Human Molecular Genetics, 2001, Vol. 10, No. 19 2123-2131
© 2001 Oxford University Press
CLN3 protein is targeted to neuronal synapses but excluded from synaptic vesicles: new clues to Batten disease
National Public Health Institute, Department of Molecular Medicine, Biomedicum Helsinki, PO Box 104, Haartmaninkatu 8, FIN-00251 Helsinki, Finland and 1Department of Neurology, Kuopio University Hospital, 70211 Kuopio, Finland
Received May 30, 2001; Revised and Accepted July 16, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL), the most common neurodegenerative disease of childhood, is caused by mutations in the CLN3 gene encoding a putative transmembrane protein. The function of CLN3 is currently unknown but it has been shown to localize in the endosomal/lysosomal compartments of non-neuronal cells. In addition, several other intracellular localizations have been proposed and the controversy of the reports suggests that CLN3 may have different intracellular localization in different cell types. Batten disease severely affects neuronal cells but leaves other organs clinically unaffected, and thus it is of utmost importance to approach the disease mechanism by studying the expression and localization of CLN3 in the brain and neuronal cells. We have analysed here CLN3 in the mouse brain using in situ hybridization, immunohistochemical staining and western blot analysis of subcellular fractions. As visual deterioration is the hallmark of Batten disease we have set up primary retinal cultures from the mouse and analysed both endogenous mouse CLN3 and Semliki Forest virus-mediated human CLN3 localization using immunofluorescence staining and confocal microscopy. We demonstrate that CLN3 is abundantly expressed in neuronal cells, especially in the cortex, hippocampus and cerebellum of the adult mouse brain. Furthermore, our results indicate that in neurons CLN3 is not solely a lysosomal protein. It is localized in the synaptosomes but, interestingly, is not targeted to the synaptic vesicles. The novel localization of CLN3 directs attention towards molecular alterations at the synapses. This should yield important clues about the mechanisms of neurodegeneration in Batten disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Batten disease (juvenile-onset neuronal ceroid lipofuscinosis or Spielmeyer–Vogt–Sjögren disease) is the most common neurodegenerative disease of childhood with an incidence of 1 in 21 000 (1). This autosomal recessively inherited disease is caused by mutations in the CLN3 gene, identified in 1995 (2). It belongs to a group of at least eight inherited progressive neurodegenerative diseases called neuronal ceroid lipofuscinoses (NCLs) (for a review see see refs 1 and 3). NCL diseases are marked by two histopathological findings: degeneration of nerve cells, foremost in the cerebral cortex, and accumulation of autofluorescent ceroid-lipopigment in both neural and peripheral tissues (4). In Batten disease, the autofluorescent material has been identified as mitochondrial ATP synthase subunit c (5).
The clinical features of Batten disease include visual failure, epileptic seizures and progressive dementia, which lead to premature death at the age of 18–30 years (6). Most patients carry a 1.02 kb deletion removing nucleotides 461–677 from the CLN3 coding region; in addition, 24 mutations and two polymorphisms have been characterized (3). The CLN3 protein is an integral transmembrane protein with several very hydrophobic domains (7). It is conserved among different species indicating a fundamental role in cell metabolism. Studies using the yeast cln3 homologue BTN1p knockout strain have indicated that the CLN3 protein may have a role in the regulation of the vacuolar pH (8). How this putative finding relates to the pathology of Batten disease still remains elusive.
We have previously demonstrated the endosomal/lysosomal localization of CLN3 protein in non-neuronal cells (9). In addition, several other intracellular localizations have been proposed for CLN3; endoplasmic reticulum (10), Golgi (11) and cell membranes and nucleus (12) in non-neuronal cells. In neurons CLN3 localization has been just tentatively examined and it seems that the protein is trafficked along the neural extensions (13) and one report indicates mitochondrial localization in retinal cells as detected by immunochemical analyses (14). The fact that the reports on the intracellular localization of CLN3 protein have been quite controversial suggest that CLN3 may have different intracellular localization in different cells.
Due to the fact that Batten disease severely affects neuronal cells and leaves other organs clinically unaffected, it is of utmost importance to approach the disease mechanism by studying the expression and localization of CLN3 in the brain and neuronal cells. We have analysed here, CLN3 in mouse brain using in situ hybridization, immunohistochemical staining and western blot analysis of subcellular fractions. Furthermore, as visual deterioration is the hallmark of Batten disease as well as all NCL disorders, we have set up primary retinal cultures from the mouse and analysed both endogenous mouse CLN3 and Semliki Forest virus (SFV)-mediated human CLN3 localization using immunofluorescence staining and confocal microscopy. Our results indicate that CLN3 has alternative trafficking routes depending on the cell type. In neuronal cells CLN3 is not specifically targeted to lysosomes, but the bulk of the protein is found in the synaptosomal fraction. CLN3 protein is not, however, targeted to synaptic vesicles (SVs) such as the other NCL protein palmitoyl protein thioesterase (PPT1) (15), indicating a yet unknown membrane compartment for CLN3 in neurons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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CLN3 is expressed in neural cells of mouse brain
We first studied the expression and localization of the CLN3 gene and CLN3 protein in adult mouse brain (2.5- and 6-month-old animals) using in situ hybridization and immunohistochemistry. Both methods resulted in a very similar distribution pattern showing that most expression was concentrated on the neuronal cells. No difference between the 2.5- and 6-month-old mice was detected in terms of the intensities or distribution of CLN3 expression (results only for the 2.5-month-old mice shown). CLN3 mRNA and protein were detected throughout the brain, for example, in the cerebral cortex, hippocampus, cerebellum and several different cerebral nuclei (Figs 1 and 2). In the cerebral cortex, CLN3-positive cells were found in all cortical layers, although not all neurons were labelled (Figs 1A and 2A). In the hippocampus, the most prominent CLN3 expression was detected in the granule cells in the dentate gyrus and the pyramidal cells of the hippocampus proper (Figs 1B and 2A and B). In the cerebellum intense mRNA signal as well as protein immunolabelling was detected in the granular and molecular layers, and in the Purkinje cell layer (Figs 1C and 2C). In terms of intracellular localization, neurons were immunopositive near the plasma membrane, and especially in some granularly stained large neurons the immunostaining was also seen in the neurites (Fig. 2D).
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CLN3 co-localizes with presynaptic proteins in retinal cells
In order to elucidate the localization of endogenous CLN3, double immunostaining with mouse CLN3-specific peptide antibody and several antibodies against specific cellular compartments in mouse primary retinal cultures was performed. Endogenous CLN3 is clearly visible in retinal cultures prepared from embryonic day (E)14.5–16.5 mice. Firstly, CLN3 protein was seen to co-localize clearly with a specific neuronal marker, mouse anti-tubulin ß III isoform (Fig. 3A). More specifically, CLN3 protein is seen in the neural extensions and synaptic terminals. This is supported by a significant degree of co-localization with pre-synaptic markers SV2 (Fig. 3B) and SYP (similar to SV2 staining, data not shown). In the axonal parts of the neurons, CLN3 protein can be seen to co-localize partially with the growth-associated protein, GAP-43 (Fig. 3D–F). GAP-43 is a major protein of neuronal growth cones and certain presynaptic terminals, and a candidate for involvement in both axon growth and synaptic plasticity. The horizontal XZ-projection of the confocal image, however, indicates that CLN3 and GAP-43 are not localized in the same subcellular compartment (Fig. 3F, insert). On the other hand, there was no obvious co-localization of CLN3 with the lysosomal marker Lamp1 (Fig. 3C). Lamp1-positive vesicles are predominant in the soma whereas CLN3 is found in the neural extensions. In the soma, CLN3 can be seen to localize near the plasma membrane. In embryonic cortical neurons endogenous CLN3 was not detectable (data not shown) further indicating that retinal cultures provide an optimal model for the analysis of CLN3 protein trafficking.
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We further assessed the localization of overexpressed human CLN3 in retinal cultures using SFV-CLN3-mediated expression. The results of double immunostaining with the human CLN3-specific peptide antibody (9) and several specific organelle markers were similar to those seen in the endogenous mouse CLN3 staining. Overexpressed CLN3 protein is present in the neurites in beads-on-a-string fashion in contrast to uniform ß-tubulin staining (Fig. 4A–C). Partial co-localization of the human CLN3 with SV2 is very similar to that of the endogenous CLN3 (Fig. 4D–F). However, the CLN3-SV2 double immunostaining is not completely overlapping suggesting that these proteins are not found entirely in the same subcellular compartment. With the lysosomal marker Lamp1 no significant co-localization was found (Fig. 4G–I).
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Mouse brain tissue fractionation demonstrates synaptosomal localization of CLN3
Immunofluorescence studies with confocal microscopy revealed that CLN3 is present along the neuronal extensions and partially co-localizes with the presynaptic marker SV2. We have recently demonstrated that PPT1, involved in the pathogenesis of the infantile form of NCL, is localized in the synaptic vesicle fraction (15). To further explore the localization of neuronal CLN3, a systematic subcellular fractionation of mouse brain tissue was performed. Abundant CLN3 immunoreactivity was detected in the 1000 g postnuclear supernatant (PNS) and in the 14 500 g supernatant (S1) (Fig. 5A). A significant fraction of the CLN3 protein was also present in the 14 500 g pellet (P1). The S1 fraction was further subjected to 100 000 g centrifugation after which a major portion of the CLN3 protein was detected in the pellet (P2). The crude synaptosomal fraction (P1) was purified by a discontinuous sucrose gradient to obtain the synaptosomal fraction (S) (Fig. 5B). SVs were also purified from the synaptosomal fraction. Western blot analysis indicated that CLN3 is present in the purified synaptosomal fraction but virtually no CLN3 protein can be detected in the synaptic vesicle fraction where the organelle marker synaptophysin was enriched. Synaptophysin was also found in P2 (Fig. 5A). Interestingly, synaptophysin and CLN3 were both found as a monomeric as well as a high-molecular weight complex in P2. This may indicate that CLN3, like synaptophysin, forms SDS-resistant, insoluble complexes. The lysosomal membrane protein Lamp1 showed different enrichment in the subcellular fractionation compared with CLN3, as most of the lysosomal protein resided in P1. The lysosomal proteins could be found enriched in the Percoll step gradient for lysosomes and CLN3 protein was present to a lesser extent in the lysosomal fraction (L, Fig. 5C).
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Western blot analysis of the mouse brain pelleting experiments showed the different distribution pattern of CLN3 and Lamp1. Remarkably, CLN3 is not identical to SYP either. Further fractionation studies of adult mice showed that CLN3 protein is present in the PNS and in the synaptosomal fraction. In the SVs and lysosomes CLN3 protein was not enriched as compared to PNS. This indicates that CLN3 is not solely a lysosomal protein but has a yet unknown function at the neuronal synapses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In order to unravel the puzzle of the function for the CLN3 protein defective in Batten disease, it is crucial to investigate its role in the nervous system. As blindness is the first symptom and common to all Batten disease patients (6,16), retinal cell cultures provide an excellent research model. Co-localization with ß-tubulin indicates that CLN3 is expressed particularly in neural cells. In addition, both immunofluorescence and mouse tissue fractionation studies clearly associate CLN3 protein with the presynaptic terminals and synaptosomes within the neural cells. This is in contrast to previous studies in non-neural cells, in which CLN3 was found mainly in the early and late endosomes and lysosomes (9 and unpublished data). Lysosomes of neural cells have been detected mainly in the cell soma where they form a very distinct punctuate pattern in contrast to CLN3 that is mainly detected in SVs along the neural extensions. In the retinal neurons CLN3 was seen to accumulate in similar neuronal varicosities as GAP-43, indicating possible axonal targeting.
The specific targeting of CLN3 to neurites might indicate that CLN3 is involved in the neuronal endocytosis or exocytosis. However, CLN3 is not enriched in the actual SVs suggesting its role to be different from synaptic vesicle membrane proteins. Pelleting of the mouse brain PNS demonstrated that unlike the lysosomal membrane protein Lamp1, CLN3 is present, apart from the isolated synaptosomes, in both 100 000 g pellet (P2) and supernatant (S2). This finding indicates its presence also in some yet unidentified microvesicular compartment. The absence of CLN3 protein from SVs but its presence in the synaptosomes and microvesicles may indicate a circulating role, which is further supported by the previous findings of CLN3 residing in several different subcellular compartments in non-neuronal cells (9–12). One possible function of CLN3 protein could be transporting some cargo to be delivered to the SVs.
The biogenesis of SVs through the endosomal AP3 mediated pathway has only recently been demonstrated (17). This pathway contains a bulk of yet unidentified protein components. Steps toward understanding the exact routing of CLN3 could be taken by exploring the potential sorting signals within CLN3 protein. There is a putative farnesylation motif CQLS in the C-terminus, which does not appear to affect the targeting of the CLN3 (18). C-terminus of CLN3 at amino acids 425 and 426 also contains a dileucine lysosomal targeting motif, which does not seem to be functional either (10). Furthermore, a tyrosine-based targeting motif YXX
(Y is tyrosine, X any amino acid, an amino acid with a bulky hydrophobic side chain) is present in CLN3; however, so far it has not been demonstrated to be functional. Tyrosine-based sorting signals have several known functions; mediating rapid internalization from the cell surface, lysosomal targeting, localization to specialized endosomal–lysosomal organelles, delivery to the basolateral plasma membrane of polarised epithelial cells or transport to SVs (19). It will be of great interest to investigate whether CLN3 protein interacts with the AP3 coat.
In comparison, PPT1 mutated in the infantile form of NCL has been reported to be present in the neurites and localized in synaptosomes as well as in the actual SVs (15). In peripheral cells PPT1 is localized in the lysosomes (15,20) and, analogously to CLN3, this NCL protein has a different localization in neuronal cells. PPT1 has been closely connected to neuronal development and especially to synaptogenesis (21,22). Localization of other NCL proteins has been studied in non-neuronal cells as well and it has been reported that the protein encoded by the CLN2 gene is a lysosomal tripeptidyl peptidase (TTP1) (23). In comparison, CLN8 is a membrane protein trafficking between the ER and Golgi intermediate compartment (24). It will be of great interest to see whether the trafficking of these proteins in neuronal cells is different from that in non-neuronal cells. Eventually, the whole set of eight different NCL proteins may define some specific transport route in neurons.
In terms of the brain pathology of Batten disease patients, documented early lesions are the loss of small stellate neurons in layer II and the loss of pyramidal neurons of layer V in the cerebral cortex (25,26). Immunohistochemistry of mouse brain sections also showed a prominent staining of CLN3 in the hippocampus and cerebellum. Nonetheless, more thorough characterization of neurodegeneration of these areas in Batten patients has not been conducted. Our current results indicate that synapses might be an important site of pathology; however, analysis of the synapses in Batten disease patients has not been documented either. In a canine model of juvenile Batten disease, loss of GABAergic neurons and synapses in the cortex and cerebellum of affected dogs is demonstrated (27). Synaptic function is very sensitive to alterations in the levels of receptors, ion channels and transporters, and an accumulation of abnormal or mistargeted proteins at the synaptic site might be responsible for neurodysfunction and neurodegeneration. This is well exemplified in Alzheimer’s disease, where a decrease in synaptic density can be demonstrated (28).
Recently, a Cln3-deficient mouse model for studying Batten disease has been developed (29) and it will be of great interest to analyse the possible defect in the endocytic pathway of the Batten mouse neurons. Involvement of the endocytic pathway in Batten disease has also been implicated by studies using the yeast knockout model for the CLN3 homologue BTN1p. In yeast, an absence of BTN1p causes changes in the expression profile of the endocytic HOOK protein and a change in the yeast vacuolar pH (8). This observation together with the novel synaptosomal localization finding direct the functional analysis of protein away from the lysosomes towards the endocytic and exocytic neuronal transport pathways. Molecular alterations at the synapses of Batten knockout mice, such as analysing whether accumulation of synaptic proteins occur, should yield important clues toward the mechanisms of neurodegeneration or presynaptic damage in Batten disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Tissue preparation
Tissue samples used were intracardially perfusion-fixed (4% PFA) and paraffin embedded 2.5-month-old mouse brain samples. The tissue was cut into 5 µm sections and mounted on a Super Frost (Menzel Gläzer) objective glass.
In situ hybridization
For the generation of probes, PCR fragment corresponding to nucleotides 262–584 of the mouse Cln3 cDNA (EST AA274409) was cloned into pBluescript"II SK(±) vector (Stratagene). Digoxigenin (DIG)-labelled anti-sense and sense probes were generated using DIG RNA labelling kit (Boehringer Mannheim) according to the manufacturer’s instructions. The nucleotide sequences of the probes were confirmed by sequencing with ABI PRISM 377 (Applied Biosystems). Non-radioactive in situ hybridization was performed as described previously (30). The sense probe was used as a negative control.
Antibodies
The polyclonal rabbit h385/CLN3 antibody against a synthetic peptide corresponding to human amino acid sequence 242–258 of the full-length CLN3 cDNA was produced as described previously (9). A novel polyclonal rabbit antibody m385/CLN3 was produced against the corresponding mouse Cln3 amino acid sequence 242–258 (Invitrogen). For rabbit immunizations, 500 µg of peptide was emulsified in complete Freund’s adjuvant and injected intradermally at multiple sites. Injections were performed at 3 week intervals for total of 9 weeks. The antiserum was tested by immunofluorescence staining of CLN3-transfected COS-1 and HeLa cells. In addition, testing by western blot analysis of CLN3-transfected COS-1 cells was performed using the pre-immune serum as a negative control.
Immunohistochemical staining
After deparaffinization with xylene and decreasing graded series of ethanol solutions endogenous peroxidase activity was blocked with 5% H2O2 for 5 min. Unspecific binding was blocked with 10% fetal calf serum (FCS, Gibco-BRL) for 1 h. Primary antiserum m385 was diluted in the blocking mixture (1:100) and the sections were incubated overnight at 4°C. Sections were washed four times with 1% FCS in Tris-buffered saline (TBS) and incubated with biotinylated goat anti-rabbit IgG (Vector; 1:200) for 1 h at room temperature, followed by incubation with peroxidase-conjugated avidin–biotin complex (Vectastain Standard ABC reagent, Vector, 1:200 in TBS) for 40 min. Specific binding was detected by using 0.05% 3,3'-diaminobenzidine (DAB Fast kit, Sigma) as the chromogen and 0.3% H2O2 as the substrate. Counterstain was produced by Mayer’s hemalum solution (Merck) and 0.75% NH3.
Dissection and culturing of retinal neurons
Retinal neuron cultures were prepared from 14.5–17.5-day-old mouse embryos (E14.5–17.5 C57BL) as follows: the eyes were removed and transferred into ice-cold 0.1 M phosphate-buffered saline (PBS) without Ca2+ and Mg2+ pH 7.4, supplemented with 20 mM glucose and incubated at 37°C for 20 min in order to weaken cation-dependent interactions between the retina and the retinal pigment epithelium. For each eye the retinal pigment epithelium and the vitreous humour were peeled away and the lens was removed. Ten to fourteen retinas were pooled in 0.5 ml PBS and trypsinized by adding DNase I and trypsin-EDTA to final concentrations of 0.02 µg/ml and 0.08 mM, respectively, followed by an incubation for 3 min at 37°C. Enzyme activity was inhibited with FCS at a final concentration of 10%, and the tissue was further mechanically dissected by triturating 10 times. The sample was then spun down gently and the pellet was resuspended in a filtered medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL) with antibiotics, 0.5 mM glutamine, 2.5 µM glutamic acid, 10 mM HEPES (pH 7.4), 1–2% FCS and 1x B27 (Gibco-BRL). Cells were plated on Matrigel-coated (1:50 in DMEM; Becton Dickinson) 5 cm2 dish with coverslips. After 4 days of incubation, culturing was continued in the medium without glutamic acid and with or without an antimitotic agent, cytosine arabinoside (AraC 5 µM, Sigma).
Recombinant SFV and infections
The recombinant SFV (SFV-CLN3) was constructed and prepared as described previously (13). The infections of the retinal cells were performed with the viral solution diluted in medium consisting of Eagle’s minimal essential medium (MEM) with 0.2% bovine serum albumin (BSA), 2 mM glutamine and 20 mM HEPES. Retinal cells to be infected had been maintained in culture for 6–7 days. The infection was carried out for 1 h at 37°C in 5% CO2, then the infection medium was removed and the incubation was continued for 10 h.
Immunofluorescence staining and confocal microscopy
Retinal cells were fixed with fresh 4% paraformaldehyde in PBS for 15 min at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 (Sigma) in 0.5% BSA (Sigma)/PBS for 15 min. Unspecific binding was blocked with 0.5% BSA in PBS for 30 min. For double immunostainings of the non-infected retinal cells, the polyclonal antibody m385 was combined either with mouse anti-tubulin ß III isoform (Chemicon, dilution 1:200), rat anti-mouse Lamp1 (developed by J.T.August, Johns Hopkins University School of Medicine, Baltimore, MD; 1:50), mouse anti-SVs (SV2) (developed by K.M.Buckley, Harvard Medical School, Boston, MA; 1:50) or mouse anti-growth associated protein 43 (GAP-43) (Boehringer Mannheim; 1:1000). Lamp1 and SV2 were obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Department of Biological Sciences, Iowa City, IA, USA under contract NO1-HD-7-3263. SFV-CLN3 infected retinal cells were double immunostained with h385 (1:500) combined with either mouse anti-tubulin ß III isoform, rat anti-mouse Lamp1, or mouse anti-SV2. Primary antibodies were diluted in blocking buffer and incubated for 1 h at room temperature. Secondary antibody incubation was performed with Rhodamine Red (TRITC)-conjugated goat anti-rabbit IgG (Immunotech; 1:200) combined either with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG+IgM (the Jackson Laboratories; 1:200) or FITC-conjugated goat anti-rat IgG (Immunotech; 1:150). The coverslips were mounted in GelMount (Biomeda Corp.) and viewed with a Leica DMR confocal microscope with TCS NT software (Leica Microscope and Scientific Instruments Group). The confocal images were completed with Adobe Illustrator 8.0 software.
Tissue fractionation
To provide the PNS 10 cortices from 2-month-old mice were gently homogenized in 60 ml SolA buffer (320 mM sucrose, 5 mM Na-HEPES/HCl pH 7.4) with 12 strokes at 800 r.p.m. in a glass-Teflon homogenizer. The homogenate was centrifuged (1000 g, SS34) for 5 min. For the pelleting analyses, the PNS was centrifuged at 14 500 g for 20 min in a Beckman Tabletop Ultracentrifuge. The supernatant (S1) was retained and the pellet (P1) was washed with Sol A and recentrifuged at 100 000 g for 1 h twice to provide the pellet P2 and supernatant S2. The pellets were resuspended in 0.2% SDS in HEPES pH 7.4. Isolation of synaptosomes of mouse brain was performed as described by Gordon-Weeks (31,32) and Lehtovirta (15). The PNS was centrifuged twice at 12 000 g SS34 for 20 min and the crude synaptosomal fraction was resuspended in SolA buffer. This sample was layered on a discontinuous sucrose gradient of 1.2 M/1.0 M/0.85 M. After centrifugation for 2 h at 110 000 g. in a SW28 rotor (Beckman) the synaptosomes were collected at the 1.0M/1.2M interphase, and resuspended in Krebs solution [NaCl, KCl, CaCl2(H2O)2, MgCl2, NaH2PO4, D-glucose, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)]. SVs were isolated as described previously (33,34). Ten cortices from 2-month-old mice were homogenized in 50 ml buffered sucrose (320 mM sucrose/4 mM Na-HEPES/HCl pH 7.3). The crude SV fraction obtained by differential centrifugation was resuspended in 40 mM sucrose and layered on a linear continuous sucrose gradient 800/50 mM. Gradients were centrifuged for 5 h at 90 000 g in a SW28 rotor (Beckman). Gradients were collected in 1 ml fractions and the refractive index of each fraction was examined. Corresponding fractions of two identical gradients were pooled to a final volume of 2 ml and 1.5 ml of each fraction containing vesicles was resuspended in 10 mM Na-HEPES/HCl pH 7.4, 150 mM NaCl, and sedimented by centrifugation for 13 h at 205 000 g in a 70Ti rotor (Beckman). The pellets were dissolved in Krebs solution. Lysosomes were isolated by homogenizing two 2-month-old mice cortices in 2 ml 250 mM sucrose, 3 mM imidazole-HCL pH 7.4 (buffer A), and passaging them through a 21-gauge needle (35). The homogenate was centrifuged and the PNS was layered on top of a step gradient of the following composition: 2 M sucrose, Percoll (Amersham) in buffer A with the density adjusted to 1.090 g/ml, and with a density of 1.075 g/ml. Centrifugation was performed in a Beckman 65.1 Vti rotor for 40 min at 45 000 g and stopped without braking. Fractions of 0.5 ml were collected. The purity of all organelle fractions was assessed by immunoblotting with organelle specific markers.
Western blot analysis
The protein concentration was measured by Bio-Rad Protein Assay System (Bio-Rad Laboratories) and 5 µg of each organelle fraction was electrophoresed in 14% SDS–PAGE (36). The proteins were transferred to nitrocellulose membrane (Hybond‘ECL‘, Amersham Pharmacia Biotech) by electroblotting at 100 mA overnight. Primary antibodies used for immunostaining were m385 antiserum (1:100), anti-mouse monoclonal synaptophysin (SYP, DAKO; 1:1000), rat anti-mouse Lamp1 and, as a negative control, m385 preimmune serum (1:100).
Secondary antibodies were peroxidase-conjugated swine anti-rabbit, anti-mouse or anti-rat immunoglobulins (1:10000; DAKO). To visualize the enhanced chemiluminescence 1.26 mM luminol (Fluka) and 0.2 mM p-coumaric acid (Sigma) were used.
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ACKNOWLEDGEMENTS |
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We thank Anne Nyberg and Carina von Schantz for their excellent technical assistance. Mikko Jalanko, Jaakko Patrakka and Laura Walliander are thanked for their help with the figures and Tanja Meriluoto for the help with preparing the manuscript. This study was financially supported by the Academy of Finland (Center of Excellence in Disease Genetics, grants 44870 and 45289), the Hjelt Fund, the Sigrid Juselius Foundation and MD PhD program of the Helsinki University Medical School.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +358 9 47448392; Fax: +358 9 47448480; Email: anu.jalanko@ktl.fi
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REFERENCES |
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1 Santavuori, P., Lauronen, L., Kirveskari, E., Åberg, L., Sainio, K. and Autti, T. (2000) Neuronal ceroid lipofuscinoses in childhood. Neurol. Sci., 21, S35–S41.[ISI][Medline]
2 The International Batten Disease Consortium. (1995) Isolation of a novel gene underlying Batten disease, CLN3. Cell, 82, 949–957.[ISI][Medline]
3 Mole, S. and Gardiner, M. (1999) Molecular genetics of the neuronal ceroid lipofuscinoses. Epilepsia, 40, 29–32.
4 Goebel, H.H. (1997) Morphologic diagnosis in neuronal ceroid lipofuscinosis. Neuropediatrics, 28, 67–69.[ISI][Medline]
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.[ISI][Medline]
6 Santavuori, P. (1988) Neuronal ceroid-lipofuscinoses in childhood. Brain Dev., 10, 80–83.[ISI][Medline]
7 Janes, R.W., Munroe, P.B., Mitchison, H.M., Gardiner, R.M., Mole, S.E. and Wallace, B.A. (1996) A model for Batten disease protein CLN3: functional implications from homology and mutations. FEBS Lett., 399, 75–77.[ISI][Medline]
8 Pearce, D.A., Nosel, S.A. and Sherman, F. (1999) Studies of pH regulation by Btn1p, the yeast homolog of human Cln3p. Mol. Genet. Metab., 66, 320–323.[ISI][Medline]
9 Järvelä, I., Sainio, M., Rantamäki, T., Olkkonen, V.M., Carpen, O., Peltonen, L. and Jalanko, A. (1998) Biosynthesis and intracellular targeting of the CLN3 protein defective in Batten disease. Hum. Mol. Genet., 7, 85–90.
10 Kida, E., Kaczmarski, W., Golabek, A.A., Kaczmarski, A., Michalewski, M. and Wisniewski, K.E. (1999) Analysis of intracellular distribution and trafficking of the CLN3 protein in fusion with the green fluorescent protein in vitro. Mol. Genet. Metab., 66, 265–271.[ISI][Medline]
11 Kremmidiotis, G., Lensink, I.L., Bilton, R.L., Woollatt, E., Chataway, T.K., Sutherland, G.R. and Callen, D.F. (1999) The Batten disease gene product (CLN3p) is a Golgi integral membrane protein. Hum. Mol. Genet., 8, 523–531.
12 Margraf, L.R., Boriack, R.L., Routheut, A.A., Cuppen, I., Alhilali, L., Bennett, C.J. and Bennett, M.J. (1999) Tissue expression and subcellular localization of CLN3, the Batten disease protein. Mol. Genet. Metab., 66, 283–289.[ISI][Medline]
13 Järvelä, I., Lehtovirta, M., Tikkanen, R., Kyttälä, A. and Jalanko, A. (1999) Defective intracellular transport of CLN3 is the molecular basis of Batten disease (JNCL). Hum. Mol. Genet., 8, 1091–1098.
14 Katz, M.L., Gao, C.L., Prabhakaram, M., Shibuya, H., Liu, P.C. and Johnson, G.S. (1997) Immunochemical localization of the Batten disease (CLN3) protein in retina. Invest. Ophthalmol. Vis. Sci., 38, 2375–2386.
15 Lehtovirta, M., Kyttälä, A., Eskelinen, E.L., Hess, M., Heinonen, O. and Jalanko, A. (2001) Palmitoyl protein thioesterase (PPT) localizes into synaptosomes and synaptic vesicles in neurons: implications for infantile neuronal ceroid lipofuscinosis (INCL). Hum. Mol. Genet., 10, 69–75.
16 Munroe, P.B., Mitchison, H.M., O’Rawe, A.M., Anderson, J.W., Boustany, R.M., Lerner, T.J., Taschner, P.E., de Vos, N., Breuning, M.H., Gardiner, R.M. et al. (1997) Spectrum of mutations in the Batten disease gene, CLN3. Am. J. Hum. Genet., 61, 310–316.[ISI][Medline]
17 Salem, N., Faundez, V., Horng, J.T. and Kelly, R.B. (1998) A v-SNARE participates in synaptic vesicle formation mediated by the AP3 adaptor complex. Nat. Neurosci., 1, 551–556.[ISI][Medline]
18 Haskell, R.E., Carr, C.J., Pearce, D.A., Bennett, M.J. and Davidson, B.L. (2000) Batten disease: evaluation of CLN3 mutations on protein localization and function. Hum. Mol. Genet., 9, 735–744.
19 Bonifacino, J.S. and Dell’Angelica, E.C. (1999) Molecular bases for the recognition of tyrosine-based sorting signals. J. Cell Biol., 145, 923–926.
20 Hellsten, E., Vesa, J., Olkkonen, V.M., Jalanko, A. and Peltonen, L. (1996) Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis. EMBO J., 15, 5240–5245.[ISI][Medline]
21 Isosomppi, J., Heinonen, O., Hiltunen, J.O., Greene, N.D., Vesa, J., Uusitalo, A., Mitchison, H.M., Saarma, M., Jalanko, A. and Peltonen, L. (1999) Developmental expression of palmitoyl protein thioesterase in normal mice. Brain Res. Dev. Brain Res., 118, 1–11.
22 Heinonen, O., Kyttälä, A., Lehmus, E., Paunio, T., Peltonen, L. and Jalanko, A. (2000) Expression of palmitoyl protein thioesterase in neurons. Mol. Genet. Metab., 69, 123–129.[ISI][Medline]
23 Lin, L., Sohar, I., Lackland, H. and Lobel, P. (2001) The human CLN2 protein/tripeptidyl-peptidase I is a serine proteaseT that autoactivates at acidic pH. J. Biol. Chem., 276, 2249–2255.
24 Lonka, L., Kyttälä, A., Ranta, S., Jalanko, A. and Lehesjoki, A.E. (2000) The neuronal ceroid lipofuscinosis CLN8 membrane protein is a resident of the endoplasmic reticulum. Hum. Mol. Genet., 9, 1691–1697.
25 Braak, H. and Goebel, H.H. (1978) Loss of pigment-laden stellate cells: a severe alteration of the isocortex in juvenile neuronal ceroid-lipofuscinosis. Acta Neuropathol., 42, 53–57.[Medline]
26 Braak, H. and Goebel, H.H. (1979) Pigmentoarchitectonic pathology of the isocortex in juvenile neuronal ceroid-lipofuscinosis: axonal enlargements in layer IIIab and cell loss in layer V. Acta Neuropathol., 46, 79–83.[Medline]
27 March, P.A., Wurzelmann, S. and Walkley, S.U. (1995) Morphological alterations in neocortical and cerebellar GABAergic neurons in a canine model of juvenile Batten disease. Am. J. Med. Genet., 57, 204–212.[ISI][Medline]
28 Masliah, E. (2000) The role of synaptic proteins in Alzheimer’s disease. Ann. N. Y. Acad. Sci., 924, 68–75.
29 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. Neurobiol. Dis., 6, 321–334.[ISI][Medline]
30 Jaquet, V., Gow, A., Tosic, M., Suchanek, G., Breitschopf, H., Lassmann, H., Lazzarini, R.A. and Matthieu, J.M. (1996) An antisense transgenic strategy to inhibit the myelin oligodendrocyte glycoprotein synthesis. Brain Res. Mol. Brain Res., 43, 333–337.[Medline]
31 Gordon-Weeks, P.R. (1987) The cytoskeletons of isolated, neuronal growth cones. Neuroscience, 21, 977–989.[ISI][Medline]
32 Gordon-Weeks, P.R. (1997) Isolation of synaptosomes, growth cones and their subcellular components. In Turner, A.J. and Bachelard, H.S. (eds), Neurochemistry. A Practical Approach. 2nd edn. IRL Press, Oxford, UK.
33 Huttner, W.B., Schiebler, W., Greengard, P. and De Camilli, P. (1983) Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation. J. Cell Biol., 96, 1374–1388.
34 Beher, D., Elle, C., Underwood, J., Davis, J.B., Ward, R., Karran, E., Masters, C.L., Beyreuther, K. and Multhaup, G. (1999) Proteolytic fragments of Alzheimer’s disease-associated presenilin 1 are present in synaptic organelles and growth cone membranes of rat brain. J. Neurochem., 72, 1564–1573.[ISI][Medline]
35 Diettrich, O., Gallert, F. and Hasilik, A. (1996) Purification of lysosomal membrane proteins from human placenta. Eur. J. Cell Biol., 69, 99–106.[ISI][Medline]
36 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[Medline]
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