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

Abstract

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 γ-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. 

Materials and Methods

Animals

Ten-week-old wild-type control 129S6/SvEv and homozygous Cln3-knockout mice on a 129S6/SvEv background ¹¹ 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 4× objective captured the whole eye, and the 40× 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). 

Results

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. 

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. 

Discussion

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, β-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. 

 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.

 

Table 1.

 

Primers Used for RT-PCR Validation of Expression Levels

Transcript	Primers
GAPDH	5′-GCTCCCTAGGCCCCTCCTG-3′
	5′-CAAGAAGGTGGTGAAGCAGGCCAC-3′
Glutaminase C	5′-TAACTGCTAGTATCTGTGCA-3′
	5′-AAGAGATAAAGGGTATGTT-3′
Lipidosin	5′-GAAGCTCGGCCTAGAGCGTG-3′
	5′-TATGAGCTCCTCCATCGTGT-3′
ELF41A	5′-TTATATGGGAGCAACTTGTC-3′
	5′-TTGGTCACTTCTAGCACATC-3′
NEDF	5′-AGTGAAAGATGCAGCCAAGA-3′
	5′-ATCGCTCCTGCGGGTTCA-3′
TC36735	5′-GAAACTTGTCTAATACCAG-3′
	5′-GACAGTTGTCACCATGAGA-3′
ATP-synthase subunit B	5′-TATGTGCTTGGAACTGGACT-3′
	5′-CACTAAGTGGACCTTGATCT-3′

Figure 1.

 

(a) Comparison of gross morphologic changes in 10-week-old cln3-knockout and wild-type control mice. Micrographs were taken with a 40× 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 40× 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.

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
−3.05	TC37346_g_at	L-asparaginase
−4.925	TC28329_g_at	NBAT
−5.325	TC30372_f_at	Glutamate dehydrogenase
−6.15	TC40291_at	Tryptophan dioxygenase
−7.1	TC33164_at	Glycine amidotransferase
−8.375	TC31560_s_at	D-aminoacid oxidase
−20.175	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
−19.725	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
−31.325	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
−3.425	TC40415_at	Cox1, cytochrome oxidase I
−3.475	TC30467_at	NADH dehydrogenase subunit 5
−4.35	TC38446_g_at	Cytochrome oxidase subunit 1
−4.85	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
−2.9	TC21250_g_at	Serine/threonine phosphatase 2B
−2.95	TC25040_at	p54, JNK-2, stress activated kinase
−3.05	TC15005_s_at	SHIP2, inositol polyphosphate phosphatase
−8.1	TC38592_g_at	cGMP phosphodiesterase type A9
−8.5	TC16846_s_at	bfgf-r, basic fibroblast growth factor receptor-1
−9.3	TC15262_at	Phosphodiesterase I
−10	TC34425_g_at	fyn(T), tyrosine kinase
−11.725	TC25522_s_at	PCTAIRE-I serine/threonine kinase
−12.1	TC15645_s_at	Inositol 5-phosphatase
−12.425	TC18155_s_at	fyn(T), tyrosine kinase
−24.65	TC33667_at	SPOT14, thyroid hormone inducible protein
−54.25	TC36931_at	Phosphatallic acid phosphotase 2c
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
−4.875	TC38739_at	Diacylglycerol kinase
−5.05	TC15835_at	Oxysterol binding protein
−5.85	TC37906_s_at	Delta-G fatty acid desaturase
−5.875	TC22730_s_at	b-fabp, brain fatty acid binding protein
−58.775	TC33071_s_at	AcylcoA desaturase 2
Protease
93.35	TC26009_at	Stefin 3
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)
−3.125	TC23291_s_at	HUD paraneoplastic antigen
−3.35	TC36543_at	Similar to paraneoplastic antigen, KIAA0883
−3.7	TC33860_at	Neuronal IL-16
−3.875	TC37323_at	Similar to Iga regulatory protein
−4.1	TC32923_s_at	Corticosteroid II-beta dehydrogenase
−5.15	TC17814_at	Corticotropin releasing hormone receptor 2
−49.575	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
−2.925	TC38522_at	KIAA0916 human protein, highwine in drosophila
−3.05	TC14267_i_at	RKIP, phosphoatidylethandlumne binding protein
−3.175	TC17976_at	rsk2, calcium activated K-channel
−3.2	TC32675_s_at	Myelin gene expression factor, myef-2
−3.225	TC37684_at	Kinesin light subunit B, (KLC)
−3.5	TC26270_s_at	KIFI, kinesin like
−3.525	TC39656_f_at	Neuritin
−3.55	TC41874_at	ndr2
−3.725	TC22759_at	Calcineurin b.
−3.975	TC38290_at	Neuroendocrine differentiation factor
−4.05	TC16990_s_at	Neural vision like protein
−4.275	TC34140_s_at	Alpha-catenin (alpha -e)
−4.3	TC26822_at	Acrogranin
−4.675	TC24292_at	ATN2, Na/K transporter subunit 2
−4.8	TC30767_at	SCHIP-1
−6.325	TC37996_at	DARPP-32
−6.45	TC31890_g_at	Secretogranin III
−7.275	TC39376_at	Pro SSAS
−7.4	TC18154_at	Brain development protein BDM1
−7.8	TC39160_g_at	SCG10
−8.475	TC22283_s_at	PLP, myelin proteilipid
−9.85	TC36359_at	TREK-1 K+ channel subunit
−9.95	TC29998_at	Myelin basic protein
−10.025	TC39061_at	Hippocampal amyloid precursor protein (beta)
−11.65	TC14881_at	SCG10
−13.825	TC24570_at	Na/K transporting ATPase, alpha 2 subunit
−15	TC30390_s_at	p21, neuron specific protein
−16.925	TC18867_at	Alpha synuclein
−18.1	TC39041_at	Nel proptein
−18.9	TC26003_at	Synaplogamin XI
−19.175	TC37308_at	Double cortin like protein
−19.625	TC22741_g_at	Neuronal specific septin-3
−20.625	TC30389_f_at	p21, neuron specific protein
−20.925	TC35356_at	Neurilin
−21.25	TC18648_at	NFI-x nucleus factor
−22.35	TC36040_at	Like GCAP1, granule cell marker protein
−29.375	TC15940_g_at	Coat protein gamma cap.
−48.05	TC22429_s_at	Myelin proteolipid protein (PLP)
−74.375	TC17234_s_at	Calbindin
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
−3.225	TC18165_g_at	Prefoldin subunit
−3.575	TC21995_g_at	Microtubule associated protein, tan
−4.15	TC18613_at	Like novel protein c380A1,2.2.
−4.775	TC20757_s_at	ANK-3
−28.625	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
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
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
−2.8	TC14392_f_at	Stathmin
−2.85	TC18861_at	Unknown
−2.85	TC24550_at	Unknown
−2.925	TC39474_f_at	Lactate dehydrogynase
−2.975	TC33078_at	Unknown
−3	TC41056_at	Unknown
−3.125	TC32289_a_at	ABC2
−3.175	TC18736_g_at	Protein synthesis initiation factor 4A, ELF41A
−3.175	TC29636_at	Unknown
−3.175	TC41293_at	Unknown
−3.2	TC41345_at	Unknown
−3.225	TC17259_g_at	Unknown
−3.225	TC20592_at	Small nuclear ribonucleo protein, SmO
−3.25	TC34446_at	Unknown
−3.275	TC16825_at	Unknown
−3.275	TC24812_at	Unknown
−3.3	TC39752_at	Unknown
−3.3	TC40014_at	Unknown
−3.35	TC25091_at	Transcription repressor RPS8
−3.35	TC38493_at	Unknown
−3.35	TC22066_at	Unknown
−3.4	TC33205_at	SERCA 2n
−3.425	TC28327_at	Similar to EXTL2 human
−3.5	TC15898_f_at	Similar to LDOC1 human
−3.5	TC22782_g_at	Similar to cadherin 22
−3.525	TC22801_at	PAK-1 threoniae/serine kinase p21 activated
−3.575	TC35044_at	Unknown
−3.65	TC25610_s_at	Neodin
−3.7	TC37065_at	Same as FCJ10856 human protein
−3.875	TC27925_i_at	Plallet activating factor PAF acatythydroinse
−3.95	TC29662_at	Unknown
−3.975	TC22817_at	Brain protein 154
−4.05	TC38604_at	Unknown
−4.075	TC18152_at	Unknown
−4.075	TC38584_at	KIA1583 human protein
−4.125	TC34542_at	Ribonucleoprotein A2/B1
−4.15	TC19732_at	Unknown
−4.3	TC33029_g_at	Unknown
−4.3	TC37489_a_at	Mouse brain protein, BH5, peanut-like
−4.475	TC39750_at	Unknown
−4.525	TC39858_at	Unknown
−4.55	TC34778_at	Unknown
−4.575	TC38646_at	KIA0436, human, like oligopeptidase
−4.6	TC26109_at	Unknown
−4.6	TC36755_at	Neural membrane glyco protein, M6–8
−4.7	TC39581_at	Unknown
−4.725	TC22193_at	Transcriptional repressor RP58
−4.8	TC38235_at	Unknown
−4.86667	TC22191_at	Unknown
−4.95	TC16013_g_at	Unknown
−4.95	TC41433_at	Human KIA0436 like
−5.075	TC32823_at	Unknown
−5.1	TC40705_at	Unknown
−5.175	TC35606_at	Unknown
−5.2	TC33812_at	Unknown
−5.25	TC38211_at	EXCM1 gene, regulator SP2 transcription
−5.25	TC38294_at	Unknown
−5.325	TC34814_at	Unknown
−5.325	TC29508_at	Unknown
−5.4	TC37388_at	Transcriptional repressor RP58
−5.475	TC40986_at	Unknown
−5.55	TC21739_at	Unknown
−5.7	TC41479_at	Unknown
−5.7	TC15800_at	Unknown
−5.7	TC38214_at	Unknown
−5.725	TC16088_at	Unknown
−5.75	TC34430_at	Unknown
−5.825	TC21730_at	Unknown
−5.85	TC25709_g_at	Unknown
−5.675	TC26422_at	Unknown
−6.05	TC28579_at	Unknown
−6.475	TC25709_at	Unknown
−6.675	TC31827_at	Unknown
−6.7	TC16888_at	Unknown
−6.8	TC38692_s_at	Unknown
−8.85	TC35006_at	Unknown
−7.025	TC39106_at	Unknown
−7.675	TC23195_g_at	Unknown
−7.725	TC21739_g_at	Unknown
−7.775	TC39973_at	Unknown
−8.075	TC18151_at	Like globulin-7 (zeamayas)
−8.25	TC32823_g_at	Unknown
−8.475	TC20619_at	Similar to alpha/beta hydrolase-1
−8.675	TC40812_at	Unknown
−8.875	TC20884_at	Unknown
−8.975	TC21031_at	Unknown
−9.225	TC34440_g_at	p00071, plakophilin related protein
−9.25	TC15280_a_at	Unknown
−9.275	TC41038_at	Small GTP binding protein
−9.35	TC31813_at	Similar to (highly) GEF protein
−9.475	TC39216_at	HSPC322
−10.6	TC17986_at	Unknown
−10.6	TC41979_at	KIA1424
−11.275	TC41373_at	Unknown
−11.475	TC18252_at	Guanina nucleotide binding protein like
−12.075	TC15777_at	Unknown
−12.1	TC23934_at	RP58, transcriptional repressor
−12.25	TC42156_at	Unknown
−12.775	TC40806_at	Unknown
−13.075	TC41228_at	Unknown
−13.325	TC32742_at	Unknown
−13.875	TC34340_at	Unknown
−14.075	TC40224_at	KIA0898 like
−14.325	TC17313_at	Unknown
−14.475	TC28738_g_at	p53 inducible protein
−14.525	TC21034_at	KIAA0631 like
−14.575	TC22935_at	Unknown
−15.875	TC30317_g_at	VAV-3 protein (GEF protein)
−15.925	TC39390_at	Unknown
−16.325	TC19908_at	Formyltetrahydrofolute dehudrogenase
−16.05	TC38088_at	Unknown
−19.2	TC16183_at	HSPC328
−19.225	TC40805_at	Unknown
−20.025	TC42007_at	Unknown
−27.3	TC16885_at	Angiotensinogen precursor
−29.425	TC23193_a_at	HSPC251
−33.025	TC26229_at	Unknown
−35.675	TC29945_at	Unknown
−38.65	TC16736_at	Unknown
−45.925	TC19832_a_at	Stathmin like protein RB3
−48.375	TC41060_at	Beta 2 chimerin
−73.95	TC33089_at	Unknown
−76.825	TC29995_at	Unknown
−83.45	TC16446_at	Unknown
−116.975	TC39645_g_at	Unknown
−277.125	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.

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
−3.0	TC41056_at	Unknown
−3.1	TC14267_i_at	RKIP protein
−3.2	TC32675_s_at	MYEF-2
−3.2	TC17259_g_at	Unknown
−3.4	TC38493_at	Unknown
−3.4	TC40415_at	Cytochrome oxidase I
−3.5	TC30467_at	Cytochrome B
−4.0	TC38290_at	NEDF
−4.1	TC34542_at	Ribonucleoprotein A2/B1
−4.9	TC19304_at	ATP Synthase B
−5.2	TC35606_at	Unknown
−14.5	TC21034_at	Lipidosin
−38.7	TC16736_g_at	ELF41A

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

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.

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.