Altered Gene Expression in the Eye of a Mouse Model for Batten Disease

Subrata Chattopadhyay,1 Evan Kingsley,1 Andrew Serour,1 Timothy M. Curran,1 Andrew I. Brooks,2,3 and David A. Pearce1,4,5

PURPOSE. Juvenile neuronal ceroid lipofuscinosis (JNCL or Bat- order is characterized initially by visual deterioration at age 5 to ten Disease) is one of the most common progressive neurode- 7 years that ultimately results in blindness. After the loss of generative disorders of childhood, resulting from autosomal vision, other neurologic characteristics are seizures, mental recessive inheritance of mutations in the CLN3 gene. Patholog- retardation, and loss of motor function.1,2 Batten disease is ically, Batten disease is characterized by lysosomal storage of always fatal. The CLN3 gene responsible for Batten disease was autofluorescent material in all tissue types. Although charac- positionally cloned in 1995,3 with most individuals with the terized by seizures, mental retardation, and loss of motor skills, disease harboring a 1-kb deletion of this gene. The disease is the first presenting symptom of Batten disease is vision loss. characterized by the accumulation of autofluorescent hydro- METHODS. High-density oligonucleotide arrays were used to phobic material in the lysosomes of neurons and other cell profile approximately 19,000 mRNAs in the eye of 10-week-old types. A predominant component of the lysosomal storage material has been identified as mitochondrial ATP synthase Cln3-knockout and normal mice, and the data were compared 4–7 with that for the cerebellum in the same model as a means to subunit c. One of the paradoxes of Batten disease is that the identify gene expression changes that are specific to the eye. accumulation of this lysosomal storage material does not ap- parently lead to disease in non-neuronal cell types. The CLN3 RESULTS. A detailed list was compiled of 285 functionally cate- protein has been localized to late endosomes and lysosomes in gorized genes that have altered expression in the eye of Cln3- non-neuronal cell types, and has been shown to co-localize knockout mice before the appearance of the characteristic with synaptic vesicle proteins in neuronal cell types 8–10.Itis lysosomal storage material. Furthermore, 18 genes were iden- apparent that, because loss of vision is the first presenting tified and 6 validated by semiquantitative RT-PCR that have symptom of Batten disease, the eye is an ideal system for the altered expression in the eye, but not in the cerebellum of study of the primary molecular events associated with the Cln3-knockout mice. The genes that have altered expression CLN3 defect. specific to the eye of the Cln3-knockout mouse may be of Cln3-knockout mice homozygous for a targeted deletion of importance in understanding the function of CLN3 in different exon1to6intheCln3 gene have been reported to show tissues. characteristic accumulation of autofluorescent lipopigments CONCLUSIONS. Downregulation of genes associated with energy containing mitochondrial ATP synthase subunit c in neural production in the mitochondria appears to be specific to the tissue and selective loss of ␥-aminobutyric acid (GABA)ergic eye. The CLN3 defect may result in altered mitochondrial neurons.11 We have previously reported gene expression function in eye but not other tissue. More detailed experimen- changes in the cerebellum of 10-week-old Cln3-knockout tation is needed to understand the contribution of these mice.12,13 To gain insight into gene expression changes that changes in expression to disease state, and whether these associate with the CLN3 defect, we repeated our gene expres- changes are specific for certain cell types within the eye. sion study in the whole eye of the Cln3-knockout mouse. We (Invest Ophthalmol Vis Sci. 2004;45:2893–2905) DOI: classified genes displaying an altered expression pattern into 10.1167/iovs.04-0143 13 functional categories based on functional information associated with the gene product. The altered gene expression atten disease or JNCL, is the juvenile form of neuronal pattern in Cln3-knockout eye shows that there are 285 genes Bceroid lipofuscinosis (NCL). Batten disease is inherited as that are either up- or downregulated. By comparing these gene an autosomal recessive condition and is the most common expression data with those previously reported in the cerebel- progressive neurodegenerative disease of childhood. The dis- lum we were are able to identify 18 genes with a change in expression that is specific to the eye. This data set provides an important comparative analysis of the CLN3 defect. From the 1Center for Aging and Developmental Biology, Aab Institute of Biomedical Sciences; the Departments of 3Environmental Medicine, 4Biochemistry and Biophysics, and 5Neurology; and the MATERIALS AND METHODS 2Center for Functional Genomics, University of Rochester School of Medicine and Dentistry, University of Rochester, Rochester, New York. Animals Supported by NIH NS40580, the EJLB Foundation, a Herbert H. Ten-week-old wild-type control 129S6/SvEv and homozygous Cln3- DeGraff Batten disease research grant, and the JNCL Research Fund. knockout mice on a 129S6/SvEv background11 were used in the study. Submitted for publication February 11, 2004; revised April 9 and All procedures were performed in accordance with NIH guidelines and May 13, 2004; accepted June 2, 2004. Disclosure: S. Chattopadhyay, None; E. Kingsley, None; A. University of Rochester Animal Care and Use Committee Guidelines. Serour, None; T.M. Curran, None; A.I. Brooks, None; D.A. Pearce, Furthermore, all research conformed to ARVO Standards for the Use of None Animals in Ophthalmic and Vision Research. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- Gene Expression Studies and Data Analysis ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Corresponding author: David A. Pearce, Center for Aging and For comparative gene expression studies whole eyes from three each Developmental Biology, Department of Biochemistry and Biophysics, of the 10-week-old wild-type control and Cln3-knockout mice were Box 645, University of Rochester, School of Medicine and Dentistry, pooled and homogenized by standard procedures (TRIzol; Invitrogen- Rochester, NY 14642; [email protected]. Gibco, Grand Island, NY) for mRNA extraction. Total RNA (10 ␮g)

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TABLE 1. Primers Used for RT-PCR Validation of Expression Levels cleared with xylenes and mounted (Permount; Fisher Scientific, Pitts- burgh, PA). Digital images were taken with a camera (Spot camera; Transcript Primers Diagnostic Instruments, Sterling Heights, MI) mounted on a micro- scope (Olympus Corp. of America, Lake Success, NY). A 4ϫ objective 5Ј-GCTCCCTAGGCCCCTCCTG-3Ј GAPDH captured the whole eye, and the 40ϫ objective was used to obtain 5Ј-CAAGAAGGTGGTGAAGCAGGCCAC-3Ј Glutaminase C 5Ј-TAACTGCTAGTATCTGTGCA-3Ј detailed images of the peripheral retina. Fluorescence images were 5Ј-AAGAGATAAAGGGTATGTT-3Ј taken through a 488-nm filter. Images were cropped and background Lipidosin 5Ј-GAAGCTCGGCCTAGAGCGTG-3Ј removed (Photoshop; Adobe Systems, Mountain View, CA). 5Ј-TATGAGCTCCTCCATCGTGT-3Ј ELF41A 5Ј-TTATATGGGAGCAACTTGTC-3Ј 5Ј-TTGGTCACTTCTAGCACATC-3Ј RESULTS NEDF 5Ј-AGTGAAAGATGCAGCCAAGA-3Ј 5Ј-ATCGCTCCTGCGGGTTCA-3Ј Batten disease is a lysosomal storage disease with accumulation TC36735 5Ј-GAAACTTGTCTAATACCAG-3Ј of autofluorescent lipopigment in the lysosomes of individuals 5Ј-GACAGTTGTCACCATGAGA-3Ј with the disorder. Homozygous Cln3-knockout mice have ATP-synthase subunit B 5Ј-TATGTGCTTGGAACTGGACT-3Ј been confirmed to have similar accumulation within the brain 5Ј-CACTAAGTGGACCTTGATCT-3Ј and eye.11,14,15 As the eye is one of the first regions of the central nervous system (CNS) to deteriorate in Batten disease, we compared gene expression in the eye of 10-week-old nor- from each sample was used to generate a high-fidelity cDNA, which is mal and Cln3-knockout mice. In this initial study we took the modified at the 3Ј end to contain an initiation site for T7 RNA poly- whole eye from Cln3-knockout and wild-type control mice and merase, as per the manufacturer’s protocol (SuperChoice; Invitrogen- examined gene expression changes associated with the CLN3 Gibco). On completion of cDNA synthesis, 1 ␮g of product was used defect. To maximize interpretation of data obtained on in an in vitro transcription (IVT) reaction that contained biotinylated changes in gene expression that are associated with the CLN3 UTP and CTP, which will be used for detection after hybridization to defect, we compared the data obtained from this study to those the microarray as per the manufacturer’s protocol (Enzo Biochemicals, in a previous experiment, in which we compared gene expres- Farmingdale, NY). Full-length IVT product (20 ␮g) was subsequently sion changes in cerebellum of 10-week-old normal and Cln3- fragmented in 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, and 150 knockout mice. mM MgOAc at 94°C for 35 minutes. After fragmentation, all compo- nents generated throughout the processing procedure (cDNA, full- Characteristics of 10-Week-Old length cRNA, and fragmented cRNA) were analyzed by gel electro- Cln3-Knockout Eyes phoresis to assess the appropriate size distribution before microarray It has been shown that cln3-knockout mice demonstrate the analysis. characteristic presence of autofluorescent storage material in All samples represented were subjected to gene expression analysis the retina at 12 months of age.15 Although wild-type control using the a high-density oligonucleotide array set (Mu19K; Affymetrix, animals also demonstrate the presence of storage material, Santa Clara, CA), at the University of Rochester Microarray Core Facil- there is a clear elevation in the cln3-knockout retina. We 12 ity, as previously described. The mathematical definitions for the considered that an ideal time to perform gene expression algorithms can be found in the Microarray Suite Analysis manual in the studies in the eye of cln3-knockout mice would be at a point algorithm tutorial. The change ratio of expression of any transcript when this pathologically characteristic storage material first between baseline and experimental is calculated after global scaling. appeared in cln3-knockout, but not in wild-type, animals. Fig- All data represented from this first approach are from pair-wise com- ure 1a shows that the retina of 10-week-old cln3-knockout parison analyses. mice seemed to be normal and resembled that of an age- matched wild-type control. The thickness of the each cell layer Reverse Transcription–Polymerase appeared to be the same, with no obvious cell loss. In Figure Chain Reaction 1b, autofluorescent storage material was beginning to appear Validation of gene expression changes was performed for GAPDH, or accumulate in the retina, particularly in the inner nuclear glutaminase C, lipidosin, protein synthesis initiation factor 4A layer and ganglion cell layer, at 10 weeks of age in cln3- (ELF41A), neuroendocrine differentiation factor (NEDF), ATP-synthase knockout, but not in the wild-type control. subunit B, and the unknown transcript identified as TC36735 by probe set numbering (Affymetrix). RNA extracts prepared for the gene chip Altered Gene Expression in Cln3-Knockout Eye studies (GeneChip; Affymetrix) were used. Amplification of a portion Gene expression was assessed in the eye of 10-week-old mice of each gene was performed with 1 ␮g total RNA (SuperScript Two- to explore changes in transcription that may precede degen- Step RT-PCR system with SYBR green; Invitrogen-Life Technologies, erative changes. To minimize variation, eyes were collected Gaithersburg, MD, on a Prism system; Applied Biosystems [ABI], Foster from three 10-week-old male Cln3-knockout and three age- City CA), according to the manufacturers’ guidelines. Primers used for matched male wild-type control mice, and the material from amplification are described in Table 1. each genotype was pooled for extraction of mRNA. Duplicate independent samples for both control and Cln3-knockout mice Histology were also prepared from another six animals. This essentially Eyes were removed from the mice and fixed in 4% paraformaldehyde provided two sets of mRNA from both wild-type control and for 3 hours, to harden the tissue and prepare it to be sectioned. The Cln3-knockout eyes, to allow a four-way comparison of gene eyes were then dehydrated 100% in an ethanol gradient and placed in expression between control and the Cln3-knockout. The re- xylenes. They were put into a 1:1 mixture of melted paraffin and sultant probes derived from these mRNA sets were hybridized xylenes at 60°C and were then moved to pure paraffin, where it to high-density Mu19 subarrays A, B, and C (Affymetrix). permeated the tissue. The cassette containing the embedded eye was Comparison of two Cln3-knockout samples to both wild- placed on a paraffin microtome (Leica Microsystems, Bannockburn, IL) type samples allows for a four-way comparison for statistical and cut into 6-␮m sections. Sections were deparaffinized and allowed evaluation. Previous studies have validated changes in gene to dry overnight, hydrated to 70% ethanol, and stained with hematox- expression in this system obtained using gene chips (Af- ylin I and eosin Y (H&E). For autofluorescence imaging, sections were fymetrix) by semiquantitative RT-PCR.13 We therefore focused

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one functional class based on information in the literature on the function of each gene product. These functional classes are based on the protein having a function in (1) signaling or cell growth; (2) cell structure, cell adhesion, or at the cell surface; (3) proteolysis or inhibition of proteolysis; (4) neuronal cell development and function; (5) lipid metabolism; (6) immune or inflammatory response; (7) energy metabolism; (8) detoxification or stress; (9) cytoskeleton; (10) cell death; (11) vascular and blood; (12) amino acid metabolism; (13) unknown, based on there being no known function of the protein, or in a small number of cases, our inability to fit the protein into any of the 12 classes. The classification of gene products and data on their altered expression is presented in Table 2. This table presents the mean change ratio, and does not include the data on the change ratio for each comparison. The entire data set can be viewed on the Internet at http://dbb.urmc.rochester. edu/laboratories/pearce/microarray.html.

Genes with Altered Expression Specific to the Eye To aid our understanding of the expression changes we see in the eye of the Cln3-knockout mice we compared the data to that obtained for expression changes in the cerebellum. We previously reported gene expression changes of twofold or more in cerebellum of 10-week-old Cln3-knockout mice.1 In Table 3, we list the 18 genes that are unique to the expression data obtained from the eye only. In other words, genes that had altered expression in the cerebellum as well as the eye have been excluded from this list. We selected six of these genes for validation by RT-PCR compared with control GAPDH expression, which is unchanged in cln3-knockout compared with normal (Fig. 2). We demonstrated that transcripts for glutaminase C and an unknown transcript designated TC36735, which had had a 6.2- and 14.0-fold increase in expression by microarray analysis, respectively, had a 3.6- and 7.3-fold increase in expression by RT-PCR, respectively. Similarly, NEDF, lipidosin, ELF4A, and ATP-synthase subunit B which had respective decreases in expression of Ϫ4.0, Ϫ15.5, Ϫ38.7, and Ϫ4.9 by microarray analysis had decreased expression of Ϫ1.9, Ϫ8.0, Ϫ8.3, and Ϫ3.5 by RT-PCR, respectively. Collectively, each validation confirms a significant change in expression of these FIGURE 1. (a) Comparison of gross morphologic changes in 10-week- transcripts in the same direction as predicted by the microar- old cln3-knockout and wild-type control mice. Micrographs were ray. We tested expression of each of these transcripts in cere- taken with a 40ϫ objective and show a representative subsection of bellum and whole brain of 10-week-old cln3-knockout and the retina with the ganglion cell layer (GCL) on the bottom of the wild-type control mice and saw no difference in expression. image. (b) Comparison of autofluorescence in 10-week-old cln3-knockout and wild-type control. Photographs were taken with a 40ϫ objective in representative subsection of the retina with the GCL at the ISCUSSION bottom of the image. The inner and outer segments of the photore- D ceptors, which autofluoresce naturally, have been cropped from these Batten disease is a devastating neurologic disorder, and, as with images. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. many disorders, the mechanism that results in vision loss and the neurodegenerative course is poorly understood. Although the Cln3-knockout mouse has many of the pathologic charac- our analysis on comparing the molecular profiles of the CLN3 teristics of Batten disease, and we show in this report the defect between eye and cerebellum. appearance of the characteristic storage material at 10-weeks of age, it does not have an apparent loss of vision.15 The gene Functional Classification of Genes with expression data we report will prove valuable as we gain Altered Expression further insight into the mechanisms that may predicate the appearance of autofluorescent storage material and perhaps We assigned a functional class to all 285 genes that had a vision loss in this and other diseases. The functional classes of reproducible change in expression of twofold or more (Table genes that have altered expression in Cln3-knockout eyes com- 2). We chose a twofold change in expression as our cutoff, pared with wild-type control eyes can be used to hypothesize because we considered at least a doubling or halving of the about the biological differences that may be present. With 285 level of a transcript to be an arbitrary way of determining reproducible changes in expression reported, many biological significance.1 In characterizing the genes with altered expres- processes are disturbed in the eyes of 10-week-old Cln3-knock- sion, many could clearly be assigned to more than one func- out mice. Moreover, it is very interesting that their eyes have tional class, due to having either more than one function, or such a shift in gene expression compared with wild-type con- because there is overlap between the functional classes them- trol mice. If we look at each functional group, most categories selves. For the sake of clarity, we assigned each gene to only have more transcripts upregulated than downregulated. For

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TABLE 2. Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Amino acid metabolism 22.475 TC39031_at Arginase 16.925 TC29651_at Possible deaminase 4.125 TC19442_at Cysteine dioxygenase Ϫ3.05 TC37346_g_at L-asparaginase Ϫ4.925 TC28329_g_at NBAT Ϫ5.325 TC30372_f_at Glutamate dehydrogenase Ϫ6.15 TC40291_at Tryptophan dioxygenase Ϫ7.1 TC33164_at Glycine amidotransferase Ϫ8.375 TC31560_s_at D-aminoacid oxidase Ϫ20.175 TC24073_at Possible arginine decarboxylase Blood/vascular 129.45 TC27670_g_at Myoglobin 37.075 TC40225_s_at Transthyretin like protein 23.25 TC32287_at hbr3 brain carboxyesterase 13.075 TC34069_at Carbonic anhydrase III 3.425 TC35644_s_at FCK1, VEGF binding, vasculogenisis Ϫ19.725 TC24493_at Electrogenic Na bicarbonate cotransporter (NBC) Cell death 322.8 TC17513_at Death asociated protein DAP1 158.25 TC37556_at p53 protein 122.3 TC19787_at p53 apoptosis-associated target 88.625 TC24816_s_at DAP1 death associated protein 24.45 TC16227_at Similar to death receptor 6 20.275 TC15396_at Early quiescence protein-1 15.825 TC19399_at Apoptosis associated speck protein P(YCARD) Detox/stress 32.55 TC40306_f_at Glutathione transferase 22.85 TC41370_s_at HSP70 12.2 TC29966_g_at Ceruloplasmin 4.825 TC35139_at HSBX, heat shock protein, like HSP20 4.6 TC31120_f_at Plasma glutathione peroxidase 4.475 TC21222_at Unknown Ϫ31.325 TC29829_at HSC73 Energy metabolism 122.7 TC18565_i_at Cytosolic malate dehydrogenase 48.375 TC15436_s_at Cytochrome c oxidase subunit VIIa-H precursor 34.575 TC20349_at Creatine kinase 11.3 TC32389_at PHS1. glycogen phosphorylase 10.875 TC32884_f_at Lactate dehydrogenase 7.475 TC23942_i_at Creatine kinase 6.95 TC19057_g_at Phosphoglycerate mutase muscle-speciﬁc subunit 6.85 TC37832_at Glycogenin-1 2.825 TC28383_s_at Glucose-6-phosphate dehydrogenase Ϫ3.425 TC40415_at Cox1, cytochrome oxidase I Ϫ3.475 TC30467_at NADH dehydrogenase subunit 5 Ϫ4.35 TC38446_g_at Cytochrome oxidase subunit 1 Ϫ4.85 TC19304_at ATP synthase B-subunit Signaling/Cell Growth 63.2 TC36530_g_at Mnk2, map kinase interacting kinase 27.6 TC18256_at Protein kinase Dyrk2 17.5 TC29599_at TC21 protein 5.225 TC36530_at Mnk2 3.825 TC26799_i_at mPRL-3 tyrosine kinase 3.525 TC34509_g_at MEKK 1, map kinase 3.275 TC35395_at Caveolin-2 2.65 TC21379_at RhoGAP, GTPase activating protein Ϫ2.9 TC21250_g_at Serine/threonine phosphatase 2B Ϫ2.95 TC25040_at p54, JNK-2, stress activated kinase Ϫ3.05 TC15005_s_at SHIP2, inositol polyphosphate phosphatase Ϫ8.1 TC38592_g_at cGMP phosphodiesterase type A9 Ϫ8.5 TC16846_s_at bfgf-r, basic ﬁbroblast growth factor receptor-1 Ϫ9.3 TC15262_at Phosphodiesterase I Ϫ10 TC34425_g_at fyn(T), tyrosine kinase Ϫ11.725 TC25522_s_at PCTAIRE-I serine/threonine kinase Ϫ12.1 TC15645_s_at Inositol 5- phosphatase Ϫ12.425 TC18155_s_at fyn(T), tyrosine kinase Ϫ24.65 TC33667_at SPOT14, thyroid hormone inducible protein Ϫ54.25 TC36931_at Phosphatallic acid phosphotase 2c (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Lipid metabolism 382 TC27653_at 3-ketoacyl-CoA thiolase B 33.75 TC19057_at Phosphoglycerate mutase muscle-specific subunit 20.175 TC15810_s_at 3-oxoacyl-CoenzymeA thiolase, mitochondrial 15.15 TC37551_at Paraoxonase 2 (PON2) 6.35 TC14864_f_at Keratinocyate lipid-binding protein 4.85 TC16399_f_at Acyl-CoA dehydrogenase 4.35 TC33350_at Putative C-8,7 Isomerase 3.725 TC35242_s_at Heme oxygenase Ϫ4.875 TC38739_at Diacylglycerol kinase Ϫ5.05 TC15835_at Oxysterol binding protein Ϫ5.85 TC37906_s_at Delta-G fatty acid desaturase Ϫ5.875 TC22730_s_at b-fabp, brain fatty acid binding protein Ϫ58.775 TC33071_s_at AcylcoA desaturase 2 Protease 93.35 TC26009_at Stefin3 91.05 TC39719_f_at Mac-2, lactose binding lechin 39.9 TC22206_at CYT4, stefin homolog 16.8 TC31277_at Serine protease 14.675 TC35575_at Epithln serine protease 12.85 TC21695_at Similar to serine proteases 12.8 TC33498_at Nephrilysin like peptidase 12.7 TC17364_at Prostasin 8.275 TC34450_at Aminopeptidase 7.95 TC34175_at Like aminopeptidase 6.925 TC16079_at Similar to neurotrypsin 2.975 TC32493_f_at Cathepsin L Structural/adhesion/cell surface 50.9 TC25512_s_at F1SP12 protein, tissue growth factor 47 TC22866_f_at Collagen alpha-1 37.85 TC19711_s_at 57 kd keratin 30.375 TC26960_at Keratin 6 alpha 28.475 TC30949_at Claudin 7 26.4 TC24005_s_at Cytokeratin 4 21.05 TC40219_at VLA-6 16.75 TC18507_f_at Decorin 8.825 TC24347_s_at Cytokeratin 13 8.3 TC33778_i_at Collagen alpha 1 7.05 TC16852_at Periplakin 6.825 TC41987_at Desmocollin type 2 4.9 TC14593_s_at Collagen alpha -2 4.75 TC40853_f_at Collegan alpha 1 3.775 TC37852_at Junction adhesion protein Immune 137.775 TC23274_at 14-3-3 protein sigma 132.1 TC20347_at Prostate stem cell antigen 92.175 TC36394_f_at Galectin-1 80.725 TC26633_g_at UCP2 63.725 TC15466_f_at Lipocortin I 43 TC34607_s_at DBPA homolog 40.8 TC39759_at TB2 like 36.2 TC37183_s_at TRP2, dopachrome taulomerase 33.325 TC40399_s_at Hepatitus virus receptor 17.8 TC40929_s_at Beta interferon induced protein 16.575 TC34649_at Similar to IC2 10.75 TC24304_g_at Linear IgA disease antigen homolog 8.85 TC25579_at Oncostatin M receptor beta 7.05 TC14570_f_at C1 inhibitor 6.425 TC41813_f_at STAT6 6.275 TC18855_f_at Catpactin 1 light chain 6.225 TC35184_f_at Macrophage capping protein 6.2 TC24577_at XLR 4 lymphocyte regulatory protein 5.925 TC19499_s_at Myc basic motif homologue-1 5.7 TC24581_at Interferon activatable protein 4.9 TC28116_g_at DOPAchrome tautomerase 4.825 TC34110_at LYVE-1, tymphatic endolithium specific receptor 4 TC15924_at LAR, lenkocyte common anitgen protein 3.7 TC14368_f_at Lipocortin II / Annexin II (anxII) Ϫ3.125 TC23291_s_at HUD paraneoplastic antigen (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Immune (continued) Ϫ3.35 TC36543_at Similar to paraneoplastic antigen, KIAA0883 Ϫ3.7 TC33860_at Neuronal IL-16 Ϫ3.875 TC37323_at Similar to Iga regulatory protein Ϫ4.1 TC32923_s_at Corticosteroid II- beta dehydrogenase Ϫ5.15 TC17814_at Corticotropin releasing hormone receptor 2 Ϫ49.575 TC26088_at BM88 antigen Neuronal cell develepment and function 294.375 TC32454_at Similar to maxi-K channel 286.325 TC20337_at Similar to synapsin 1 75.925 TC16591_at Proleo lipid protein 2 66.05 TC33406_s_at Peripheral benzodinzepine receptor (ptr) 64.625 TC30567_s_at Syndecan-1 60.75 TC36687_at Protein 4.1G 48 TC19521_g_at Nuclear pore-targeting complex component of 58 kDa 31.85 TC37537_at Protein 4.1G 30.1 TC36143_g_at Ezrin 26.1 TC32955_s_at Els transcription factor 20.025 TC31586_at Syntaxin 3 14.575 TC25273_at Dystonin; BPAG-1 13.225 TC24557_at Dkk-3, dickkopf-3 12.375 TC30575_at SIX3 protein 11.1 TC22048_at Semaphorin IV isoform a 10.45 TC40712_at Connexin 46 6.625 TC40937_s_at UNC119/ HRG4 4.125 TC41353_at Glypican 3 3.725 TC36142_at Ezrin 3.225 TC41791_at PACSIN2 (syndapin IIaa) 3.075 TC36827_at Cerebellum postnatal protein-1 Ϫ2.925 TC38522_at KIAA0916 human protein, highwine in drosophila Ϫ3.05 TC14267_i_at RKIP, phosphoatidylethandlumne binding protein Ϫ3.175 TC17976_at rsk2, calcium activated K-channel Ϫ3.2 TC32675_s_at Myelin gene expression factor, myef-2 Ϫ3.225 TC37684_at Kinesin light subunit B, (KLC) Ϫ3.5 TC26270_s_at KIFI, kinesin like Ϫ3.525 TC39656_f_at Neuritin Ϫ3.55 TC41874_at ndr2 Ϫ3.725 TC22759_at Calcineurin b. Ϫ3.975 TC38290_at Neuroendocrine differentiation factor Ϫ4.05 TC16990_s_at Neural vision like protein Ϫ4.275 TC34140_s_at Alpha-catenin (alpha -e) Ϫ4.3 TC26822_at Acrogranin Ϫ4.675 TC24292_at ATN2, Na/K transporter subunit 2 Ϫ4.8 TC30767_at SCHIP-1 Ϫ6.325 TC37996_at DARPP-32 Ϫ6.45 TC31890_g_at Secretogranin III Ϫ7.275 TC39376_at Pro SSAS Ϫ7.4 TC18154_at Brain development protein BDM1 Ϫ7.8 TC39160_g_at SCG10 Ϫ8.475 TC22283_s_at PLP, myelin proteilipid Ϫ9.85 TC36359_at TREK-1 Kϩ channel subunit Ϫ9.95 TC29998_at Myelin basic protein Ϫ10.025 TC39061_at Hippocampal amyloid precursor protein (beta) Ϫ11.65 TC14881_at SCG10 Ϫ13.825 TC24570_at Na/K transporting ATPase, alpha 2 subunit Ϫ15 TC30390_s_at p21, neuron specific protein Ϫ16.925 TC18867_at Alpha synuclein Ϫ18.1 TC39041_at Nel proptein Ϫ18.9 TC26003_at Synaplogamin XI Ϫ19.175 TC37308_at Double cortin like protein Ϫ19.625 TC22741_g_at Neuronal specific septin-3 Ϫ20.625 TC30389_f_at p21, neuron specific protein Ϫ20.925 TC35356_at Neurilin Ϫ21.25 TC18648_at NFI-x nucleus factor Ϫ22.35 TC36040_at Like GCAP1, granule cell marker protein Ϫ29.375 TC15940_g_at Coat protein gamma cap. Ϫ48.05 TC22429_s_at Myelin proteolipid protein (PLP) Ϫ74.375 TC17234_s_at Calbindin (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Cytoskeleton factors 158.975 TC15490_s_at Troponin T fast skeletal muscle isoform 109.95 TC18963_g_at Beta-tropomyosin 66.975 TC40898_f_at Troponin 1 29.275 TC28732_s_at Myosin heavy chain 2a 19.65 TC19756_at Actin-related protein complex 1b 15.95 TC22827_at Tropomyosin alpha 14.325 TC33465_g_at Myosin light chain 1 13.85 TC15815_at Alpha-actin 13.725 TC22502_f_at Myosin light chain 2 12.925 TC28358_f_at Myosin regulatory light chain 12.5 TC40908_at Tubulin beta 5 subunit 7.825 TC32100_s_at Alpha actinin -4 4.025 TC29413_f_at Myosin heavy chain 2B 3.85 TC39600_f_at Getsolin Ϫ3.225 TC18165_g_at Prefoldin subunit Ϫ3.575 TC21995_g_at Microtubule associated protein, tan Ϫ4.15 TC18613_at Like novel protein c380A1,2.2. Ϫ4.775 TC20757_s_at ANK-3 Ϫ28.625 TC38163_f_at Tubulin beta-3 unknown 224.275 TC27018_f_at Gamma 4-crystallin (aa 85-174): gamma-A-crystallin 121.075 TC27118_at Beta-A4-crystallin 116.9 TC23815_s_at Mouse fast skeletal muscle SR calcium ATPase 94.75 TC29087_s_at Unknown 90.65 TC16275_at Ataxia-telangieclasia group D-associated protein 86.475 TC15537_f_at Folate-binding protein 1 79.925 TC23172_at Unknown 67.825 TC20950_at mCBP 62.075 TC34763_at Unknown 57.15 TC16425_s_at Unknown 52.55 TC18741_f_at Unknown 44.025 TC37929_at Unknown 42.8 TC14369_f_at Protein-tyrosine kinase substrate 39.4 TC16278_at Gamma-C-crystallin 37.725 TC15584_at Unknown 36.725 TC32177_at Unknown 36.725 TC23979_g_at Unknown 33.5 TC34356_at Unknown 33.275 TC31887_at Possible glycoprotein 33.075 TC22352_s_at s100-type calcium binding protein 32.475 TC26873_at Beta-A3/A1 crystallin 32 TC30868_f_at PABP polyA binding protein 31.3 TC31507_at 5100-type calcium binding protein 29.8 TC36511_at Unknown 28.275 TC17811_at Unknown 28.2 TC40393_s_at Calcium binding protein 27.525 TC41119_at Unknown 26.875 TC23617_f_at Histone H1 26.825 TC24011_at Unknown 26.225 TC27544_at Possible kinase 25.675 TC20429_at GSG1 25 TC42144_at Dithiolethiume induced gene 24.75 TC16184_g_at Unknown 23.7 TC33945_at Unknown 23.45 TC30861_at Hepatocellular carcinoma-associated antigen 112 23 TC37660_s_at Like undine kinase 21.875 TC37456_at Unknown 21.8 TC16584_g_at Unknown 21.3 TC20216_at Unknown 20.7 TC38463_g_at Onzin 20.525 TC37769_g_at Unknown 20 TC38105_at A mannosylglycoprotein b 1–6 acetyl glucosaminyl transferase 18.2 TC40398_g_at BGP-1 glycoprotein 17.8 TC39732_at Unknown 17.65 TC40856_at P8 protein 16.95 TC22847_s_at Aldose reductase-related protein 1 16.55 TC22660_at Unknown (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

unknown (continued) 15.825 TC21363_at Unknown 15.075 TC40330_at Unknown 14.85 TC37670_g_at Unknown 14.275 TC21338_at Unknown 14.075 TC33781_at Unknown 13.975 TC36735_g_at Unknown 13.475 TC35151_at Unknown 13.4 TC37498_g_at Similar to TITIN 13.35 TC35267_at Unknown 12.525 TC32779_at Unknown 12.275 TC22217_g_at Gamma-sarcoglycan 12.05 TC21382_at Unknown 11.75 TC38572_at Like KIA0009 11.225 TC28227_at BM-003 11.2 TC15010_at Ataxia-telangieclasia group D-associated protein 10.95 TC40893_f_at Alpha crystallin B-chain 10.8 TC35947_at Secreted modular calcium binding protein 10.7 TC42043_s_at Unknown 10.525 TC35166_at Unknown 10.35 TC31979_at Unknown 10.325 TC40743_s_at Transketolase 10.025 TC24736_at Unknown 9.8 TC32729_at Unknown 9.1 TC14992_i_at Double LIM protein-1 9.05 TC34343_at Unknown Eye unknown 8.95 TC31534_g_at RNA binding protein RBP, MS 8.7 TC37405_at Possible glycoprotein 8.575 TC34789_at Like echinoderm microtubule associated protein 8.45 TC41574_g_at Unknown 8.25 TC40445_at Unknown 8.1 TC31394_g_at Unknown 7.425 TC16738_at Cell growth regulating nuclear protein 7.175 TC35223_g_at Hopotocyte growth factor activator 6.975 TC19085_f_at Histone H2a 1-F 6.975 TC19105_g_at Unknown 6.7 TC16842_at Unknown 6.675 TC30935_at Unknown 6.65 TC15420_g_at TGF-beta-inducible protein 6.625 TC41695_at Unknown 6.6 TC41312_at Unknown 6.425 TC36884_at Signal transducer of transcription activator 6 6.375 TC33908_at Unknown 6.2 TC28305_at Unknown 6.175 TC40703_at Unknown 6.075 TC21210_at Like SIAP-2 sarolemmal associated protein 6.05 TC22993_s_at Like BM-019 6.025 TC30968_at Unknown 5.925 TC38179_at NX-17 5.75 TC16584_at Unknown 5.725 TC39835_at Unknown 5.575 TC31268_at Unknown 5.575 TC31974_s_at Hapatocyte growth factor activator inhibitor 5.525 TC35180_at UROPLAKIN IB (UP18) 5.45 TC34662_g_at FGF binding protein 1 5.45 TC39762_at Unknown 5.25 TC22472_g_at Insulin-like growth factor binding protein-4, IGFBP-4 5.1 TC32825_at Unknown 5.1 TC15512_at Unknown 5.05 TC16200_at Unknown 5.025 TC26659_f_at Gamma E crystallin 5.025 TC33445_at SM-20, growth factor responsive protein 5 TC31101_g_at NX-17 4.95 TC32986_g_at SGLT1 sodium glucose transporter-1 4.925 TC28395_at Unknown 4.75 TC39257_g_at Unknown 4.75 TC41820_f_at pnbp1, poly a binding protein (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Eye unknown (continued) 4.725 TC33957_f_at Unknown 4.7 TC16337 s at Aldose reductase-related protein 1 reductase 4.7 TC38699_at Unknown 4.55 TC39978_at Unknown 4.475 TC23656_g_at HSP 70 4.45 TC16809_at Sialomucin MGC-24 4.425 TC34081_at Unknown 4.275 TC32695_a_at TtSII B 4.15 TC25859_at Unknown 4.025 TC34228_g_at Unknown 4 TC40849_at KIAA 1561 like 3.775 TC36904_g_at Unknown 3.775 TC41912_at Unknown 3.725 TC41574_at Unknown 3.7 TC33236_f_at Collagen alpha-2 3.675 TC38758_at Similar to ribonuclease 3.65 TC40553_at Predicted osteoblast protein G53786 3.525 TC16880_at Unknown 3.5 TC41930_at ZNT4 zinc transporter 4 3.425 TC18373_at Unknown 3.425 TC36701_at MEA1 antigen 3.425 TC37498_at Like title 3.4 TC33329_at Cavaolio-1 3.35 TC28712_at Unknown 3.325 TC26918_i_at Ocant-binding transcription factor I (cbta) 3.275 TC35003_s_at Initiation factor 3.25 TC17314_at Unknown 3.175 TC28860_at Unknown 3.175 TC28860_at Unknown 3.15 TC30867_at PAB1, polyadcnytate binding protein 3.125 TC34228_at Unknown 3.1 TC33264_at Guanine nucleotide binding protein 2.925 TC22848_s_at UBC3 2.9 TC28660_g_at Unknown 2.7 TC34620_f_at Ribosomal protein 520 Ϫ2.8 TC14392_f_at Stathmin Ϫ2.85 TC18861_at Unknown Ϫ2.85 TC24550_at Unknown Ϫ2.925 TC39474_f_at Lactate dehydrogynase Ϫ2.975 TC33078_at Unknown Ϫ3 TC41056_at Unknown Ϫ3.125 TC32289_a_at ABC2 Ϫ3.175 TC18736_g_at Protein synthesis initiation factor 4A, ELF41A Ϫ3.175 TC29636_at Unknown Ϫ3.175 TC41293_at Unknown Ϫ3.2 TC41345_at Unknown Ϫ3.225 TC17259_g_at Unknown Ϫ3.225 TC20592_at Small nuclear ribonucleo protein, SmO Ϫ3.25 TC34446_at Unknown Ϫ3.275 TC16825_at Unknown Ϫ3.275 TC24812_at Unknown Ϫ3.3 TC39752_at Unknown Ϫ3.3 TC40014_at Unknown Ϫ3.35 TC25091_at Transcription repressor RPS8 Ϫ3.35 TC38493_at Unknown Ϫ3.35 TC22066_at Unknown Ϫ3.4 TC33205_at SERCA 2n Ϫ3.425 TC28327_at Similar to EXTL2 human Ϫ3.5 TC15898_f_at Similar to LDOC1 human Ϫ3.5 TC22782_g_at Similar to cadherin 22 Ϫ3.525 TC22801_at PAK-1 threoniae/serine kinase p21 activated Ϫ3.575 TC35044_at Unknown Ϫ3.65 TC25610_s_at Neodin Ϫ3.7 TC37065_at Same as FCJ10856 human protein Ϫ3.875 TC27925_i_at Plallet activating factor PAF acatythydroinse Ϫ3.95 TC29662_at Unknown Ϫ3.975 TC22817_at Brain protein 154 (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Eye unknown (continued) Ϫ4.05 TC38604_at Unknown Ϫ4.075 TC18152_at Unknown Ϫ4.075 TC38584_at KIA1583 human protein Ϫ4.125 TC34542_at Ribonucleoprotein A2/B1 Ϫ4.15 TC19732_at Unknown Ϫ4.3 TC33029_g_at Unknown Ϫ4.3 TC37489_a_at Mouse brain protein, BH5, peanut-like Ϫ4.475 TC39750_at Unknown Ϫ4.525 TC39858_at Unknown Ϫ4.55 TC34778_at Unknown Ϫ4.575 TC38646_at KIA0436, human, like oligopeptidase Ϫ4.6 TC26109_at Unknown Ϫ4.6 TC36755_at Neural membrane glyco protein, M6–8 Ϫ4.7 TC39581_at Unknown Ϫ4.725 TC22193_at Transcriptional repressor RP58 Ϫ4.8 TC38235_at Unknown Ϫ4.86667 TC22191_at Unknown Ϫ4.95 TC16013_g_at Unknown Ϫ4.95 TC41433_at Human KIA0436 like Ϫ5.075 TC32823_at Unknown Ϫ5.1 TC40705_at Unknown Ϫ5.175 TC35606_at Unknown Ϫ5.2 TC33812_at Unknown Ϫ5.25 TC38211_at EXCM1 gene, regulator SP2 transcription Ϫ5.25 TC38294_at Unknown Ϫ5.325 TC34814_at Unknown Ϫ5.325 TC29508_at Unknown Ϫ5.4 TC37388_at Transcriptional repressor RP58 Ϫ5.475 TC40986_at Unknown Ϫ5.55 TC21739_at Unknown Ϫ5.7 TC41479_at Unknown Ϫ5.7 TC15800_at Unknown Ϫ5.7 TC38214_at Unknown Ϫ5.725 TC16088_at Unknown Ϫ5.75 TC34430_at Unknown Ϫ5.825 TC21730_at Unknown Ϫ5.85 TC25709_g_at Unknown Ϫ5.675 TC26422_at Unknown Ϫ6.05 TC28579_at Unknown Ϫ6.475 TC25709_at Unknown Ϫ6.675 TC31827_at Unknown Ϫ6.7 TC16888_at Unknown Ϫ6.8 TC38692_s_at Unknown Ϫ8.85 TC35006_at Unknown Ϫ7.025 TC39106_at Unknown Ϫ7.675 TC23195_g_at Unknown Ϫ7.725 TC21739_g_at Unknown Ϫ7.775 TC39973_at Unknown Ϫ8.075 TC18151_at Like globulin-7 (zeamayas) Ϫ8.25 TC32823_g_at Unknown Ϫ8.475 TC20619_at Similar to alpha/beta hydrolase-1 Ϫ8.675 TC40812_at Unknown Ϫ8.875 TC20884_at Unknown Ϫ8.975 TC21031_at Unknown Ϫ9.225 TC34440_g_at p00071, plakophilin related protein Ϫ9.25 TC15280_a_at Unknown Ϫ9.275 TC41038_at Small GTP binding protein Ϫ9.35 TC31813_at Similar to (highly) GEF protein Ϫ9.475 TC39216_at HSPC322 Ϫ10.6 TC17986_at Unknown Ϫ10.6 TC41979_at KIA1424 Ϫ11.275 TC41373_at Unknown Ϫ11.475 TC18252_at Guanina nucleotide binding protein like Ϫ12.075 TC15777_at Unknown Ϫ12.1 TC23934_at RP58, transcriptional repressor Ϫ12.25 TC42156_at Unknown (continues)

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TABLE 2. (continued). Expression Changes Greater Than 2 in Cln3-Knockout Eye, Compared with Wild-Type Control

Average Change TIGR/Affymetrix No. Annotation

Eye unknown (continued) Ϫ12.775 TC40806_at Unknown Ϫ13.075 TC41228_at Unknown Ϫ13.325 TC32742_at Unknown Ϫ13.875 TC34340_at Unknown Ϫ14.075 TC40224_at KIA0898 like Ϫ14.325 TC17313_at Unknown Ϫ14.475 TC28738_g_at p53inducibleprotein Ϫ14.525 TC21034_at KIAA0631 like Ϫ14.575 TC22935_at Unknown Ϫ15.875 TC30317_g_at VAV-3 protein (GEF protein) Ϫ15.925 TC39390_at Unknown Ϫ16.325 TC19908_at Formyltetrahydrofolute dehudrogenase Ϫ16.05 TC38088_at Unknown Ϫ19.2 TC16183_at HSPC328 Ϫ19.225 TC40805_at Unknown Ϫ20.025 TC42007_at Unknown Ϫ27.3 TC16885_at Angiotensinogen precursor Ϫ29.425 TC23193_a_at HSPC251 Ϫ33.025 TC26229_at Unknown Ϫ35.675 TC29945_at Unknown Ϫ38.65 TC16736_at Unknown Ϫ45.925 TC19832_a_at Stathmin like protein RB3 Ϫ48.375 TC41060_at Beta 2 chimerin Ϫ73.95 TC33089_at Unknown Ϫ76.825 TC29995_at Unknown Ϫ83.45 TC16446_at Unknown Ϫ116.975 TC39645_g_at Unknown Ϫ277.125 TC29990_l_at Unknown

Each cln3-knockout sample was compared with each wild-type control to give a four-pair-wise comparison. These functional classes are based on the protein having a role in one or more of the 12 functions listed in the Methods section. This list, including the ﬁnal category, unknown, based on an unknown function of the protein or, in a small number of genes, our inability to ﬁt the protein into the above classes, can be viewed at http://dbb.urmc.rochester.edu/laboratories/pearce/microarray.html.

example, genes involved in proteolysis, both for degradation the cell surface are also up regulated, and along with the (for example, cathepsin L) and inhibition of degradation (for changes observed in proteolysis and the cytoskeleton, may example, steﬁn 3) are predominantly upregulated. Similarly, 14 predict that intracellular and extracellular integrity of the cells (74%) of 19 of transcripts associated with the cytoskeleton (for is somewhat compromised. The neuronal cell development example, ␤-tropomyosin and troponin) are upregulated. All and function class reveals that many genes are up- and down- transcripts associated with cell structure, cell adhesion, and regulated. It is of interest that a number of genes associated

TABLE 3. Annotated List of Genes with Altered Expression in the Eye Only between Wild-Type and Cln3-Knockout as Compared with the Cerebellum Data Set

Change Ratio TIGR/Affymetrix No. Annotation

Upregulated genes 14.0 TC36735_g_at Unknown 6.2 TC28305_at Glutaminase C 4.2 TC25659_at Similar to TRIP protein 3.4 TC39097_at 42-9-9 protein 2.7 TC21379_at RhoGAP, GTPase activating protein Down regulated genes Ϫ3.0 TC41056_at Unknown Ϫ3.1 TC14267_i_at RKIP protein Ϫ3.2 TC32675_s_at MYEF-2 Ϫ3.2 TC17259_g_at Unknown Ϫ3.4 TC38493_at Unknown Ϫ3.4 TC40415_at Cytochrome oxidase I Ϫ3.5 TC30467_at Cytochrome B Ϫ4.0 TC38290_at NEDF Ϫ4.1 TC34542_at Ribonucleoprotein A2/B1 Ϫ4.9 TC19304_at ATP Synthase B Ϫ5.2 TC35606_at Unknown Ϫ14.5 TC21034_at Lipidosin Ϫ38.7 TC16736_g_at ELF41A

List derived from genes that had an increase or decrease in expression of twofold or more.

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FIGURE 2. Validation of gene expression changes by RT-PCR. Expres- sion levels measured by RT-PCR for each sample that was also processed for microarray analysis. Each reaction was repeated in triplicate. Relative expression levels are normalized to GAPDH, which had identical expression in normal and cln3-knockout tissue. Transcripts for glutaminase C and an unknown transcript designated TC36735, which had 6.2- and 14.0-fold increases in expression by microarray analysis, respectively, had a 3.6- and 7.3-fold increase in expression by RT-PCR, respectively. NEDF, lipidosin, and ELF4A, which had respective decreases in expression of Ϫ4.0-, Ϫ15.5-, Ϫ38.7- and Ϫ4.9-fold by microarray had decreased expression of Ϫ1.9-, Ϫ8.0-, Ϫ8.3-, and Ϫ3.5-fold by RT-PCR, respectively.

with cell death (for example, p53 and DAP1) are also upregu- It is apparent that 13 (72%) of 18 of the genes with an lated, suggesting that a certain number of cells may have been altered expression pattern in the eye only are downregu- programmed to die. lated. However, it is important to note that this is based on All but one gene in the detoxification and stress func- those transcripts that were detectable in the samples and tional class are upregulated, which is consistent with what present on the microarrays used. Nevertheless, three of may be predicted about the diseased state resulting in cel- these are a part of complexes in the mitochondria that lular damage. Up- and downregulation of several genes as- contribute to energy production: cytochrome oxidase, cyto- sociated with energy metabolism is also apparent, some of chrome B, and ATP synthase. It has been suggested that which are associated with mitochondrial function (for ex- mitochondrial dysfunction could precipitate cell death in 17 ample, cytochrome oxidase and subunit B of ATP-synthase). NCL. If cell types in the eye are more susceptible to the The role of mitochondria and energy metabolism in Batten CLN3 defect, the changes we report in expression of mito- disease is particularly interesting in view of the emphasis chondrial proteins involved in energy production is also placed on the role of mitochondrial dysfunction in this suggestive that mitochondrial dysfunction is a part of the disease in many other studies (for review, see Ref. 16). A degenerative process. It is also fascinating that ATP synthase hallmark of Batten disease is accumulation of ATP synthase subunit B is downregulated, when another component of subunit C in the lysosome; thus, it is interesting that there is this complex, subunit C, is a major component of the stor- an apparent downregulation of a different subunit of this age material that accumulates in the lysosome. This obser- complex and ATP synthase B-subunit. Many genes associated vation fits with previous reports of decreased activity of ATP with lipid metabolism are up- and downregulated, and de- synthase and decreased mitochondrial function in NCL and that perhaps there is coordinate regulation in the expression tailed analysis of these events may contribute to our under- 18–21 standing the composition of accumulating lipopigments in of ATP-synthase subunits. As more microarray data sets the Batten disease and the molecular mechanisms underly- on the expression of genes in the eye and certain cell types ing their deposition. Overall, these findings suggest that within the eye become available, a more detailed interpre- Cln3-knockout mice have a major change in the biology of tation of the CLN3 defect on different cell types will be possible. the cells within the eye. The fact that there is such a large number of changes in gene expression compared with wild- type control animals suggests that molecular events associ- Acknowledgments ated with the CLN3 defect occur before the appearance of autofluorescent storage material in 10-week-old Cln3-knock- The authors thank Hannah Mitchison (University College of London, out mice. London, UK) and Robert Nussbaum (National Human Genome Re- Because there are several cell types in the eye, it is not search Institute) for originally providing the Cln3-knockout mice used possible to identify whether subsets of cells or all cell types to establish the colony of mice for this study. experience the gene expression changes we report. These gene expression data present an overall picture of a vast set of molecular changes that result from a lack of the CLN3 protein References and provide a valuable data set for researchers for further 1. Goebel, HH, Mole SE, Lake BD, eds. The neuronal ceroid lipofus- exploration of the pathogenesis of the disease and for cell- cinoses (Batten disease). Biomedical and Health Research. type–specific changes in gene expression. There are 18 genes Amsterdam: IOS Press: 1999:211. with altered gene expression in the eye that are not evident in 2. Wisniewski KE, Zhong N. Batten Disease: diagnosis, treatment, and the previously reported data set on altered expression in the research. In: Hall JC, ed. Advances in Genetics. San Diego: Aca- cerebellum.12 demic Press; 2001:1–34.

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