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Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses
www.elsevier.com/locate/ynbdi Neurobiology of Disease 19 (2005) 194 – 199
Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses Liisa Myllykangas,a Jaana Tyynel7,b,d Andrea Page-McCaw,c,1 Gerald M. Rubin,c Matti J. Haltia,d and Mel B. Feanya,T a
Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, NRB Room 652, Boston, MA 02115, USA b Institute of Biomedicine and Neuroscience Research Program, University of Helsinki, Helsinki, Finland c Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA d Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland Received 9 June 2004; revised 10 December 2004; accepted 22 December 2004 Available online 19 February 2005 Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest agedependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs. D 2005 Elsevier Inc. All rights reserved. Keywords: Neuronal ceroid lipofuscinoses; Drosophila; Cathepsin D; Neurodegeneration; Autofluorescence; Granular osmiophilic deposits
Introduction Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited lysosomal storage disorders characterized by accumuT Corresponding author. Fax +1 617 525 4422. E-mail address: [email protected] (M.B. Feany). 1 Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, USA. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.12.019
lation of autofluorescent storage material, particularly in neurons, and neurodegeneration. Their incidence in the United States has been estimated at 1:12,500. As a group, they represent the most common progressive neurodegenerative diseases of childhood. The human NCLs are classified into eight different forms (designated CLN1 through CLN8) based on recent molecular genetic findings. Six human genes that carry recessive mutations leading to NCLs have been identified: CLN1 and CLN2 encode the lysosomal enzymes palmitoyl protein thioesterase I (PPT1) and tripeptidyl peptidase-I, whereas CLN3, CLN5, CLN6, and CLN8 encode putative membrane proteins of unknown function (Haltia, 2003; Wisniewski et al., 2001). In addition to human forms of NCL, a congenital ovine NCL form has been described, caused by a single nucleotide mutation in the active site of a lysosomal aspartyl protease cathepsin D. This mutation leads to the production of enzymatically inactive protein and results in typical NCL pathology (Tyynela et al., 2000). Cathepsin D knockout mice also show neuronal accumulation of autofluorescent ceroid lipofuscin accompanied by neurodegeneration in retina and central nervous system (Koike et al., 2000, 2003; Nakanishi et al., 2001), further confirming that loss of normal function of cathepsin D results in NCL pathology. Modeling diseases in lower organisms provides powerful genetic tools to study their pathogenesis. In particular, the fruit fly Drosophila melanogaster has a highly developed nervous system and could thus be a suitable organism for modeling typical features of NCLs. We report here that cathepsin D mutant flies exhibit the key features related to NCL pathology: progressive accumulation of autofluorescent storage material in neurons and modest neurodegeneration. Thus, the pathogenic pathway leading to NCL appears to be highly conserved during evolution, and cathepsin D mutant flies can be exploited as a Drosophila model of NCL.
L. Myllykangas et al. / Neurobiology of Disease 19 (2005) 194–199
Methods Drosophila lines and genetics The cathD EP2151 EP element insertion line was used to generate the imprecise excision allele cathD 1 by mobilization with trans-
195
posase (Engels et al., 1990). An imprecise excision of approximately 1 kb in the 5Vend of cathepsin D was identified by PCR on pooled lines and confirmed by sequencing. A precise excision of cathD EP2151 was used as a control. Both cathD 1 and the precise excision of cathD EP2151 were crossed to deficiency line Df(2R)CA53 to confirm the results obtained from the homozygous
Fig. 1. Amino acid sequence of cathepsin D and generation of cathD 1 mutant flies. (A) Sequence alignment of fly, mouse, ovine, and human cathepsin D. Identical amino acids are labeled in gray. The high degree of conservation between different species is shown. (B) Genomic structure of the Drosophila cathD locus. The insertion site of cathD EP2151 in the 5Vuntranslated region and the breakpoints of the cathD 1 deletion are indicated. Coding regions are shown in black boxes. (C) RT-PCR products amplified from RNA isolated from different Drosophila lines. Lane 1: cathD EP2151 precise excision line (control) heads; lane 2: cathD EP2151 precise excision line (control) bodies; lane 3: cathD 1 heads; lane 4: cathD 1 bodies. cathD transcript is present in flies containing intact cathepsin D gene (lanes 1–2), whereas there is no transcript in flies bearing the cathD 1 deletion (3–4). (D) Quantitative analysis of cathepsin D RT-PCR products in different fly lines. Quantification confirms that cathD 1 flies do not contain detectable amounts of cathD transcript. Columns show average product of three independent PCR reactions compared to w 1118 control flies, which represent 100%. The scale is logarithmic. The error bars represent the standard error of the mean.
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cathD 1 and cathD EP2151 precise excision lines. All Drosophila were maintained and aged at 258C.
Results Cathepsin D is conserved
RNA isolation and RT-PCR RNA was isolated from cathD 1 flies and controls and the level of transcription of cathepsin D determined by quantitative RT-PCR. Briefly, a total of 200–300 flies were collected after eclosion, stored at 808C, and rapidly decapitated by vortexing on dry ice for 8 s. Heads and bodies were separated with a sieve of appropriate pore size. Trizol solution was used to isolate RNA according to manufacturer’s protocol. Real-time PCR was performed as described by Scherzer et al. (2003) by using primers 5V-TCCACGGATACGGTGTCCAT-3V and 5VCTGAGCGCCTCAGCGAAT-3V. Sectioning, staining, and microscopy Histological sections were prepared from paraffin-embedded material. Flies were fixed in 4% formaldehyde, dehydrated, and infiltrated with paraffin. 4 Am sections were cut and stained with standard hematoxylin and eosin, periodic acid Schiff (PAS), or luxol fast blue (LFB) stains. Autofluorescence was detected in unstained sections. TUNEL staining was performed using a commercial reagent kit (Oncogenek). For quantification, TUNEL-positive nuclei in at least 8 hemibrains were counted per genotype. Standard transmission electron microscopy was performed on brains of 45-day-old flies, after fixing the dissected brains in 2.5% glutaraldehyde overnight.
The Drosophila homolog of cathepsin D (CG1548; cathD) is located in the 43E18 region of chromosome 2R and is predicted to encode a polypeptide of 392 amino acids. The predicted protein exhibits 50% amino acid identity and 65% similarity with human cathepsin D and 47% amino acid identity and 62% similarity to sheep cathepsin D. Although other proteins homologous to mammalian cathepsin D are present in the fly genome, CG1548 is most homologous to mammalian cathepsin D. Fig. 1A shows the high degree of conservation of cathepsin D in different species. Generation of cathepsin D mutant flies The EP element insertion line cathD EP2151 line bears an EP element located 77 bp upstream from the start codon of Drosophila cathD. To create a disruption of cathD, we mobilized this element and selected for imprecise excision events that deleted DNA sequence close to the insertion. A line that bears a deletion of 916 bp in the 5Vend of the Drosophila cathD gene, extending from the EP-element to the nucleotide 847 of the coding sequence was recovered and is designated cathD 1 . The deletion line lacks over 70% of the coding sequence including the start codon (Fig. 1B) and both protease active sites. A chromosome bearing a precise excision of cathD EP2151 was used as a control in our studies. The breakpoints of the deletion and the normal sequence of the precise excision were confirmed by DNA sequencing.
Fig. 2. (A) Progressive accumulation of autofluorescent storage material in the brains of cathD 1 flies. Brain sections from the medullary cortex of cathD 1 (top) and precise excision controls (bottom) are shown at different ages as indicated. Autofluorescent storage material (arrowheads) accumulates progressively with age in the cortex of cathD 1 flies (arrowheads). Scale bar is 5 Am. (B) Autofluorescent storage material is visualized with both FITC and rhodamine filter sets (arrowheads) in brain sections of 45 day old flies. Scale bar is 5 Am.
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Fig. 3. Histochemical PAS staining of paraffin-embedded brain sections. The storage material has histochemical characteristics typical of neuronal ceroid lipofuscin, showing positive staining with PAS (A, arrow) in brain sections of 45 day old flies bearing the cathD 1 mutation over the deficiency Df(2R)CA53. The brain section of a wild-type fly (B, arrowhead indicates neuronal cytoplasm) does not show positive staining. Scale bar is 5 Am.
To confirm the absence of cathD transcript in the imprecise excision strain, we performed quantitative real-time PCR using Drosophila cathD-specific primers. Real-time PCR of RNA extracted from control (precise excision of cathD EP2151 ) line showed cathD transcript both in the heads and bodies of these flies, in similar quantities to those seen in the control w 1118 line. Amplification of RNA isolated from bodies or heads of flies bearing the deletion resulted in no detectable PCR product, confirming the lack of cathD transcript (Figs. 1C and D). Cathepsin D mutants are viable cathD 1 flies develop and eclose normally, and are fertile and viable. Lifespan assays did not reveal a substantial difference between cathD 1 mutant flies and controls. Cathepsin D mutant flies show progressive neuronal accumulation of storage material To examine effects of cathD deficiency in the brain, sections were prepared from the heads of flies aged 1, 15, 30, and 45 days. Standard hematoxylin-and-eosin-stained sections showed appropriate development and normal structure of major brain areas. However, examination of the brain sections of the cathD 1 flies under fluorescence revealed progressive accumulation of autofluorescent storage material in neurons. Autofluorescent storage material was difficult to detect at day 1 and was most abundant in the oldest flies examined (45 days) (Fig. 2A, arrowheads). Storage
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material was found throughout the cortex, mostly in the neuronal cell bodies, but it was also present in the neuropil. Control flies did not show significant autofluorescence, even at 45 days (Fig. 2A). Flies bearing the cathD 1 allele over a deficiency chromosome Df(2R)CA53 showed similar accumulation of storage material. The fluorescence of the storage material was observed using wide range of wavelengths, similar to other cathepsin D-deficient animals (Fig. 2B, arrowheads). PAS stain of the brain sections revealed that the storage material was PAS positive (Fig. 3, arrow), thus resembling the storage material found in mammalian forms of NCLs. Similar PAS-positive material was not present in controls (Fig. 3, arrowhead). Storage material was also positive with the luxol fast blue stain (data not shown). In addition to neurons, storage material was found in some other tissues, including the fat body and intestine (data not shown). Storage material was not observed in cathD 1 /w 1118 heterozygotes. To characterize the ultrastructure of the storage material, we next performed electron-microscopic examination of the brains of the cathD 1 flies and controls. Cortical neurons possessed numerous electron-dense granular inclusions in their cell bodies. The accumulated material was membrane bound and formed globular round structures (Figs. 4A and B). The storage material was finely granular with some lamellar elements (Fig. 4C). No clear fingerprint or curvilinear bodies were observed. Thus the findings closely resembled the granular osmiophilic deposits found in the human infantile and ovine congenital forms of NCL (Haltia, 2003). No storage material was seen in precise excision control flies of the same age. Cathepsin D mutants show modest neurodegeneration We next focused our analysis on possible signs of neurodegeneration in the brains of cathD 1 flies. No obvious cell loss was observed in the hematoxylin and eosin stained sections. As neuronal cell death in NCLs may occur by an apoptotic mechanism (Koike et al., 2003), we performed TUNEL staining. Significant numbers of TUNEL-positive nuclei were present in the brains of 45-day old cathD 1 over Df(2R)CA53 flies (Fig. 5A, arrowheads), whereas aged controls showed only rare TUNEL-positive nuclei (Figs. 5B and C). TUNEL-positive neurons were observed throughout the central body, but particularly in the optic medulla and lamina (Fig. 5A). Similar results were obtained with homozygous cathD 1 flies. Examination of younger cathD mutant flies (1, 15, and 30 days) did not reveal significant numbers of TUNEL-positive nuclei. 45-day-old control flies bearing the
Fig. 4. Electron micrographs showing inclusion bodies in cortical neurons from a 45-day-old cathD 1 fly. (A) A low power view demonstrates the presence of globular osmiophilic storage material (arrow) in the neuronal cytoplasm. N indicates nucleus. Scale bar is 500 nm. (B) At higher magnification membranebound nature of the globular material is apparent (arrow points to a membrane). Scale bar is 200 nm. (C) High power view demonstrates the finely granular ultrastructure of the inclusions, with some lamellar elements. No fingerprint or curvilinear bodies were observed. Scale bar is 50 nm.
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Fig. 5. TUNEL-positive neurons indicating apoptotic cell death in the brains of cathD-deficient flies. Several TUNEL-positive cells (arrowheads) are present in the optic lobes and lamina of 45-day old cathD 1 /Df(2R)CA53 flies (A), but not the controls (B). r, retina; l, lamina; m, medulla. Scale bar is 15 Am. (C) Quantification of TUNEL-positive neurons per hemibrain. The number of TUNEL-positive neurons is markedly increased in the 45-day-old cathepsin Ddeficient flies. Error bars represent the standard error of the mean.
cathD EP2151 precise excision over Df(2R)CA53 also showed very few TUNEL-positive cells.
Discussion Cathepsin D mutant sheep exhibit extreme NCL pathology including characteristic neuronal storage material and severe brain atrophy at birth (Tyynela et al., 2000). Cathepsin D knockout mice also show the characteristic neuronal storage material but relatively modest neuronal loss in the neocortex (Koike et al., 2000, 2003; Nakanishi et al., 2001). Here we report that cathepsin D deficiency induces the key features of NCL pathology in an invertebrate, Drosophila. The ultrastructure of the storage material found in fly neurons closely mimics the morphological features of the membrane-bound granular osmiophilic deposits found in the brains of the cathepsin D-deficient sheep and the human infantile NCL patients. The staining properties and the strong autofluorescence of the storage material, as well as the progressive nature of the storage deposition, further emphasize similarities between the cathepsin D mutant fly and established forms of NCLs. Furthermore, increased number of TUNEL-positive cells in the brains of aged cathepsin D mutant flies suggests that age-related neurodegeneration occurs in the central nervous system of these flies. Despite the increased TUNEL staining, marked neuronal loss was not evident in aged cathepsin D-deficient flies. More dramatic degrees of cell loss have been documented in other Drosophila models of neurodegenerative disease and are typically seen as decreased numbers of neurons in the cellular cortical layer accompanied by neuropil vacuolization (Ghosh and Feany, 2004; Wittmann et al., 2001). Similar changes were not observed in cathD mutant flies, suggesting that the degree of neurodegeneration was modest. We observed TUNEL-positive cells only in very aged cathD mutant flies. Thus, it is possible that more marked neurodegenerative changes and neuronal loss would have been seen had the lifespan of the flies been longer. The relative preponderance of neurodegenerative changes in the visual system is intriguing since the visual system is particularly sensitive in many NCL forms, including the cathepsin D-deficient mice (Koike et al., 2003).
In addition to cathepsin D, Drosophila has homologs for two other NCL genes: PPT1 (CLN1) and CLN3. Recent studies have shown that the Drosophila PPT1 has enzymatic activity (Glaser et al., 2003), and that overexpression of dPPT1 in fly eye results in retinal cell death that occurs through an apoptotic mechanism (Korey and MacDonald, 2003). These findings indicate that pathways involved in human NCLs also exist in flies and thus Drosophila might be a suitable organism to investigate these pathways. Several neurodegenerative diseases have now been modeled in Drosophila, including Parkinson’s disease, the tauopathies and polyglutamine disorders (Feany and Bender, 2000; FernandezFunez et al., 2000; Jackson et al., 1998; Warrick et al., 1998; Wittmann et al., 2001). These models have proven their power especially in genetic modifier screens, which have helped delineate cellular pathways mediating neurodegeneration in these diseases (Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Shulman and Feany, 2003). Our results suggest that a genetic approach can now be applied to the study of NCL disorders by utilizing the cathepsin D mutant flies. Large-scale drug screens and candidate gene testing are other potentially fruitful approaches that a Drosophila model of NCL provides. In summary, we have created a Drosophila model of NCL that recapitulates key pathologic features relevant to all NCLs: progressive accumulation of autofluorescent storage material in neurons and modest neurodegeneration. Thus, the metabolic pathways leading to NCLs appear to be highly conserved during evolution, and Drosophila can be exploited in studies on the pathogenesis of these diseases. Particularly, the cathepsin D mutant Drosophila may be used to elucidate the molecular mechanisms by which cathepsin D deficiency leads to neuronal degeneration.
Acknowledgments We are indebted to E. Harwood, L.J. Morse, M. Ericsson, and L. Trakimas for excellent technical assistance and C. Scherzer and other Feany lab members for discussions and support. We would also like to acknowledge the Bloomington Drosophila Stock Center for providing stocks. This work was supported by grants from NIH (AG00880 and AG019790 to MBF), Academy of
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Finland (200406 to LM and 1207016 to JT), Finnish Cultural Foundation, and Maud Kuistila Memorial Foundation.
References Engels, W.R., Johnson-Schlitz, D.M., Eggleston, W.B., Sved, J., 1990. High-frequency P element loss in Drosophila is homolog dependent. Cell 62, 515 – 525. Feany, M.B., Bender, W.W., 2000. A Drosophila model of Parkinson’s disease. Nature 404, 394 – 398. Fernandez-Funez, P., Nino-Rosales, M.L., de Gouyon, B., She, W.C., Luchak, J.M., Martinez, P., Turiegano, E., Benito, J., Capovilla, M., Skinner, P.J., McCall, A., Canal, I., Orr, H.T., Zoghbi, H.Y., Botas, J., 2000. Identification of genes that modify ataxin-1-induced neurodegeneration. Nature 408, 101 – 106. Ghosh, S., Feany, M.B., 2004. Comparison of pathways controlling toxicity in the eye and brain in Drosophila models of human neurodegenerative diseases. Hum. Mol. Genet. 13, 2011 – 2018. Glaser, R.L., Hickey, A.J., Chotkowski, H.L., Chu-LaGraff, Q., 2003. Characterization of Drosophila palmitoyl–protein thioesterase 1. Gene 312, 271 – 279. Haltia, M., 2003. The neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol. 62, 1 – 13. Jackson, G.R., Salecker, I., Dong, X., Yao, X., Arnheim, N., Faber, P.W., MacDonald, M.E., Zipursky, S.L., 1998. Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron 21, 633 – 642. Kazemi-Esfarjani, P., Benzer, S., 2000. Genetic suppression of polyglutamine toxicity in Drosophila. Science 287, 1837 – 1840. Koike, M., Nakanishi, H., Saftig, P., Ezaki, J., Isahara, K., Ohsawa, Y., Schulz-Schaeffer, W., Watanabe, T., Waguri, S., Kametaka, S., Shibata, M., Yamamoto, K., Kominami, E., Peters, C., von Figura, K., Uchiyama, Y., 2000. Cathepsin D deficiency induces lysosomal
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storage with ceroid lipofuscin in mouse CNS neurons. J. Neurosci. 20, 6898 – 6906. Koike, M., Shibata, M., Ohsawa, Y., Nakanishi, H., Koga, T., Kametaka, S., Waguri, S., Momoi, T., Kominami, E., Peters, C., Figura, K., Saftig, P., Uchiyama, Y., 2003. Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice. Mol. Cell. Neurosci. 22, 146 – 161. Korey, C.A., MacDonald, M.E., 2003. An over-expression system for characterizing Ppt1 function in Drosophila. BMC Neurosci. 4, 30. Nakanishi, H., Zhang, J., Koike, M., Nishioku, T., Okamoto, Y., Kominami, E., von Figura, K., Peters, C., Yamamoto, K., Saftig, P., Uchiyama, Y., 2001. Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice. J. Neurosci. 21, 7526 – 7533. Scherzer, C.R., Jensen, R.V., Gullans, S.R., Feany, M.B., 2003. Gene expression changes presage neurodegeneration in a Drosophila model of Parkinson’s disease. Hum. Mol. Genet. 12, 2457 – 2466. Shulman, J.M., Feany, M.B., 2003. Genetic modifiers of tauopathy in Drosophila. Genetics 165, 1233 – 1242. Tyynela, J., Sohar, I., Sleat, D.E., Gin, R.M., Donnelly, R.J., Baumann, M., Haltia, M., Lobel, P., 2000. A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration. EMBO J. 19, 2786 – 2792. Warrick, J.M., Paulson, H.L., Gray-Board, G.L., Bui, Q.T., Fischbeck, K.H., Pittman, R.N., Bonini, N.M., 1998. Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell 93, 939 – 949. Wisniewski, K.E., Kida, E., Golabek, A.A., Kaczmarski, A., Connell, F., Zhong, N., 2001. Neuronal ceroid lipofuscinoses: classification and diagnosis. In: Wisniewski, K.E., Zhong, N. (Eds.), Batten Disease: Diagnosis, Treatment, and Research. Academic Press, San Diego, pp. 1 – 34. Wittmann, C.W., Wszolek, M.F., Shulman, J.M., Salvaterra, P.M., Lewis, J., Hutton, M., Feany, M.B., 2001. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293, 711 – 714.
Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses Liisa Myllykangas,a Jaana Tyynel7,b,d Andrea Page-McCaw,c,1 Gerald M. Rubin,c Matti J. Haltia,d and Mel B. Feanya,T a
Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, NRB Room 652, Boston, MA 02115, USA b Institute of Biomedicine and Neuroscience Research Program, University of Helsinki, Helsinki, Finland c Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, CA 94720, USA d Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland Received 9 June 2004; revised 10 December 2004; accepted 22 December 2004 Available online 19 February 2005 Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest agedependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs. D 2005 Elsevier Inc. All rights reserved. Keywords: Neuronal ceroid lipofuscinoses; Drosophila; Cathepsin D; Neurodegeneration; Autofluorescence; Granular osmiophilic deposits
Introduction Neuronal ceroid lipofuscinoses (NCLs) are a group of inherited lysosomal storage disorders characterized by accumuT Corresponding author. Fax +1 617 525 4422. E-mail address: [email protected] (M.B. Feany). 1 Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180, USA. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.12.019
lation of autofluorescent storage material, particularly in neurons, and neurodegeneration. Their incidence in the United States has been estimated at 1:12,500. As a group, they represent the most common progressive neurodegenerative diseases of childhood. The human NCLs are classified into eight different forms (designated CLN1 through CLN8) based on recent molecular genetic findings. Six human genes that carry recessive mutations leading to NCLs have been identified: CLN1 and CLN2 encode the lysosomal enzymes palmitoyl protein thioesterase I (PPT1) and tripeptidyl peptidase-I, whereas CLN3, CLN5, CLN6, and CLN8 encode putative membrane proteins of unknown function (Haltia, 2003; Wisniewski et al., 2001). In addition to human forms of NCL, a congenital ovine NCL form has been described, caused by a single nucleotide mutation in the active site of a lysosomal aspartyl protease cathepsin D. This mutation leads to the production of enzymatically inactive protein and results in typical NCL pathology (Tyynela et al., 2000). Cathepsin D knockout mice also show neuronal accumulation of autofluorescent ceroid lipofuscin accompanied by neurodegeneration in retina and central nervous system (Koike et al., 2000, 2003; Nakanishi et al., 2001), further confirming that loss of normal function of cathepsin D results in NCL pathology. Modeling diseases in lower organisms provides powerful genetic tools to study their pathogenesis. In particular, the fruit fly Drosophila melanogaster has a highly developed nervous system and could thus be a suitable organism for modeling typical features of NCLs. We report here that cathepsin D mutant flies exhibit the key features related to NCL pathology: progressive accumulation of autofluorescent storage material in neurons and modest neurodegeneration. Thus, the pathogenic pathway leading to NCL appears to be highly conserved during evolution, and cathepsin D mutant flies can be exploited as a Drosophila model of NCL.
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Methods Drosophila lines and genetics The cathD EP2151 EP element insertion line was used to generate the imprecise excision allele cathD 1 by mobilization with trans-
195
posase (Engels et al., 1990). An imprecise excision of approximately 1 kb in the 5Vend of cathepsin D was identified by PCR on pooled lines and confirmed by sequencing. A precise excision of cathD EP2151 was used as a control. Both cathD 1 and the precise excision of cathD EP2151 were crossed to deficiency line Df(2R)CA53 to confirm the results obtained from the homozygous
Fig. 1. Amino acid sequence of cathepsin D and generation of cathD 1 mutant flies. (A) Sequence alignment of fly, mouse, ovine, and human cathepsin D. Identical amino acids are labeled in gray. The high degree of conservation between different species is shown. (B) Genomic structure of the Drosophila cathD locus. The insertion site of cathD EP2151 in the 5Vuntranslated region and the breakpoints of the cathD 1 deletion are indicated. Coding regions are shown in black boxes. (C) RT-PCR products amplified from RNA isolated from different Drosophila lines. Lane 1: cathD EP2151 precise excision line (control) heads; lane 2: cathD EP2151 precise excision line (control) bodies; lane 3: cathD 1 heads; lane 4: cathD 1 bodies. cathD transcript is present in flies containing intact cathepsin D gene (lanes 1–2), whereas there is no transcript in flies bearing the cathD 1 deletion (3–4). (D) Quantitative analysis of cathepsin D RT-PCR products in different fly lines. Quantification confirms that cathD 1 flies do not contain detectable amounts of cathD transcript. Columns show average product of three independent PCR reactions compared to w 1118 control flies, which represent 100%. The scale is logarithmic. The error bars represent the standard error of the mean.
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cathD 1 and cathD EP2151 precise excision lines. All Drosophila were maintained and aged at 258C.
Results Cathepsin D is conserved
RNA isolation and RT-PCR RNA was isolated from cathD 1 flies and controls and the level of transcription of cathepsin D determined by quantitative RT-PCR. Briefly, a total of 200–300 flies were collected after eclosion, stored at 808C, and rapidly decapitated by vortexing on dry ice for 8 s. Heads and bodies were separated with a sieve of appropriate pore size. Trizol solution was used to isolate RNA according to manufacturer’s protocol. Real-time PCR was performed as described by Scherzer et al. (2003) by using primers 5V-TCCACGGATACGGTGTCCAT-3V and 5VCTGAGCGCCTCAGCGAAT-3V. Sectioning, staining, and microscopy Histological sections were prepared from paraffin-embedded material. Flies were fixed in 4% formaldehyde, dehydrated, and infiltrated with paraffin. 4 Am sections were cut and stained with standard hematoxylin and eosin, periodic acid Schiff (PAS), or luxol fast blue (LFB) stains. Autofluorescence was detected in unstained sections. TUNEL staining was performed using a commercial reagent kit (Oncogenek). For quantification, TUNEL-positive nuclei in at least 8 hemibrains were counted per genotype. Standard transmission electron microscopy was performed on brains of 45-day-old flies, after fixing the dissected brains in 2.5% glutaraldehyde overnight.
The Drosophila homolog of cathepsin D (CG1548; cathD) is located in the 43E18 region of chromosome 2R and is predicted to encode a polypeptide of 392 amino acids. The predicted protein exhibits 50% amino acid identity and 65% similarity with human cathepsin D and 47% amino acid identity and 62% similarity to sheep cathepsin D. Although other proteins homologous to mammalian cathepsin D are present in the fly genome, CG1548 is most homologous to mammalian cathepsin D. Fig. 1A shows the high degree of conservation of cathepsin D in different species. Generation of cathepsin D mutant flies The EP element insertion line cathD EP2151 line bears an EP element located 77 bp upstream from the start codon of Drosophila cathD. To create a disruption of cathD, we mobilized this element and selected for imprecise excision events that deleted DNA sequence close to the insertion. A line that bears a deletion of 916 bp in the 5Vend of the Drosophila cathD gene, extending from the EP-element to the nucleotide 847 of the coding sequence was recovered and is designated cathD 1 . The deletion line lacks over 70% of the coding sequence including the start codon (Fig. 1B) and both protease active sites. A chromosome bearing a precise excision of cathD EP2151 was used as a control in our studies. The breakpoints of the deletion and the normal sequence of the precise excision were confirmed by DNA sequencing.
Fig. 2. (A) Progressive accumulation of autofluorescent storage material in the brains of cathD 1 flies. Brain sections from the medullary cortex of cathD 1 (top) and precise excision controls (bottom) are shown at different ages as indicated. Autofluorescent storage material (arrowheads) accumulates progressively with age in the cortex of cathD 1 flies (arrowheads). Scale bar is 5 Am. (B) Autofluorescent storage material is visualized with both FITC and rhodamine filter sets (arrowheads) in brain sections of 45 day old flies. Scale bar is 5 Am.
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Fig. 3. Histochemical PAS staining of paraffin-embedded brain sections. The storage material has histochemical characteristics typical of neuronal ceroid lipofuscin, showing positive staining with PAS (A, arrow) in brain sections of 45 day old flies bearing the cathD 1 mutation over the deficiency Df(2R)CA53. The brain section of a wild-type fly (B, arrowhead indicates neuronal cytoplasm) does not show positive staining. Scale bar is 5 Am.
To confirm the absence of cathD transcript in the imprecise excision strain, we performed quantitative real-time PCR using Drosophila cathD-specific primers. Real-time PCR of RNA extracted from control (precise excision of cathD EP2151 ) line showed cathD transcript both in the heads and bodies of these flies, in similar quantities to those seen in the control w 1118 line. Amplification of RNA isolated from bodies or heads of flies bearing the deletion resulted in no detectable PCR product, confirming the lack of cathD transcript (Figs. 1C and D). Cathepsin D mutants are viable cathD 1 flies develop and eclose normally, and are fertile and viable. Lifespan assays did not reveal a substantial difference between cathD 1 mutant flies and controls. Cathepsin D mutant flies show progressive neuronal accumulation of storage material To examine effects of cathD deficiency in the brain, sections were prepared from the heads of flies aged 1, 15, 30, and 45 days. Standard hematoxylin-and-eosin-stained sections showed appropriate development and normal structure of major brain areas. However, examination of the brain sections of the cathD 1 flies under fluorescence revealed progressive accumulation of autofluorescent storage material in neurons. Autofluorescent storage material was difficult to detect at day 1 and was most abundant in the oldest flies examined (45 days) (Fig. 2A, arrowheads). Storage
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material was found throughout the cortex, mostly in the neuronal cell bodies, but it was also present in the neuropil. Control flies did not show significant autofluorescence, even at 45 days (Fig. 2A). Flies bearing the cathD 1 allele over a deficiency chromosome Df(2R)CA53 showed similar accumulation of storage material. The fluorescence of the storage material was observed using wide range of wavelengths, similar to other cathepsin D-deficient animals (Fig. 2B, arrowheads). PAS stain of the brain sections revealed that the storage material was PAS positive (Fig. 3, arrow), thus resembling the storage material found in mammalian forms of NCLs. Similar PAS-positive material was not present in controls (Fig. 3, arrowhead). Storage material was also positive with the luxol fast blue stain (data not shown). In addition to neurons, storage material was found in some other tissues, including the fat body and intestine (data not shown). Storage material was not observed in cathD 1 /w 1118 heterozygotes. To characterize the ultrastructure of the storage material, we next performed electron-microscopic examination of the brains of the cathD 1 flies and controls. Cortical neurons possessed numerous electron-dense granular inclusions in their cell bodies. The accumulated material was membrane bound and formed globular round structures (Figs. 4A and B). The storage material was finely granular with some lamellar elements (Fig. 4C). No clear fingerprint or curvilinear bodies were observed. Thus the findings closely resembled the granular osmiophilic deposits found in the human infantile and ovine congenital forms of NCL (Haltia, 2003). No storage material was seen in precise excision control flies of the same age. Cathepsin D mutants show modest neurodegeneration We next focused our analysis on possible signs of neurodegeneration in the brains of cathD 1 flies. No obvious cell loss was observed in the hematoxylin and eosin stained sections. As neuronal cell death in NCLs may occur by an apoptotic mechanism (Koike et al., 2003), we performed TUNEL staining. Significant numbers of TUNEL-positive nuclei were present in the brains of 45-day old cathD 1 over Df(2R)CA53 flies (Fig. 5A, arrowheads), whereas aged controls showed only rare TUNEL-positive nuclei (Figs. 5B and C). TUNEL-positive neurons were observed throughout the central body, but particularly in the optic medulla and lamina (Fig. 5A). Similar results were obtained with homozygous cathD 1 flies. Examination of younger cathD mutant flies (1, 15, and 30 days) did not reveal significant numbers of TUNEL-positive nuclei. 45-day-old control flies bearing the
Fig. 4. Electron micrographs showing inclusion bodies in cortical neurons from a 45-day-old cathD 1 fly. (A) A low power view demonstrates the presence of globular osmiophilic storage material (arrow) in the neuronal cytoplasm. N indicates nucleus. Scale bar is 500 nm. (B) At higher magnification membranebound nature of the globular material is apparent (arrow points to a membrane). Scale bar is 200 nm. (C) High power view demonstrates the finely granular ultrastructure of the inclusions, with some lamellar elements. No fingerprint or curvilinear bodies were observed. Scale bar is 50 nm.
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Fig. 5. TUNEL-positive neurons indicating apoptotic cell death in the brains of cathD-deficient flies. Several TUNEL-positive cells (arrowheads) are present in the optic lobes and lamina of 45-day old cathD 1 /Df(2R)CA53 flies (A), but not the controls (B). r, retina; l, lamina; m, medulla. Scale bar is 15 Am. (C) Quantification of TUNEL-positive neurons per hemibrain. The number of TUNEL-positive neurons is markedly increased in the 45-day-old cathepsin Ddeficient flies. Error bars represent the standard error of the mean.
cathD EP2151 precise excision over Df(2R)CA53 also showed very few TUNEL-positive cells.
Discussion Cathepsin D mutant sheep exhibit extreme NCL pathology including characteristic neuronal storage material and severe brain atrophy at birth (Tyynela et al., 2000). Cathepsin D knockout mice also show the characteristic neuronal storage material but relatively modest neuronal loss in the neocortex (Koike et al., 2000, 2003; Nakanishi et al., 2001). Here we report that cathepsin D deficiency induces the key features of NCL pathology in an invertebrate, Drosophila. The ultrastructure of the storage material found in fly neurons closely mimics the morphological features of the membrane-bound granular osmiophilic deposits found in the brains of the cathepsin D-deficient sheep and the human infantile NCL patients. The staining properties and the strong autofluorescence of the storage material, as well as the progressive nature of the storage deposition, further emphasize similarities between the cathepsin D mutant fly and established forms of NCLs. Furthermore, increased number of TUNEL-positive cells in the brains of aged cathepsin D mutant flies suggests that age-related neurodegeneration occurs in the central nervous system of these flies. Despite the increased TUNEL staining, marked neuronal loss was not evident in aged cathepsin D-deficient flies. More dramatic degrees of cell loss have been documented in other Drosophila models of neurodegenerative disease and are typically seen as decreased numbers of neurons in the cellular cortical layer accompanied by neuropil vacuolization (Ghosh and Feany, 2004; Wittmann et al., 2001). Similar changes were not observed in cathD mutant flies, suggesting that the degree of neurodegeneration was modest. We observed TUNEL-positive cells only in very aged cathD mutant flies. Thus, it is possible that more marked neurodegenerative changes and neuronal loss would have been seen had the lifespan of the flies been longer. The relative preponderance of neurodegenerative changes in the visual system is intriguing since the visual system is particularly sensitive in many NCL forms, including the cathepsin D-deficient mice (Koike et al., 2003).
In addition to cathepsin D, Drosophila has homologs for two other NCL genes: PPT1 (CLN1) and CLN3. Recent studies have shown that the Drosophila PPT1 has enzymatic activity (Glaser et al., 2003), and that overexpression of dPPT1 in fly eye results in retinal cell death that occurs through an apoptotic mechanism (Korey and MacDonald, 2003). These findings indicate that pathways involved in human NCLs also exist in flies and thus Drosophila might be a suitable organism to investigate these pathways. Several neurodegenerative diseases have now been modeled in Drosophila, including Parkinson’s disease, the tauopathies and polyglutamine disorders (Feany and Bender, 2000; FernandezFunez et al., 2000; Jackson et al., 1998; Warrick et al., 1998; Wittmann et al., 2001). These models have proven their power especially in genetic modifier screens, which have helped delineate cellular pathways mediating neurodegeneration in these diseases (Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Shulman and Feany, 2003). Our results suggest that a genetic approach can now be applied to the study of NCL disorders by utilizing the cathepsin D mutant flies. Large-scale drug screens and candidate gene testing are other potentially fruitful approaches that a Drosophila model of NCL provides. In summary, we have created a Drosophila model of NCL that recapitulates key pathologic features relevant to all NCLs: progressive accumulation of autofluorescent storage material in neurons and modest neurodegeneration. Thus, the metabolic pathways leading to NCLs appear to be highly conserved during evolution, and Drosophila can be exploited in studies on the pathogenesis of these diseases. Particularly, the cathepsin D mutant Drosophila may be used to elucidate the molecular mechanisms by which cathepsin D deficiency leads to neuronal degeneration.
Acknowledgments We are indebted to E. Harwood, L.J. Morse, M. Ericsson, and L. Trakimas for excellent technical assistance and C. Scherzer and other Feany lab members for discussions and support. We would also like to acknowledge the Bloomington Drosophila Stock Center for providing stocks. This work was supported by grants from NIH (AG00880 and AG019790 to MBF), Academy of
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Finland (200406 to LM and 1207016 to JT), Finnish Cultural Foundation, and Maud Kuistila Memorial Foundation.
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