Human Molecular Genetics, 2002, Vol. 11, No. 22 2709-2721
© 2002 Oxford University Press
Cln3 
ex7/8 knock-in mice with the common JNCL mutation exhibit progressive neurologic disease that begins before birth
1Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA and 2The Jackson Laboratory, Bar Harbor, ME 04609, USA
Received May 17, 2002; Accepted August 16, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Juvenile-onset neuronal ceroid lipofuscinosis (JNCL; Batten disease) features hallmark membrane deposits and loss of central nervous system (CNS) neurons. Most cases of the disease are due to recessive inheritance of an
1 kb deletion in the CLN3 gene, encoding battenin. To investigate the common JNCL mutation, we have introduced an identical genomic DNA deletion into the murine CLN3 homologue (Cln3) to create Cln3 
ex7/8 knock-in mice. The Cln3 ex7/8 allele produced alternatively spliced mRNAs, including a variant predicting non-truncated protein, as well as mutant battenin that was detected in the cytoplasm of cells in the periphery and CNS. Moreover, Cln3 ex7/8 homozygotes exhibited accrual of JNCL-like membrane deposits from before birth, in proportion to battenin levels, which were high in liver and select neuronal populations. However, liver enzymes and CNS development were normal. Instead, Cln3 ex7/8 mice displayed recessively inherited degenerative changes in retina, cerebral cortex and cerebellum, as well as neurological deficits and premature death. Thus, the harmful impact of the common JNCL mutation on the CNS was not well correlated with membrane deposition per se, suggesting instead a specific battenin activity that is essential for the survival of CNS neurons. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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The juvenile-onset form of neuronal ceroid lipofuscinosis (NCL) (JNCL; Batten or Spielmeyer–Vogt disease) is a recessively inherited neurodegenerative disorder, whose symptoms, including loss of vision, seizures and psychomotor decline, become apparent at 5–10 years of age (1). JNCL is caused by mutations in CLN3, a novel 16p12.1 gene that encodes a predicted transmembrane protein (2) called battenin. While 31 different CLN3 mutations have been found (3), the vast majority of JNCL disease chromosomes (>80%) carry a common
1 kb CLN3 deletion that eliminates exons 7 and 8. Mutant mRNA, observed in JNCL tissue, was predicted to encode a frameshifted truncated product, that, consistent with recessive inheritance, presumably lacks battenin activity (2). The central nervous system (CNS) neurons and other cells of JNCL patients, including liver, lymphocytes and fibroblasts, contain autofluorescent membranous ‘fingerprint’ inclusions (4) enriched in ATP synthase subunit c, a mitochondrial inner membrane protein (5). These deposits, which are also formed in Cln3 knockout mice as a consequence of complete battenin deficiency (6,7), have implicated battenin in membrane turnover. However, battenin's activity remains to be identified. Importantly, a synaptosomal localization in neuronal cells, rather than the association with other secretory membranes in non-neuronal cells, may imply neuron-specific activities (8,9).
Moreover, while battenin is not essential for development (6,7), atrophy of cortical neurons in Cln3 knockout mice due to complete battenin deficiency (7) suggests a role in neuronal survival that may be disrupted in JNCL patients. This may, but need not, involve the formation of deposits that result from loss of battenin. However, because ‘knockout’ alleles are not faithful genetic JNCL replicas, Cln3 knockout mice do not reveal the impact of CLN3 mutations that actually lead to onset of disease in JNCL patients. This fundamental knowledge will be critical for the development of effective therapeutic strategies for this tragic disorder.
Therefore, to assess the consequences of the common JNCL mutation, we have used gene targeting in embryonic stem (ES) cells to create Cln3ex7/8 knock-in mice bearing the common 1 kb CLN3 deletion at the murine Cln3 locus. Our analyses have revealed Cln3ex7/8 mRNAs and mutant battenin products, membrane deposition and recessively inherited neurologic disease, extending data from JNCL patients and providing a unique resource with which to investigate and modify the early consequences of the JNCL mutation.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of JNCL common mutation Cln3
ex7/8 knock-in miceTo reproduce the common
1 kb CLN3 deletion mutation in a precise genetic JNCL mouse model, we ‘knocked-in’ an identical 1 kb genomic DNA deletion, eliminating exons 7 and 8 and surrounding non-coding DNA (ex7/8 allele). The gene targeting strategy is summarized in Figure 1A. Homologous recombination in embryonic stem cells replaced the 1 kb segment of Cln3 DNA with a ‘floxed’ PGKneo cassette, generating Cln3ex7/8neo mice. Matings with hCMV–cre transgenic mice, expressing Cre recombinase, then produced progeny in which the floxed neo cassette was excised. Subsequent breeding segregated the cre transgene to yield the Cln3ex7/8 knock-in mouse line, carrying the common JNCL mutation. As shown in Figure 1B, hybridization of Southern blots of EcoRv- and SpeI-digested tail-clip DNAs with probes for Cln3 sequences outside the targeting vector, at 5' (probe A) and 3' (probe B) locations, revealed the expected restriction fragments, confirming proper targeting and excision of the neo cassette.
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The common mutation yields multiple stable mRNA splice variants
Northern blot analyses of Poly(A)+ RNA isolated from peripheral tissue and brain of wild-type and homozygous Cln3
ex7/8 mice was performed to confirm, in the latter, the exon 7 and 8 deletion and to assess the impact of the deletion mutation at the mRNA level. As shown in Figure 2A, the full-length Cln3 cDNA probe revealed an 1.7 kb band in the wild-type mRNAs, with levels in brain lower than in either liver or kidney, consistent with reported expression of human CLN3 mRNA (2). Furthermore, mRNA from Cln3ex7/8 homozygotes yielded a band of similar size, although at reduced signal intensity. Densitometry of Cln3-specific signal, normalized to ß-actin levels (not shown), indicated Cln3ex7/8 mRNA levels that were decreased by 10.8-, 9.3- and 4.5-fold in kidney, liver and brain, respectively, indicating stable mutant mRNA, at reduced levels. As the 200 bp mRNA mobility shift, due to loss of exons 7 and 8, was difficult to discern, the northern blot was hybridized with an exon 7/8 probe. This revealed the wild-type mRNA band but not the mRNA transcripts in Cln3ex7/8 homozygote tissues, thereby confirming the common JNCL deletion.
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To identify the Cln3
ex7/8 mRNA transcripts, we performed RT–PCR amplification of mRNA from peripheral tissue and brain, with exon 1 forward and exon 15 reverse primers. cDNA products shown in Figure 2B were cloned and sequenced. The major 1.6 kb band amplified from wild-type mRNA represented full-length Cln3 cDNA. In contrast, all Cln3ex7/8 products lacked exons 7 and 8. The 1.4 kb product missed only exons 7 and 8, while two 1.5 kb products retained either intron 10 or 11, encoding an 19 kDa truncated, frameshifted product (2). The 1.6 kb product missed exons 7 and 8 but retained intron 1, encoding a more severe truncation (25 amino acids). However, the 1.3 kb Cln3ex7/8 cDNA was deleted for exons 7, 8 and 5. This mutant cDNA encoded an 38 kDa protein comprising the N terminus (exons 1–4) and C terminus (exons 9–15) of battenin, with a novel 29-residue midsection encoded by frameshifted exon 6 (Fig. 2C). Notably, database searches (BLASTN) revealed a human homologue of this non-truncating species in GenBank (accession no. AF015598), cloned from a JNCL patient carrying the common deletion mutation.
Cln3ex7/8 homozygote tissues exhibit battenin immunoreactivity
As the latter Cln3ex7/8 mRNA suggested a mutant protein that might retain the portion of battenin C-terminal of the exon 7/8 deletion, we performed analyses with affinity-purified polyclonal reagents batp1 and batp2, which detect downstream epitopes (depicted in Fig. 2C). In a variety of immunoblot formats, batp1 and batp2, like the majority of battenin reagents (8,10), did not reliably detect endogenous battenin, but each reagent specifically revealed an 44 kDa doublet of battenin overexpressed in extracts of a tetracycline (Tet-off) inducible CLN3 cDNA neuroglioma cell line (Fig. 2D). Preabsorption with appropriate peptide eliminated detection of both bands, demonstrating the specificity of these reagents. The slowly migrating batp1 (and batp2) reactive species represented a glycosylated form of battenin, since this band was not detected when induced cell extracts were pretreated with endoglycosidases (not shown).
However, as reported for other battenin reagents (8,10), batp1 and batp2 yielded specific signal when used in immunohistochemistry formats with fixed cells and tissue. As shown in Figure 3, Cln3ex7/8 heterozygote and wild-type tissue sections (not shown) exhibited batp1 staining in the cytoplasm of cells in the liver, with a halo of darkly stained hepatocytes surrounding arteries. A punctate cytoplasmic staining pattern was found in cells with neuronal morphology in all brain regions surveyed, but most prominently in hippocampus, cerebellum, cortex and thalamus (not shown). Notably, the cytoplasmic batp1 reactivity, although not the nuclear staining was eliminated by preabsorption with the antigenic peptide. Moreover, as shown in Figure 3 for cerebellum and cortex, Cln3ex7/8 heterozygote sections stained with batp2 reagent exhibited a distribution and pattern of cytoplasmic reactivity closely similar to batp1, which also was eliminated by preabsorption with the antigen peptide (data not shown). Notably, the specific batp1 and batp2 staining mirrored the subcellular and regional distribution of battenin reported with an independent reagent in wild-type C57BL mice (9).
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Remarkably, as shown in Figure 3, Cln3
ex7/8 homozygote tissues exhibited batp1 and batp2 immunostaining that was similar to the wild-type or heterozygous Cln3ex7/8 pattern. However, as shown for batp1, the intensity of staining was markedly decreased in liver and slightly reduced in brain, consistent with decreased Cln3ex7/8 mRNA levels. Reduced levels of staining in homozygous mutant brain sections were also detected by batp2, presented for cerebellum and cortex in Figure 3. Notably, the punctate cytoplasmic batp1 and batp2 staining was eliminated by preabsorption with the appropriate peptide antigen. Thus, cytoplasmic battenin immunostaining detected in Cln3ex7/8 periphery and CNS neurons by independent affinity-purified reagents strongly suggests stable mutant protein, with epitopes C-terminal to the exon 7/8 deletion.
JNCL-like membrane deposition in the periphery and CNS
To determine whether the common JNCL mutation leads to membrane deposition, we surveyed liver and CNS of Cln3ex7/8 homozygotes and littermates at 10 months of age (Fig. 4). All Cln3ex7/8 homozygote tissues examined exhibited abundant autofluorescent accumulations, evident by confocal microscopy, and ATPase subunit c-reactive deposits, detected by immunostaining. The most extensively affected cells were hepatocytes in the liver, the only peripheral tissue examined, CA2 and CA3 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, cortical neurons and thalamic neurons (not shown). Retinal neurons possessed deposits in the retinal ganglion cell layer (RGC) and the inner nuclear layer (INL), with small punctate granules in the outer nuclear layer (ONL), the location of photoreceptor cell bodies.
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Ultrastructural electron-microscopic examination revealed a classic JNCL-like ‘fingerprint’ inclusion profile, shown in Figure 5. The cytoplasm of hippocampal CA2 pyramidal cells and retinal ganglion cells exhibited electron-dense membranous perinuclear inclusions, not found in wild-type cells. The deposits in Cln3
ex7/8 homozygotes, therefore, conformed to the classic features of membranous inclusions in JNCL tissue.
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Cln3
ex7/8 deposits onset before birthWe next investigated the early temporal course of membrane protein deposition, using ATPase subunit c-immunostaining to monitor liver and CNS of Cln3
ex7/8 homozygotes at embryonic day 19.5 (E19.5), postnatal days 1.5 and 8 (P1.5 and P8) and 1 month of age. The results (Fig. 6) revealed extensive deposits in liver cells as early as E19.5, presaging the huge inclusion burden at 10 months of age. In the CNS, accumulates were detected at E19.5 in cerebellar Purkinje cells, in neurons of the dentate gyrus of the hippocampus and in the subventricular zone at the lateral ventricle. For the latter two regions, reactive deposits were not apparent from P8 onward, likely due to stage-appropriate migration of deposit-laden neurons, consistent with normal brain morphology in adult Cln3ex7/8 homozygotes. Hippocampal CA2/CA3 pyramidal neurons, with severe inclusions at 10 months, first exhibited subunit c-reactive deposits at P8, increasing with age. Retinal deposits were first observed at P1.5, within the neuroblastic layer (NBL), where progenitor neurons that will form the ONL and the INL of the mature retina are localized. Punctate subunit c-reactive granules were evident within the ONL and INL in the mature retina at P8 and 1 month.
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Cln3
ex7/8 homozygotes exhibit neurologic deficits and decreased survivalMicroscopic examination of immunostained or hematoxylin stained (not shown) sections did not reveal changes in brain or retinal architecture that distinguished Cln3
ex7/8 homozygotes. However, as illustrated in Figure 7A, at 10–17 months, a reduction in photoreceptors stained with the cone marker peanut agglutinin (11) was evident. Retinas of hypopigmented Cln3ex7/8 homozygote mice exhibited significantly fewer (P<0.0001) cone cells per field than retinas from hypopigmented Cln3ex7/8 heterozygote littermates (n=3; mean±SD=60±18 versus n=3; mean±SD=94±19), an 37% decrease that indicated retinal cell degeneration. Interestingly, retinas from pigmented Cln3ex7/8 homozygote littermates (n=2) did not exhibit obvious cone cell loss at this age, suggesting that hypopigmentation may hasten retinal degeneration.
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Furthermore, glial fibrillary acidic protein (GFAP) immunostaining revealed striking reactive gliosis in the motor cortex (Fig. 7A), white matter tracts of the cerebellum and midbrain, including the inferior colliculus (not shown), of all 10-month homozygous mutant but no 10-month wild-type or heterozygous Cln3
ex7/8 mice. This finding is indicative of a response to neuronal cell injury, although TUNEL stain did not reveal apoptotic changes (not shown).
To test for neurologic impairment, we performed behavioral assays with Cln3ex7/8 homozygotes and littermate controls, at 10–12 months, revealing recessive phenotypes that may not be fully penetrant at these ages. The results of a tail hang/clasping assay (Fig. 7B) revealed clasping in 42% of Cln3ex7/8 homozygotes, compared with 10% of wild-type and Cln3ex7/8 heterozygote mice. Gait analyses, measuring hind and fore paw tracks left during tunnel walks (Fig. 2C), revealed shortened average stride length (rear paw) for homozygous mutant mice (5.78±0.08 cm versus 6.11±0.08 cm for controls; P=0.004), and increased base width (rear paw stance) (3.06±0.02 cm, versus 2.95±0.03 cm for controls; P=0.005).
Furthermore, as shown in Figure 7D, when followed until 12 months of age, Cln3ex7/8 homozygotes exhibited a reduced lifespan (80% survival; P=0.01), compared with their littermates (heterozygote and wildtype), with the earliest death at 7 months of age. The proximal cause of premature death is not evident, since the homozygous mutant mice did not exhibit handling-induced seizures or other obvious life-threatening abnormalities.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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To investigate the consequences of the CLN3 mutation that is associated with the vast majority of JNCL cases, we have created precise genetic JNCL Cln3
ex7/8 knock-in mice carrying the common 1 kb genomic DNA deletion. Homozygous mutant mice displayed membrane accumulation in peripheral cells and CNS neurons, evident in embryos, followed by degeneration of retinal neurons, CNS reactive astrocytosis, neurologic deficits and reduced survival. These findings provide experimental support for a chronic recessive disease process that begins before birth (12). Remarkably, this process did not obviously perturb normal CNS development, but instead caused the demise of mature neurons. The common JNCL mutation did not abrogate Cln3 expression, consistent with mRNA in JNCL patient tissue (2). We identified exon 7/8-deleted mRNAs that encode severely truncated polypeptides and an alternatively spliced mRNA, ex5/7/8del, encoding a non-truncated mutant protein. Immunohistochemistry results strongly suggest a non-truncated mutant protein that merits investigation to determine whether it retains battenin activity. An examination of this mutant variant and other battenin isoforms will be required for a full understanding of the impact of the common JNCL mutation. However, as endogenous battenin has not been reliably detected in immunoblot format with antibody reagents (8,10), the subcellular distribution and stability of these mutant variants may need to be studied in overexpression transfected cell systems.
The level of battenin activity that is sufficient to forestall neurologic disease is not yet known, although the threshold must lie between 50% and 0%. Heterozygous Cln3ex7/8 mice and human JNCL carriers do not exhibit overt disease, while complete deficiency causes cortical neuron atrophy (7) and low levels of retinal cell apoptosis (0.2%) in Cln3 knockout mice (13). Thus, if non-truncated mutant battenin, or other mutant isoforms detected in Cln3ex7/8 knock-in mice retain battenin activity, it is possible that the disease might be attacked in the majority of JNCL patients by increasing the levels of expression of the deleted CLN3 gene.
The normal battenin levels may anticipate the severity of membrane deposition in the disease. This suggests that the physiologic requirement for battenin activity, which varies from cell to cell (14), may determine the rate of membrane accrual. Liver cells exhibited abundant battenin staining, while different populations of CNS neurons displayed graded levels, The most prominent were neurons of the hippocampus, cortex, thalamus and cerebellum, in agreement with the findings of Luiro et al. (9). Moreover, in Cln3ex7/8 homozygotes, battenin-reactivity was not localized to membrane deposits in brain, although the largest accumulates in liver were stained (not shown). Thus, if mutant staining accurately reflects wild-type protein, battenin may not be a normal component of the membrane that forms neuronal deposits in the disease. This suggests that battenin may not act at the inner mitochondrial membrane but rather at some other step in mitochondrial autophagy that when disrupted leads to ATP synthase subunit c deposits.
The inclusion burden did not accurately predict harmful cellular consequences of the common JNCL mutation. For example, membrane deposits in the liver of homozygote mutant embryos increased to prodigious size with age, but serum transaminase (aminotransferase) levels were unchanged, suggesting relatively unimpaired liver function (not shown). On the other hand, retinal neurons in the outer nuclear layer displayed only small inclusions but exhibited neurodegenerative changes by 10 months of age. Thus, despite less obvious membrane deposition, the consequences of lack of battenin function were more severe for CNS neurons. This apparent paradox suggests that membrane deposits themselves may not be a ‘toxic’ entity. Instead, the disease process may involve a specific battenin-dependent pathway that is essential for neuronal cell survival, consistent with battenin in neurons, in a novel axonal syntaptosome compartment (9).
The premature death of Cln3ex7/8 homozygotes, although not fully penetrant by 12 months of age, revealed the vulnerability of the whole organism to the common JNCL mutation. The immediate cause of death was not apparent; development appeared to be normal, as found with complete battenin deficiency (6,7), and major organ systems, such as the liver, did not exhibit dysfunction. Given the neurodegenerative changes, it is possible that death may result from CNS dysfunction, although seizures, prominent in JNCL patients, were not apparent.
Disease phenotypes in individual Cln3ex7/8 mice exhibited variability in onset. This may reflect incomplete penetrance at these ages, or, as observed in JNCL patients, the disease phenotypes elicited by the Cln3ex7/8 mutation may display variable expressivity. For example, although reactive gliosis was found in all mutant homozygotes by 10 months, only a proportion exhibited neurologic dysfunction and retinal neuron dropout at this age. The latter phenotype was evident in hypopigmented mice, suggesting that placing the Cln3ex7/8 allele on inbred strain backgrounds that differ in pigmentation may modify the pathologic process in retinal neurons. Cln3ex7/8 strains on standard genetic backgrounds would facilitate analyses to determine whether retinal cell loss is associated with impaired visual function. Thus, Cln3ex7/8 mice can be used to identify disease modifiers, providing candidates for influencing disease in JNCL patients.
CLN3 is one of several genes [CLN1 (15), CLN2 (16), CLN5 (17), CLN6 (18,19) and CLN8 (20)] that when disrupted cause recessively inherited NCL in humans. The similarities among the human disease phenotypes, animals with spontaneous mutations in disease gene homologues (21–24), and Cln1 and Cln3 knockout mice (6,7,25) suggest that battenin and the CLN-encoded proteins may act in a shared pathway or complex (10). However, phenotypic differences produced by distinct loci or mutations may also provide clues to the disease process. Compared with Cln3 knockout mice, the common JNCL mutation appears to cause a more severe neurologic disease, with evident loss of photoreceptors (37% compared with 0.2%), neurologic motor deficits and decreased survival, Thus, Cln3ex7/8 mice, precise genetic replicas of the mutation that causes disease in most JNCL patients, provides a resource for identifying early events in the disease process and an optimal resource for assessing potential therapeutics.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Gene targeting in ES cells and generation of knock-in mice
Cln3 genomic DNA fragments, comprising a 5'
4.5 kb BamHI–EcoRI fragment (upstream of exon1 through intron 6) and a 3' 3.8 kb HindIII fragment (intron 8 through intron 13), were ligated into the pPNT1ox2 vector, with negative (PGKtk) and positive (PGKneo) selection, with lox P sites flanking the PGKneo. The targeting vector was linearized by digestion with NotI and electroporated into R1 ES cells. ES cell clones were selected for G418 resistance and screened for correct targeting by Southern blot analysis of EcoRv- and SpeI-digested genomic DNA using DNA probes 5' (probe A) and 3' (probe B) of the targeting vector (Fig. 1A).
Correctly targeted ES cell clones were microinjected into blastocysts to generate a line of Cln3ex7/8neo mice from one male founder. The Cln3ex7/8 allele was achieved by mating Cln3ex7/8neo mice with hCMV–cre transgenic males for excision of PGKneo, as described previously (26). Excision of the PGKneo in somatic tissues (brain, eyes, liver, kidney and spleen) of Cln3ex7/8 mice at the F2 generation was confirmed by Southern blot using probe A. Intercross matings and subsequent matings of cre-negative offspring to CD1 mice segregated the hCMV–cre transgene, confirmed by specific PCR assay (27). The finished Cln3ex7/8 allele was maintained on an outbred 129Sv/Ev/CD1 background, which produces white (hypopigmented) and agouti progeny.
Northern blot analysis and RT–PCR
Total RNA was extracted from dissected tissues using the RNAqueous RNA Isolation Kit (Ambion). mRNA was further purified from total RNA extracts with the MicroPoly(A) Purist kit (Ambion). For northern blotting, poly(A) RNAs were separated on a 1.2% denaturing agarose gel and transferred to nylon, and blots were hybridized with a 32P-labeled probe in ExpressHyb hybridization solution (Clontech), for 14 h at 65°C. Blots were washed according to instructions, and visualized by autoradiography using XAR film (Kodak). For multiple probings, blots were stripped using Clontech's protocol. Densitometry was performed on scanned blots using Scion Image software (Scion Corporation).
RT–PCR using total or poly(A) RNA as template was performed using the Thermoscript RT–PCR system (Invitrogen). OligodT-primed cDNA was PCR-amplified using primers ‘exonl-forward’ (5'-ACCCACCTGTCCCAGACTTTA-3') and ‘exon 15-reverse’ (5'-AGGGCAGAGGGTAGGAGAAA-3') with the following cycling parameters: 1 cycle= 94°C, 2 min; 35 cycles=94°C, 1 min, 55°C 30 s, 72°C, 3 min; 1 cycle=72°C, 15 min PCR reactions were separated on a 2% agarose gel and cloned using the TOPO PCR Cloning Kit (Invitrogen). Sequencing was performed on an ABI Prism 377 automated sequencer.
Anti-battenin reagents
Batp1 (human battenin amino acids 236–252) and batp2 (human battenin amino acids 391–409) peptides, with an added N-terminal cysteine, were synthesized, and used for polyclonal antibody production in rabbits (coupled to keyhole limpet hemocyanin) and affinity-purification by Quality Controlled Biochemicals/Biosource International and Research Genetics, respectively. Extracts of a stably transfected CLN3 cDNA neuroglioma cell line, with tetracycline (Tet-off)-inducible (Clontech) battenin expression, were generated by lysis in RIPA buffer of pelleted uninduced (1 mg/ml Tet, 8 h) and induced (0 mg/mlTet, 8 h) cells. Proteins were separated on 10% Tris–glycine SDS–PAGE and transferred to nitrocellulose. Immunoblots were probed with batp1 (1 : 400) or batp2 (1 : 50), incubated with peroxidase-conjugated anti-rabbit IgG (1 : 10 000, Amersham Pharmacia) and developed by Western Lightning chemiluminescence (Perkin Elmer). Preabsorption was with 20 µg/ml of appropriate antigen peptide. In an immunostaining format, affinity-purified batp1 and batp2 stained paraformaldehyde-fixed cultured neuronal cells in a punctate cytoplasmic pattern, extending into long projections as varicosities and focal adhesions, in a pattern also described by Luiro et al. (9) in primary retinal cultures (data not shown). In an immunohistochemistry format, with fixed sections of brain and peripheral tissue, the affinity-purified batp1 and batp2 reagents detected specific reactivity that was not detected by pre-immune sera. To confirm specificity in this format, affinity-purified antibody was pre-incubated at room temperature for 1 h with 20 µg/ml of the antigen peptide conjugated to agarose beads, and the depleted supernatant, recovered by centrifugation, was incubated with tissue sections.
Histology and immunostaining
For light and fluorescent microscopic analysis, adult mice were deeply anesthetized by xylazine/ketamine and sacrificed by cardiac perfusion of ice-cold phosphate-buffered saline (PBS, PH 7.4), followed by periodate–lysine–phosphate (PLP) fixative (2% paraformaldehyde, 0.01 M sodium periodate and 0.1 M lysine phosphate buffer, pH 7.4). Tissues were dissected, and fixation was continued overnight in PLP fixative at 4°C. For embryo studies, vaginal plug appearance was considered E0.5. Tissues were collected from E19.5, P1.5 and P8 mice and were fixed by incubation in PLP overnight at 4°C. All tissues were then rinsed, processed, paraffin-embedded. and sectioned at a 7 µm thickness.
For autofluorescence experiments, paraffin sections were dewaxed in xylene, rehydrated through an ethanol series (2x100%, 2x95%, 2x75%, 50%, 30%), and rinsed briefly in dH2O. Sections were coverslipped in Vectashield mounting medium (Vector Laboratories) to prevent photobleaching. Autofluorescence was detected at 568 nm on a BioRad Radiance 2100 confocal system (Biorad).
Immunostaining was performed as follows. Paraffin sections were dewaxed and rehydrated, as above, and washed in Tris-buffered saline (TBS, pH 7.5). Antigen retrieval was performed by boiling in citric acid buffer, pH 6 (2x boil 5 min, cool 20 min), followed by incubation in 1% sodium dodecyl sulfate (SDS) at ambient temperature for 5 min. Sections were extensively rinsed in TBS for removal of SDS and processed for immunostaining using Vectastain ABC-peroxidase staining kits, according to the manufacturer's protocol (Vector Laboratories). Antibodies and dilutions were as follows: batp1, 1 : 400; batp2, 1 : 50; anti-subunit c, 1 : 500 (a gift from Dr Kominami) (28); and anti-glial fibrillary acidic protein (GFAP), 1 : 2000 (DAKO Corporation, Carpinteria, CA). Biotinylated peanut agglutinin (PNA, 5 µg/ml, Vector Laboratories) was used to specifically label cone photoreceptors (11).
TUNEL staining was performed on dewaxed and rehydrated paraffin sections, according to the procedure of Namura et al. (29). Incorporated biotinylated dUTP was detected using the avidin biotin detection system (Vectastain ABC Kit, Vector Laboratories).
Electron microscopy
Mouse tissues for electron microscopic analysis were prepared as above, except that perfusion was with 1.25% paraformaldehyde, 2.5% glutaraldehyde and 0.03% picric acid in 100 mM cacodylate buffer, pH 7.4. Tissues were postfixed for 2 h (1% osmium tetroxide and 1.5% potassium ferrocyanide) and embedded in an Epon–Araldite mixture (Electron Microscopy Sciences) for ultrathin sectioning (95 nm). Sections were mounted on formvar-coated grids and counterstained with 2% uranyl acetate followed by 0.2% lead citrate. Transmission electron microscopy (TEM) was performed on a JEOL 1200EX electron microscope.
Retinal cell counts
Peanut agglutinin-stained retinal cross-sections were viewed under a 20x objective on a light microscope for cell count determination. For each section, five or six fields were counted. Values are given as average cells per field±standard deviation, Only white eyes were considered in the cell counts. Statistical significance was determined by a two-tailed Student's t-test using Microsoft Excel software (Microsoft Corporation).
Behavioral analyses
Mice were tested for clasping behavior in a 1 min tail-hang assay. Individuals were scored positive for clasping if limbs were clenched towards the belly for 
5 s. Three trials, on separate days, were performed by an investigator blinded to the genotypic status of the mice. Gait traces were produced by walks in a paper-lined tunnel apparatus, of mice with differentially painted hind- and forepaws. Four trials, on two separate days, were performed for each mouse, and measurements were determined by blinded investigators. Statistical significance was tested in a two-tailed Student's t-test using Microsoft Excel software (Microsoft Corporation).
Serum transaminase assay
For serum collection, mice were deeply anesthetized by isofluorane and sacrificed by decapitation. Blood was collected and serum was separated using Microtainer serum separator tubes, according to the manufacturer's recommendations (Becton Dickinson). To assess liver function, serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) were measured using the ALT and AST manual assay kit (Sigma Diagnostics).
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ACKNOWLEDGEMENTS |
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The authors thank Dr E. Kominami for antibody to subunit c of mitochondrial ATPase, L. Trakimas and M. Ericsson of the Harvard Medical School Electron Microscope Facility for assistance with TEM studies, and the MGH Genomics Core Facility for DNA sequencing. We also thank Drs R. Wetzel and S. Gustincich for helpful discussions. This work was supported by the JNCL Research Fund and by NIH/NINDS Grants NS 32099 and NS 33648. S.L.C. is the recipient of a Fellowship from the Batten Disease Support and Research Association (BDSRA).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Molecular Neurogenetics Unit, Massachusetts General Hospital, Building 149, 13th Street, Charlestown, MA 02129 USA. Tel: +1 6177265089; Fax: +1 6177265735; Email: macdonam{at}helix.mgh.harvard.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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1 Kohlschutter, A., Gardiner, R.M. and Goebel, H.H. (1993) Human forms of neuronal ceroid-lipofuscinosis (Batten disease): consensus on diagnostic criteria, Hamburg 1992. J. Inherit. Metab. Dis., 16, 241–244.[Web of Science][Medline]
2 International Batten Disease Consortium (1995) Isolation of a novel gene underlying Batten disease, CLN3. Cell, 82, 949–957.[Web of Science][Medline]
3 Mole, S.E., Zhong, N.A., Sarpong, A., Logan, W.P., Hofmann, S., Yi, W., Franken, P.F., van Diggelen, O.P., Breuning, M.H., Moroziewicz, D. et al. (2001) New mutations in the neuronal ceroid lipofuscinosis genes. Eur. J. Paediatr. Neurol., 5 (Suppl. A), 7–10.[Medline]
4 Wisniewski, K.E., Rapin, I. and Heaney-Kieras, J. (1988) Clinico-pathological variability in the childhood neuronal ceroid-lipofuscinoses and new observations on glycoprotein abnormalities. Am. J. Med. Genet., 5(Suppl), 27–46.
5 Palmer. D.N., Fearnley, I.M., Walker, J.E., Hall, N.A., Lake, B.D., Wolfe, L.S., Haltia, M., Martinus, R.D. and Jolly, R.D. (1992) Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease). Am. J. Med. Genet., 42, 561–567.[Web of Science][Medline]
6 Katz, M.L., Shibuya, H., Liu, P.C., Kaur, S., Gao, C.L. and Johnson, G.S. (1999) A mouse gene knockout model for juvenile ceroid-lipofuscinosis (Batten disease). J. Neurosci. Res., 57, 551–556.[Web of Science][Medline]
7 Mitchison, H.M., Bernard, D.J., Greene, N.D., Cooper, J.D., Junaid, M.A., Pullarkat, R.K., de Vos, N., Breuning, M.H., Owens, J.W., Mobley, W.C. et al. (1999) Targeted disruption of the Cln3 gene provides a mouse model for Batten disease. The Batten Mouse Model Consortium [corrected]. [Erratum (2000) Neurobiol. Dis., 7, 127.] Neurobiol. Dis., 6, 321–334.[Web of Science][Medline]
8 Pearce, D.A. (2000) Localization and processing of CLN3, the protein associated to Batten disease: Where is it and what does it do? J. Neurosci. Res., 59, 19–23.[Web of Science][Medline]
9
Luiro, K., Kopra, O., Lehtovirta, M. and Jalanko, A. (2001) CLN3 protein is targeted to neuronal synapses but excluded from synaptic vesicles: new clues to Batten disease. Hum. Mol. Genet., 10, 2123–2131.
10
Vesa, J., Chin, M.H., Oelgeschläger, K., Isosomppi, J., DellAngelica, E.C., Jalanko, A. and Peltonen, L. (2002) Neuronal ceroid lipofuscinoses are connected at molecular level: Interaction of CLN5 protein with CLN2 and CLN3. Mol. Biol. Cell, 13, 2410–2420.
11
Chen, J., Tucker, C.L., Woodford, B., Szel, A., Lem, J., Gianella-Borradori, A., Simon, M.I. and Bogenmann, E. (1994) The human blue opsin promoter directs transgene expression in short-wave cones and bipolar cells in the mouse retina. Proc. Natl Acad. Sci. USA, 91, 2611–2615.
12 Munroe, P.B., Rapola, J., Mitchison, H.M., Mustonen, A., Mole, S.E., Gardiner, R.M. and Jarvela, I. (1996) Prenatal diagnosis of Batten's disease. Lancet, 347, 1014–1015.[Web of Science][Medline]
13 Seigel, G.M., Loery, A., Kummer, A., Bernard, D.J., Greene, N.D.E., Turmaine, M., Derksen, T., Nussbaum, R.L., Davidson, B.A., Wagner, J. et al. (2002) Retinal pathology and function in a Cln3 knockout mouse model of juvenile neuronal ceroid lipofuscinosis (Batten disease). Mol. Cell. Neurosci., 19, 515–527.[Web of Science][Medline]
14 Chattopadhyay, S. and Pearce, D.A. (2000) Neural and extraneural expression of the neuronal ceroid lipofuscinoses genes CLN1, CLN2, and CLN3: functional implications for CLN3. Mol. Genet. Metab., 71, 207–211.[Web of Science][Medline]
15 Vesa, J., Hellsten, E., Verkruyse, L.A., Camp, L.A., Rapola, J., Santavuori, P., Hofmann, S.L. and Peltonen, L. (1995) Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature, 376, 584–587.[Medline]
16
Sleat, D.E., Donnelly, R.J., Lackland, H., Liu, C.G., Sohar, I., Pullarkat, R.K. and Lobel, P. (1997) Association of mutations in a lysosomal protein with classical late-infantile neuronal ceroid lipofuscinosis. Science, 277, 1802–1805.
17 Savukoski, M., Klockars, T., Holmberg, V., Santavouri, P., Lander, E.S. and Peltonen, L. (1998) CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis. Nat. Genet., 19, 286–288.[Web of Science][Medline]
18 Gao, H., Boustany, R.M., Espinola, J.A., Cotman, S.L., Srinidhi, L., Antonellis, K.A., Gillis, T., Qin, X., Liu, S., Donahue, L.R. et al. (2002) Mutations in a novel CLN6-encoded transmembrane protein cause variant neuronal ceroid lipofuscinosis in man and mouse. Am. J. Hum. Genet., 70, 324–335.[Web of Science][Medline]
19 Wheeler, R.B., Sharp, J.D., Schultz, R.A., Joslin, J.M., Williams, R.E. and Mole, S.E. (2002) The gene mutated in variant late-infantile neuronal ceroid lipofuscinosis (CLN6) and in nclf mutant mice encodes a novel predicted transmembrane protein. Am. J. Hum. Genet., 70, 537–542.[Web of Science][Medline]
20 Ranta, S., Zhang, Y., Ross, B., Lonka, L., Takkunen, E., Messer, A., Sharp, J., Wheeler, R., Kusumi, K., Mole, S. et al. (1999) The neuronal ceroid lipofuscinoses in human EPMR and mnd mutant mice are associated with mutations in CLN8. Nat. Genet., 23, 233–236.[Web of Science][Medline]
21 Pardo, C.A., Rabin, B.A., Palmer, D.N. and Price, D.L. (1994) Accumulation of the adenosine triphosphate synthase subunit C in the mnd mutant mouse. A model for neuronal ceroid lipofuscinosis. Am. J. Pathol., 144, 829–835.[Abstract]
22 Bronson, R.T., Donahue, L.R., Johnson, K.R., Tanner, A., Lane, P.W. and Faust, J.R. (1998) Neuronal ceroid lipofuscinosis (nclf), a new disorder of the mouse linked to chromosome 9. Am. J. Med. Genet., 77, 289–297.[Web of Science][Medline]
23 Koppang, N. (1973) Canine ceroid-lipofuscinosis—a model for human neuronal ceroid-lipofuscinosis and aging. Mech. Ageing Dev., 2, 421–445.[Web of Science][Medline]
24 Jolly, R.D., Martinus, R.D. and Palmer, D.N. (1992) Sheep and other animals with ceroid-lipofuscinoses: their relevance to Batten disease. Am. J. Med. Genet., 42, 609–614.[Web of Science][Medline]
25
Gupta, P., Soyombo, A.A., Atashband, A., Wisniewski, K.E., Shelton, J.M., Richardson, J.A., Hammer, R.E. and Hofmann, S.L. (2001) Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice. Proc. Natl Acad. Sci. USA, 98, 13566–13571.
26 White, J.K., Auerbach, W., Duyao, M.P., Vonsattel, J.P., Gusella, J.F., Joyner, A.L. and MacDonald, M.E. (1997) Huntingtin is required for neurogenesis and is not impaired by the Huntington's disease CAG expansion. Nat. Genet., 17, 404–410.[Web of Science][Medline]
27 Tsien, J.Z., Chen, D.F., Gerber, D., Tom, C., Mercer, E.H., Anderson, D.J., Mayford, M., Kandel, E.R. and Tonegawa, S. (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell, 87, 1317–1326.[Web of Science][Medline]
28
Kominami, E., Ezaki, J., Muno, D., Ishido, K., Ueno, T. and Wolfe, L.S. (1992) Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease). J. Biochem., 111, 278–282.
29
Namura, S., Zhu, J., Fink, K., Endres, M., Srinivasan, A., Tomaselli, K.J., Yuan, J. and Moskowitz, M.A. (1998) Activation and cleavage of caspase-3 in apoptosis induced by experimental cerebral ischemia. J. Neurosci., 18, 3659–3668.
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