ppt1 enzyme activity, which is known to be deleterious (KOREY and MACDONALD 2003). The reduction in viability caused by Ppt1 overexpression precludes the ability to detect rescue of the reduced life span caused by Ppt1 deficiency.

View larger version (15K): In this window In a new window Download PPT slide   FIGURE 8.— Ppt1 deficiency reduces adult viability. Survival curves of Df(1)446-20/Ppt1S77F (triangles), Df(1)446-20/Ppt1A179T (squares), Ppt1A179T/Ppt1S77F (diamonds), and w1118 (circles) are shown. The number of flies that survived was determined every 3–4 days for the length of the experiment. Mean, median, and maximum life spans and P- and n-values calculated from these curves are shown in Table 1.

 

View this table: In this window In a new window   TABLE 1 Ppt1-deficient flies have reduced life span

 
To determine whether Ppt1-mutant flies might also have a neurodegeneration phenotype that correlates with the reduction in life span, the gross morphology of brains isolated from Ppt1-mutant flies was assessed in hemotoxylin/eosin-stained paraffin sections and in whole brains from Ppt1-mutant animals containing GFP marker genes that highlight specific brain structures such as the mushroom bodies. No consistent alteration of brain morphology was observed in Ppt1-mutant flies, and no overt evidence of neurodegeneration was detected, such as vacuolization, brain shrinkage, or a reduction in the number of nuclei (data not shown). Furthermore, we found no evidence of elevated levels of apoptosis in flies as old as 60 days as assayed by terminal deoxynucleotidyltransferase-mediated nick end labeling or by immunolocalization of H2A.X phosphorylation (data not shown; MADIGAN et al. 2002). There was also no evidence for degradation of ommatidial structure, as assayed by the deep pseudopupal technique (data not shown). These results suggest that loss of Ppt1 in flies does not cause neurodegeneration and that neurodegeneration does not cause the observed reduction in life span.

Ppt1-mutant flies accumulate abnormal storage material:

Mutation of the Drosophila Ppt1 gene causes the age-dependent accumulation of abnormal storage material, a characteristic feature of all NCLs, and of lysosomal storage diseases in general. The storage material that accumulates in the brains of Ppt1-mutant flies, however, has a very different morphology from that of the GROD that accumulates in human INCL. The deposits are highly laminar in structure, most similar in morphology to the membranous cytoplasmic bodies that accumulate in Tay–Sachs and Sandhoff diseases (see images in ITOH et al. 1984; PERL 2001). In addition, the curvilinear deposits detected in the thoracic ganglion are most similar to the curvilinear profiles characteristic of late infantile NCL (Figure 1E). It is not unknown for human NCL patients to have deposits of differing morphologies in different cell types. Most interestingly, there is even a case of adult-onset NCL in which laminar deposits similar in structure to those reported here were intermingled with, and continuous with, curvilinear deposits in neurons of the patient's spinal anterior horn (ISEKI et al. 1987). In addition to being morphologically different, the storage material in Ppt1-mutant flies may also be biochemically different from the deposits seen in INCL. The storage material in Ppt1-mutant flies could not be detected using a variety of lipophilic stains or periodic acid Schiff, stains that readily detect the storage material produced in all NCLs, including INCL (our unpublished observations). The differences in morphology and biochemical composition between the storage material produced in Ppt1-mutant flies and the GROD seen in INCL patients suggest that the loss of Ppt1 has different biochemical consequences in flies and in mammals. Such differences could arise from differences either in the substrates of Ppt1 or in more general aspects of lysosome function and homeostasis that indirectly affect the downstream consequences of Ppt1 loss. The latter possibility is supported by the observation that the upregulation of other lysosomal enzymes, typically seen in NCLs, does not appear to occur in Ppt1-mutant flies. Levels of ß-glucuronidase and hexosaminidase A are significantly elevated in INCL patients and in Ppt1-knockout mice, but they are unchanged in Ppt1-mutant flies (our unpublished observation; PRASAD and PULLARKAT 1996; GRIFFEY et al. 2004). Finally, the general idea that Ppt1-mutant phenotypes vary among organisms because of species-specific differences in Ppt1 substrates and/or lysosomal catabolic pathways is also supported by the phenotype of Ppt1 mutants in C. elegans. Loss of Ppt1 in C. elegans causes mitochondrial pathology without accumulation of aberrant storage material, a phenotype very different from the phenotypes observed in either flies or mammals and suggestive of unique functions for Ppt1 in worms (PORTER et al. 2005).

No evidence of a neurodegeneration phenotype was found in Ppt1-mutant flies. Gross brain morphology and rates of apoptotic cell death in the brain were unaltered even in aged Ppt1 mutants. The reason why Ppt1 deficiency leads to neural cell death in mammals but not in flies is unknown. It is possible that flies simply do not live long enough for some pathological byproduct or process caused by the loss of Ppt1 to reach toxic levels. Alternatively, Ppt1 might have an essential function in mammalian neurons that is lacking in flies. For example, PPT1 may localize to synaptic vesicles rather than to lysosomes in cultured mouse neurons, suggesting that PPT1 plays an important role in turnover of palmitoylated signaling molecules at the synapse (HEINONEN et al. 2000; LEHTOVIRTA et al. 2001; AHTIAINEN et al. 2003; see, however, VIRMANI et al. 2005). The subcellular localization of Ppt1 in Drosophila is not known; however, if this neural-specific function of Ppt1 were lacking or nonessential in flies, then loss of Ppt1 might be without consequence for synaptic function. While we have generated Ppt1-specific antibodies to address this question, we have thus far been unable to detect endogenous Ppt1 on Western blots or by immunocytology. The absence of an obvious neurodegeneration phenotype does not preclude the possibility of more subtle abnormalities in neural function. Ppt1-mutant flies have a shortened life span, suggesting that loss of Ppt1 has pathological consequences sufficient to cause an age-dependent reduction in viability (Figure 8 and Table 1). In this context, it is interesting to note that abnormal storage material accumulates almost exclusively in the CNS, suggesting that fly neurons, like human neurons, are preferentially sensitive to Ppt1 loss (Figures 1 and 5). Thus, it would not be surprising if the observed reduction in viability of Ppt1-mutant flies is a consequence of pathology specifically in the CNS as opposed to other tissues. A primary focus of future studies will be to determine whether Ppt1-mutant flies have phenotypes indicative of age-dependent loss of neural function.

Drosophila NCL models:

Mutation to two lysosomal genes of Drosophila, in addition to Ppt1, causes disease phenotypes associated with NCLs. Cathepsin D (cathD) encodes a lysosomal aspartic proteinase that, when mutated in sheep, causes ovine NCL. Ovine NCL exhibits phenotypes like those of human NCLs, including accumulation of autofluorescent storage material and neurodegeneration. cathD-mutant flies accumulate autofluorescent storage material and show an age-dependent increase in the number of apoptotic cells in the brain, although the increased cell death does not produce a reduction in life span (MYLLYKANGAS et al. 2005). The fact that mutations in two different NCL-related homologs in Drosophila, Ppt1 and cathD, both recapitulate at least some phenotypes associated with human disease suggests that the molecular mechanisms that cause those phenotypes in NCLs may be conserved in flies. The Drosophila benchwarmer gene (bnch; allelic to the gene spinster) encodes a lysosomal transmembrane carbohydrate transporter that, when mutated, causes accumulation of autofluorescent storage material as well as neurodegeneration. The latter effect is likely a consequence, at least in part, of disrupted endosome-to-lysosome transport pathways (NAKANO et al. 2001; SWEENEY and DAVIS 2002; DERMAUT et al. 2005). Human homologs of bnch are not known to be NCL genes but are mutated in lysosomal storage diseases caused by defective efflux of lysosomal substrates, particularly as occurs in sialic acid storage disease (discussed in DERMAUT et al. 2005). The abnormal deposits that accumulate in Ppt1-mutant flies are similar in structure to a class of deposits seen in bnch mutants (DERMAUT et al. 2005). The fact that mutations in three different lysosomal proteins in Drosophila—Ppt1, cathD, and bnch—all cause the accumulation of abnormal autofluorescent storage material suggests that at least some consequences of perturbation to lysosomal function are conserved between flies and humans. Storage material accumulation alone, however, is clearly not sufficient to cause neurodegeneration, as evidenced by the lack of neurodegeneration and minimal cell death in Ppt1 and cathD mutants, respectively (this report; MYLLYKANGAS et al. 2005). A lack of correlation between the severity of storage material accumulation and the degree of neurodegeneration has also been noted in human NCL disease and has been used to suggest that accumulation of storage material and neurodegeneration are separate manifestations caused by the loss of particular CLN genes (see discussion in OSWALD et al. 2005).

A primary focus for future studies will be the identification of genes that can dominantly modify the phenotypes produced by Ppt1 mutations. Identification of second-site modifier genes may provide insight into the normal functions of Ppt1, as well as into how the loss of those functions could contribute to disease. In addition, determination of whether such loci also modify phenotypes caused by mutations in cathD or bnch will help us to differentiate the modifiers specific for Ppt1-related functions from the modifiers that affect phenotypes common to lysosomal storage disease mutations in general.

We acknowledge technical support by the Wadsworth Center's Electron Microscopy and DAI Light Microscopy Core Facilities, and we thank the Bloomington Drosophila Stock Center for fly stocks and the anonymous reviewers for helpful comments. This work was supported by National Institutes of Health grants NS44572 to R.L.G. and NS33648 to M.E.M.

¹ Present address: Albany Medical College, Albany, NY 12208.

² Present address: Department of Biology, College of Charleston, Charleston, SC 29424.

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Communicating editor: T. H. EICKBUSH

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