(Investigative Ophthalmology and Visual Science. 2004;45:2893-2905.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI: 10.1167/iovs.04-0143
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
1From the Center for Aging and Developmental Biology, Aab Institute of Biomedical Sciences; the
3Departments of Environmental Medicine,
4Biochemistry and Biophysics, and
5Neurology; and the
2Center for Functional Genomics, University of Rochester School of Medicine and Dentistry, University of Rochester, Rochester, New York.
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Abstract
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PURPOSE. Juvenile neuronal ceroid lipofuscinosis (JNCL or Batten Disease) is one of the most common progressive neurodegenerative disorders of childhood, resulting from autosomal recessive inheritance of mutations in the CLN3 gene. Pathologically, Batten disease is characterized by lysosomal storage of autofluorescent material in all tissue types. Although characterized by seizures, mental retardation, and loss of motor skills, the first presenting symptom of Batten disease is vision loss.
METHODS. High-density oligonucleotide arrays were used to profile approximately 19,000 mRNAs in the eye of 10-week-old Cln3-knockout and normal mice, and the data were compared with that for the cerebellum in the same model as a means to identify gene expression changes that are specific to the eye.
RESULTS. A detailed list was compiled of 285 functionally categorized genes that have altered expression in the eye of Cln3-knockout mice before the appearance of the characteristic lysosomal storage material. Furthermore, 18 genes were identified and 6 validated by semiquantitative RT-PCR that have altered expression in the eye, but not in the cerebellum of Cln3-knockout mice. The genes that have altered expression specific to the eye of the Cln3-knockout mouse may be of importance in understanding the function of CLN3 in different tissues.
CONCLUSIONS. Downregulation of genes associated with energy production in the mitochondria appears to be specific to the eye. The CLN3 defect may result in altered mitochondrial function in eye but not other tissue. More detailed experimentation is needed to understand the contribution of these changes in expression to disease state, and whether these changes are specific for certain cell types within the eye.
Batten disease or JNCL, is the juvenile form of neuronal ceroid lipofuscinosis (NCL). Batten disease is inherited as an autosomal recessive condition and is the most common progressive neurodegenerative disease of childhood. The disorder is characterized initially by visual deterioration at age 5 to 7 years that ultimately results in blindness. After the loss of vision, other neurologic characteristics are seizures, mental retardation, and loss of motor function.1 2 Batten disease is always fatal. The CLN3 gene responsible for Batten disease was positionally cloned in 1995,3 with most individuals with the disease harboring a 1-kb deletion of this gene. The disease is characterized by the accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and other cell types. A predominant component of the lysosomal storage material has been identified as mitochondrial ATP synthase subunit c.4 5 6 7 One of the paradoxes of Batten disease is that the accumulation of this lysosomal storage material does not apparently lead to disease in non-neuronal cell types. The CLN3 protein has been localized to late endosomes and lysosomes in non-neuronal cell types, and has been shown to co-localize with synaptic vesicle proteins in neuronal cell types 8 9 10 . It is apparent that, because loss of vision is the first presenting symptom of Batten disease, the eye is an ideal system for the study of the primary molecular events associated with the CLN3 defect.
Cln3-knockout mice homozygous for a targeted deletion of exon 1 to 6 in the Cln3 gene have been reported to show characteristic accumulation of autofluorescent lipopigments containing mitochondrial ATP synthase subunit c in neural tissue and selective loss of 
-aminobutyric acid (GABA)ergic neurons.11 We have previously reported gene expression changes in the cerebellum of 10-week-old Cln3-knockout mice.12 13 To gain insight into gene expression changes that associate with the CLN3 defect, we repeated our gene expression study in the whole eye of the Cln3-knockout mouse. We classified genes displaying an altered expression pattern into 13 functional categories based on functional information associated with the gene product. The altered gene expression pattern in Cln3-knockout eye shows that there are 285 genes that are either up- or downregulated. By comparing these gene expression data with those previously reported in the cerebellum 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.
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Materials and Methods
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Animals
Ten-week-old wild-type control 129S6/SvEv and homozygous Cln3-knockout mice on a 129S6/SvEv background11 were used in the study. All procedures were performed in accordance with NIH guidelines and University of Rochester Animal Care and Use Committee Guidelines. Furthermore, all research conformed to ARVO Standards for the Use of Animals in Ophthalmic and Vision Research.
Gene Expression Studies and Data Analysis
For comparative gene expression studies whole eyes from three each of the 10-week-old wild-type control and Cln3-knockout mice were pooled and homogenized by standard procedures (TRIzol; Invitrogen-Gibco, Grand Island, NY) for mRNA extraction. Total RNA (10 µg) from each sample was used to generate a high-fidelity cDNA, which is modified at the 3' end to contain an initiation site for T7 RNA polymerase, as per the manufacturer’s protocol (SuperChoice; Invitrogen-Gibco). On completion of cDNA synthesis, 1 µg of product was used in an in vitro transcription (IVT) reaction that contained biotinylated UTP and CTP, which will be used for detection after hybridization to the microarray as per the manufacturer’s protocol (Enzo Biochemicals, Farmingdale, NY). Full-length IVT product (20 µg) was subsequently fragmented in 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, and 150 mM MgOAc at 94°C for 35 minutes. After fragmentation, all components generated throughout the processing procedure (cDNA, full-length cRNA, and fragmented cRNA) were analyzed by gel electrophoresis to assess the appropriate size distribution before microarray analysis.
All samples represented were subjected to gene expression analysis using the a high-density oligonucleotide array set (Mu19K; Affymetrix, Santa Clara, CA), at the University of Rochester Microarray Core Facility, as previously described.12 The mathematical definitions for the algorithms can be found in the Microarray Suite Analysis manual in the algorithm tutorial. The change ratio of expression of any transcript between baseline and experimental is calculated after global scaling. All data represented from this first approach are from pair-wise comparison analyses.
Reverse Transcription–Polymerase Chain Reaction
Validation of gene expression changes was performed for GAPDH, glutaminase C, lipidosin, protein synthesis initiation factor 4A (ELF41A), neuroendocrine differentiation factor (NEDF), ATP-synthase subunit B, and the unknown transcript identified as TC36735 by probe set numbering (Affymetrix). RNA extracts prepared for the gene chip studies (GeneChip; Affymetrix) were used. Amplification of a portion of each gene was performed with 1 µg total RNA (SuperScript Two-Step RT-PCR system with SYBR green; Invitrogen-Life Technologies, Gaithersburg, MD, on a Prism system; Applied Biosystems [ABI], Foster City CA), according to the manufacturers’ guidelines. Primers used for amplification are described in Table 1 .
Histology
Eyes were removed from the mice and fixed in 4% paraformaldehyde for 3 hours, to harden the tissue and prepare it to be sectioned. The eyes were then dehydrated 100% in an ethanol gradient and placed in xylenes. They were put into a 1:1 mixture of melted paraffin and xylenes at 60°C and were then moved to pure paraffin, where it permeated the tissue. The cassette containing the embedded eye was placed on a paraffin microtome (Leica Microsystems, Bannockburn, IL) and cut into 6-µm sections. Sections were deparaffinized and allowed to dry overnight, hydrated to 70% ethanol, and stained with hematoxylin I and eosin Y (H&E). For autofluorescence imaging, sections were cleared with xylenes and mounted (Permount; Fisher Scientific, Pittsburgh, PA). Digital images were taken with a camera (Spot camera; Diagnostic Instruments, Sterling Heights, MI) mounted on a microscope (Olympus Corp. of America, Lake Success, NY). A 4x objective captured the whole eye, and the 40x objective was used to obtain detailed images of the peripheral retina. Fluorescence images were taken through a 488-nm filter. Images were cropped and background removed (Photoshop; Adobe Systems, Mountain View, CA).
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Results
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Batten disease is a lysosomal storage disease with accumulation of autofluorescent lipopigment in the lysosomes of individuals with the disorder. Homozygous Cln3-knockout mice have been confirmed to have similar accumulation within the brain 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 normal and Cln3-knockout mice. In this initial study we took the whole eye from Cln3-knockout and wild-type control mice and examined gene expression changes associated with the CLN3 defect. To maximize interpretation of data obtained on changes in gene expression that are associated with the CLN3 defect, we compared the data obtained from this study to those in a previous experiment, in which we compared gene expression changes in cerebellum of 10-week-old normal and Cln3-knockout mice.
Characteristics of 10-Week-Old Cln3-Knockout Eyes
It has been shown that cln3-knockout mice demonstrate the characteristic presence of autofluorescent storage material in the retina at 12 months of age.15 Although wild-type control animals also demonstrate the presence of storage material, there is a clear elevation in the cln3-knockout retina. We considered that an ideal time to perform gene expression studies in the eye of cln3-knockout mice would be at a point when this pathologically characteristic storage material first appeared in cln3-knockout, but not in wild-type, animals. Figure 1a shows that the retina of 10-week-old cln3-knockout mice seemed to be normal and resembled that of an age-matched wild-type control. The thickness of the each cell layer appeared to be the same, with no obvious cell loss. In Figure 1b , autofluorescent storage material was beginning to appear or accumulate in the retina, particularly in the inner nuclear layer and ganglion cell layer, at 10 weeks of age in cln3-knockout, but not in the wild-type control.
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FIGURE 1. (a) Comparison of gross morphologic changes in 10-week-old cln3-knockout and wild-type control mice. Micrographs were taken with a 40x objective and show a representative subsection of the retina with the ganglion cell layer (GCL) on the bottom of the image. (b) Comparison of autofluorescence in 10-week-old cln3-knockout and wild-type control. Photographs were taken with a 40x objective in representative subsection of the retina with the GCL at the bottom of the image. The inner and outer segments of the photoreceptors, which autofluoresce naturally, have been cropped from these images. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.
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Altered Gene Expression in Cln3-Knockout Eye
Gene expression was assessed in the eye of 10-week-old mice to explore changes in transcription that may precede degenerative changes. To minimize variation, eyes were collected from three 10-week-old male Cln3-knockout and three age-matched male wild-type control mice, and the material from each genotype was pooled for extraction of mRNA. Duplicate independent samples for both control and Cln3-knockout mice were also prepared from another six animals. This essentially provided two sets of mRNA from both wild-type control and Cln3-knockout eyes, to allow a four-way comparison of gene expression between control and the Cln3-knockout. The resultant probes derived from these mRNA sets were hybridized to high-density Mu19 subarrays A, B, and C (Affymetrix).
Comparison of two Cln3-knockout samples to both wild-type samples allows for a four-way comparison for statistical evaluation. Previous studies have validated changes in gene expression in this system obtained using gene chips (Affymetrix) by semiquantitative RT-PCR.13 We therefore focused our analysis on comparing the molecular profiles of the CLN3 defect between eye and cerebellum.
Functional Classification of Genes with Altered Expression
We assigned a functional class to all 285 genes that had a reproducible change in expression of twofold or more (Table 2) . We chose a twofold change in expression as our cutoff, because we considered at least a doubling or halving of the level of a transcript to be an arbitrary way of determining significance.1 In characterizing the genes with altered expression, many could clearly be assigned to more than one functional class, due to having either more than one function, or because there is overlap between the functional classes themselves. For the sake of clarity, we assigned each gene to only 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 –4.0, –15.5, –38.7, and –4.9 by microarray analysis had decreased expression of –1.9, –8.0, –8.3, and –3.5 by RT-PCR, respectively. Collectively, each validation confirms a significant change in expression of these transcripts in the same direction as predicted by the microarray. We tested expression of each of these transcripts in cerebellum and whole brain of 10-week-old cln3-knockout and wild-type control mice and saw no difference in expression.
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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
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FIGURE 2. Validation of gene expression changes by RT-PCR. Expression 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 –4.0-, –15.5-, –38.7- and –4.9-fold by microarray had decreased expression of –1.9-, –8.0-, –8.3-, and –3.5-fold by RT-PCR, respectively.
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Discussion
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Batten disease is a devastating neurologic disorder, and, as with 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 characteristics of Batten disease, and we show in this report the appearance of the characteristic storage material at 10-weeks of age, it does not have an apparent loss of vision.15 The gene expression data we report will prove valuable as we gain further insight into the mechanisms that may predicate the appearance of autofluorescent storage material and perhaps vision loss in this and other diseases. The functional classes of genes that have altered expression in Cln3-knockout eyes compared with wild-type control eyes can be used to hypothesize about the biological differences that may be present. With 285 reproducible changes in expression reported, many biological processes are disturbed in the eyes of 10-week-old Cln3-knockout mice. Moreover, it is very interesting that their eyes have such a shift in gene expression compared with wild-type control mice. If we look at each functional group, most categories have more transcripts upregulated than downregulated. For example, genes involved in proteolysis, both for degradation (for example, cathepsin L) and inhibition of degradation (for example, stefin 3) are predominantly upregulated. Similarly, 14 (74%) of 19 of transcripts associated with the cytoskeleton (for example, ß-tropomyosin and troponin) are upregulated. All transcripts associated with cell structure, cell adhesion, and the cell surface are also up regulated, and along with the changes observed in proteolysis and the cytoskeleton, may predict that intracellular and extracellular integrity of the cells is somewhat compromised. The neuronal cell development and function class reveals that many genes are up- and downregulated. It is of interest that a number of genes associated with cell death (for example, p53 and DAP1) are also upregulated, suggesting that a certain number of cells may have been programmed to die.
All but one gene in the detoxification and stress functional class are upregulated, which is consistent with what may be predicted about the diseased state resulting in cellular damage. Up- and downregulation of several genes associated with energy metabolism is also apparent, some of which are associated with mitochondrial function (for example, cytochrome oxidase and subunit B of ATP-synthase). The role of mitochondria and energy metabolism in Batten disease is particularly interesting in view of the emphasis placed on the role of mitochondrial dysfunction in this disease in many other studies (for review, see Ref. 16 ). A hallmark of Batten disease is accumulation of ATP synthase subunit C in the lysosome; thus, it is interesting that there is an apparent downregulation of a different subunit of this complex and ATP synthase B-subunit. Many genes associated with lipid metabolism are up- and downregulated, and detailed analysis of these events may contribute to our understanding the composition of accumulating lipopigments in the Batten disease and the molecular mechanisms underlying their deposition. Overall, these findings suggest that Cln3-knockout mice have a major change in the biology of 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 associated with the CLN3 defect occur before the appearance of autofluorescent storage material in 10-week-old Cln3-knockout mice.
Because there are several cell types in the eye, it is not possible to identify whether subsets of cells or all cell types 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 and provide a valuable data set for researchers for further exploration of the pathogenesis of the disease and for cell-type–specific changes in gene expression. There are 18 genes with altered gene expression in the eye that are not evident in the previously reported data set on altered expression in the cerebellum.12
It is apparent that 13 (72%) of 18 of the genes with an altered expression pattern in the eye only are downregulated. However, it is important to note that this is based on those transcripts that were detectable in the samples and present on the microarrays used. Nevertheless, three of these are a part of complexes in the mitochondria that contribute to energy production: cytochrome oxidase, cytochrome B, and ATP synthase. It has been suggested that mitochondrial dysfunction could precipitate cell death in NCL.17 If cell types in the eye are more susceptible to the CLN3 defect, the changes we report in expression of mitochondrial proteins involved in energy production is also suggestive that mitochondrial dysfunction is a part of the degenerative process. It is also fascinating that ATP synthase subunit B is downregulated, when another component of this complex, subunit C, is a major component of the storage material that accumulates in the lysosome. This observation fits with previous reports of decreased activity of ATP synthase and decreased mitochondrial function in NCL and that perhaps there is coordinate regulation in the expression of ATP-synthase subunits.18 19 20 21 As more microarray data sets on the expression of genes in the eye and certain cell types within the eye become available, a more detailed interpretation of the CLN3 defect on different cell types will be possible.
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Acknowledgements
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The authors thank Hannah Mitchison (University College of London, London, UK) and Robert Nussbaum (National Human Genome Research Institute) for originally providing the Cln3-knockout mice used to establish the colony of mice for this study.
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Footnotes
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Supported by NIH NS40580, the EJLB Foundation, a Herbert H. DeGraff Batten disease research grant, and the JNCL Research Fund.
Submitted for publication February 11, 2004; revised April 9 and May 13, 2004; accepted June 2, 2004.
Disclosure: S. Chattopadhyay, None; E. Kingsley, None; A. Serour, None; T.M. Curran, None; A.I. Brooks, None; D.A. Pearce, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: David A. Pearce, Center for Aging and Developmental Biology, Department of Biochemistry and Biophysics, Box 645, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642; david_pearce{at}urmc.rochester.edu.
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Abstract
Materials and Methods
