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IME1, which is required for the initiation of meiosis, is regulated by Cln3:Cdc28 kinase, which activates the G1-to-S transition, and Snf1 kinase, which mediates glucose repression. Here we examine the pathway by which Cln3:Cdc28p represses IME1 and the relationship between Cln3:Cdc28p and Snf1p in this regulation. When wild-type yeast cease growth, they express IME1 to moderate levels, intermediate between the low levels expressed during growth and the high levels expressed during sporulation. Moderate IME1 expression occurred in cln3Delta, cln1Delta cln2Delta, cdc28-4 and swi6Delta mutants, even during growth. These mutants also induced IME1 expression more rapidly than the wild-type. CLN3 required SWI6 and CLN2 to repress IME1 and IME2, but CLN1 was much less active than CLN2 in this repression. The phenotype of the cln3Delta snf1Delta double mutant indicated that Cln3:Cdc28p regulates IME1 independently of SNF1. Entry into meiosis involves two independent but sequential controls, which regulate IME1 via a three position switch: (i) during growth IME1 is repressed by the CLN3/SWI6/CLN2 pathway, (ii) once growth ceases, this repression is released and IME1 is expressed at moderate levels, and (iii) subsequently, nutritional conditions that activate Snf1p allow high IME1 expression. Show less

The neuronal ceroid-lipofuscinoses (NCL) are the most common group of progressive neurodegenerative diseases in children, with an incidence as high as one in 12,500 live births. The main features of this disease are failure of psychomotor development, impaired vision, seizures, and premature death. Many biochemical and physiological studies have been initiated to determine the cellular defect underlying the disease, although only a few traits have been truly associated with the disorders. One of the paradox's of the NCL-diseases is the characteristic accumulation of autofluorescent hydrophobic material in the lysosomes of neurons and other cell types. However, the accumulation of this lysosomal storage material, which no doubt contributes to the neurologic disease, does not apparently lead to disease outside the CNS, and how these cellular alterations relate to the neurodegeneration in NCLs is unknown. Mutations have been identified in six distinct genes/proteins, namely CLN1, which encodes PPT1, a protein thiolesterase; CLN2, which encodes TPP1, a serine protease; and CLN3, CLN5, CLN6, and CLN8, which encode novel transmembrane proteins. Mutation in any one of these CLN-proteins results in a distinct type of NCL-disease. However, there are many shared similarities in the pathology of these diseases. The most obvious connection between PPT1, TPP1, CLN3, CLN5, CLN6, and CLN8 is their subcellular localization. To date, three of the four proteins whose subcellular localization has been confirmed, namely PPT1, TPP1, and CLN3, reside in the lysosome. We review the function of the CLN-proteins and discuss the possibility that a disruption in a common biological process leads to an NCL-disease. Show less

Mutations in the CLN3 gene are responsible for the neurodegenerative disorder Batten disease; however, the molecular basis of this disease remains unknown. In studying a mouse model for Batten disease, we report the presence of an autoantibody to glutamic acid decarboxylase (GAD65) in cln3-knockout mice serum that associates with brain tissue but is not present in sera or brain of normal mice. The autoantibody to GAD65 has the ability to inhibit the activity of glutamic acid decarboxylase. Furthermore, brains from cln3-knockout mice have decreased activity of glutamic acid decarboxylase as a result of the inhibition of this enzyme by the autoantibody, resulting in brain samples from cln3-knockout mice having elevated levels of glutamate as compared with normal. This elevated glutamate in the brain of cln3-knockout mice co-localizes with presynaptic markers. The decreased activity of GAD65 and increased levels of glutamate may have a causative role in astrocytic hypertrophy evident in cln3-knockout mice, and in altered expression of genes involved in the synthesis and utilization of glutamate that underlie a shift from synthesis to utilization of glutamate. An autoantibody to GAD65 is also present in sera of 20 out of 20 individuals tested who have Batten disease. Postmortem tissue shows decreased reactivity to an anti-GAD65 antibody that may be due to loss of GAD65-positive neurons or due to the reactive epitope being blocked by the presence of the autoantibody. We propose that an autoimmune response to GAD65 may contribute to a preferential loss of GABAergic neurons associated with Batten disease. Show less

Batten disease or JNCL, is the juvenile form of Neuronal Ceroid Lipofuscinosis (NCL) an autosomal recessive neurodegenerative disorder. Since retinal degeneration is an early consequence of Batten disease, we examined the eyes of Cln3 knockout mice (1-20 months of age), along with heterozygotes and appropriate controls, to determine whether or not the Cln3 defect would lead to characteristic retinal degeneration and visual loss. Accumulation of autofluorescent material and intracellular inclusions were markedly increased in Cln3 knockout retinal ganglion cells, as well as most other nuclear layers. Nerve fiber density was also significantly decreased in Cln3 knockout retinae. Apoptosis was observed in the photoreceptor layer of Cln3 knockout. However, the degree of retinal degeneration up to age 20 months was not extensive. Fundus examinations of Cln3 knockout mice showed no significant abnormalities, while electroretinograms remained robust through 11 months of age. In summary, it appears that accumulation of autofluorescent material, carbohydrate storage material, as well as apoptotic cell death are retinal manifestations of the Cln3 defect that do not appear to extinguish retinal function in this mouse model of Batten disease. Show less

Multiple gene defects cause Batten disease. Accelerated apoptosis accounts for neurodegeneration in the late infantile and juvenile forms that are due to defects in the CLN3 and CLN2 genes. Extensive neuronal death is seen in CLN2- and CLN3-deficient human brain as well as in CLN6-deficient sheep brain and retina. When neurons in late infantile and juvenile brain survive, they manage to do so by upregulating the neuroprotective molecule Bcl-2. The CLN3 gene has antiapoptotic properties at the molecular level. We show that the CLN2 gene is neuroprotective: it enhances growth of NT2 cells and maintains survival of human postmitotic hNT neurons. Conversely, blocking CLN3 or CLN2 expression in hNT neurons with adenoviral antisense-CLN3 or antisense-CLN2-AAV2 constructs causes apoptosis. The drug flupirtine is a triaminopyridine derivative that acts as a nonopioid analgesic. Flupirtine upregulates Bcl-2, increases glutathione levels, activates an inwardly rectifying potassium channel, and delays loss of intermitochondrial membrane calcium retention capacity. We show that flupirtine aborts etoposide-induced apoptosis in CLN1-, CLN2-, CLN3-, and CLN6-deficient as well as normal lymphoblasts. Flupirtine also prevents the death of CLN3- and CLN2-deficient postmitotic hNT neurons at the mitochondrial level. We show that a mechanism of neuroprotection exerted by flupirtine involves complete functional antagonism of N-methyl-D-aspartate or N-methyl-D-aspartate-induced neuronal apoptosis. Flupirtine may be useful as a drug capable of halting the progression of neurodegenerative diseases caused by dysregulated apoptosis. Show less

Transcription of the CLN3 G(1) cyclin in Saccharomyces cerevisiae is positively regulated by glucose in a process that involves a set of DNA elements with the sequence AAGAAAAA (A(2)GA(5)). To identify proteins that interact with these elements, we used a 1-hybrid approach, which yielded a nuclear zinc finger protein previously identified as Azf1. Gel shift and chromatin immunoprecipitation experiments show that Azf1 binds to the A(2)GA(5) CLN3 regulatory sequences in vitro and in vivo, thus identifying a transcriptional regulatory protein for CLN3 and a DNA sequence target for Azf1. We show that glucose-induced expression of a reporter gene driven by the A(2)GA(5) CLN3 regulatory sequences is dependent upon the presence of AZF1. Furthermore, deletion of AZF1 markedly reduces the transcriptional induction of CLN3 by glucose. In addition, Azf1 can induce reporter expression in a glucose-specific manner when artificially tethered to a promoter via the DNA-binding domain from Gal4. We conclude that AZF1 is a glucose-dependent transcription factor that interacts with the CLN3 A(2)GA(5) repeats to play a positive role in the regulation of CLN3 mRNA expression by glucose. Show less

Juvenile Batten disease is a neurodegenerative disease caused by accelerated apoptotic death of photoreceptors and neurons attributable to defects in the CLN3 gene. CLN3 is antiapoptotic when overexpressed in NT2 neuronal precursor cells. CLN3 negatively modulates endogenous ceramide levels in NT2 cells and acts upstream of ceramide generation. Because defects in regulation of apoptosis are involved in the development of cancer, we evaluated the expression of CLN3 on both mRNA and protein levels in a variety of cancer cell lines and solid colon cancer tissue. We also observed the effect of the blocking of CLN3 protein expression on cancer cell growth, survival, ceramide production, and apoptosis by using an adenovirus-bearing antisense CLN3 construct. We show that CLN3 mRNA and protein are overexpressed in glioblastoma (U-373G and T98g), neuroblastoma (IMR-32 and SK-N-MC), prostate (Du145, PC-3, and LNCaP), ovarian (SK-OV-3, SW626, and PA-1), breast (BT-20, BT-549, and BT-474), and colon (SW1116, SW480, and HCT 116) cancer cell lines but not in pancreatic (CAPAN and As-PC-1) or lung (A-549 and NCI-H520) cancer cell lines. CLN3 is also up-regulated in mouse melanoma and breast carcinoma cancer cell lines. We found CLN3 expression is 22-330% higher than in corresponding normal colon control tissue in 8 of 10 solid colon tumors. An adenovirus-expressing antisense CLN3 (Ad-AS-CLN3) blocks CLN3 protein expression in DU-145, BT-20, SW1116, and T98g cancer cell lines as seen by Western blot. Blocking of CLN3 expression using Ad-AS-CLN3 inhibits growth and viability of cancer cells. It also causes elevation in endogenous ceramide production through de novo ceramide synthesis and results in increased apoptosis as shown by propidium iodide and JC-1 staining. This suggests that Ad-AS-CLN3 may be an option for therapy in some cancers. More importantly these results suggest that CLN3 is a novel molecular target for cancer drug discovery. Show less

We derived novel, testable predictions from a mathematical model of the budding yeast cell cycle. A key qualitative prediction of bistability was confirmed in a strain simultaneously lacking cdc14 and G1 cyclins. The model correctly predicted quantitative dependence of cell size on gene dosage of the G1 cyclin CLN3, but it incorrectly predicted strong genetic interactions between G1 cyclins and the anaphase-promoting complex specificity factor Cdh1. To provide constraints on model generation, we determined accurate concentrations for the abundance of all nine cyclins as well as the inhibitor Sic1 and the catalytic subunit Cdc28. For many of these we determined abundance throughout the cell cycle by centrifugal elutriation, in the presence or absence of Cdh1. In addition, perturbations to the Clb-kinase oscillator were introduced, and the effects on cyclin and Sic1 levels were compared between model and experiment. Reasonable agreement was obtained in many of these experiments, but significant experimental discrepancies from the model predictions were also observed. Thus, the model is a strong but incomplete attempt at a realistic representation of cell cycle control. Constraints of the sort developed here will be important in development of a truly predictive model. Show less

The retinoblastoma family of proteins are key cell cycle regulatory molecules important for the differentiation of various mammalian cell types. The retinoblastoma protein regulates transcription of a variety of genes either by blocking the activation domain of various activators or by active repression via recruitment to appropriate promoters. We show here that the retinoblastoma family of proteins functions as direct transcriptional repressors in a heterologous yeast system when fused to the DNA binding domain of Gal4. Mapping experiments indicate that either the A or the B domain of the pocket region is sufficient for repression in vivo. As is the case in mammalian cells, a phosphorylation site mutant of the retinoblastoma protein is a stronger transcriptional repressor than the wild type protein. We show that transcriptional repression by pRb is dependent on CLN3 in vivo. Furthermore, the yeast histone deacetylase components, RPD3 and SIN3, are required for transcriptional repression. Show less

In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G(1) is promoted by the G(1) cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28. We show here that all known aspects of the function of Cln3 in G(1) phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6. Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G(1)-S transition. The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28. The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter. The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6. Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein. Show less

The transcription factor Mcm1 is regulated by adjacent binding of a variety of different factors regulating the expression of cell-type-specific, cell cycle-specific, and metabolic genes. In this work, we investigate a new class of Mcm1-regulated promoters that are cell cycle regulated and peak in late M-early G(1) phase of the cell cycle via a promoter element referred to as an early cell cycle box (ECB). Gel filtration experiments indicate that the ECB-specific DNA binding complex is over 200 kDa in size and includes Mcm1 and at least one additional protein. Using DNase I footprinting in vitro, we have observed protection of the ECB elements from the CLN3, SWI4, CDC6, and CDC47 promoters, which includes protection of the 16-bp palindrome to which Mcm1 dimers are known to bind as well as protection of extended flanking sequences. These flanking sequences influence the stability and the variety of complexes that form on the ECB elements, and base substitutions in the protected flank affect transcriptional activity of the element. Chromatin immunoprecipitations show that Mcm1 binds in vivo to ECB elements throughout the cell cycle and that binding is sensitive to carbon source changes. Show less

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 approximately 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( Deltaex7/8) knock-in mice. The Cln3( Deltaex7/8) 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( Deltaex7/8) 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( Deltaex7/8) 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. Show less

As yeast colonies ceased growth, cells at the edge of these colonies transited from the cell division cycle into meiosis at high efficiency. This transition occurred remarkably synchronously and only at late stages of colony maturation. The transition occurred on medium containing acetate or low concentrations of glucose, but not on medium containing high glucose. The repression by high glucose was overcome when IME1 was overexpressed from a plasmid. Experiments with different growth media imply that meiosis in colonies is triggered by changes in the nutrient environment as colonies mature. HAP2 is required to sporulate in any carbon source, whereas GRR1 is required for glucose repression of sporulation. CLN3 is required to repress meiosis in colonies but not in liquid cultures, indicating that the regulators that mediate the transition to meiosis in colonies are not identical to the regulators that mediate this transition in liquid cultures. Show less

Btn2p, a novel coiled-coil protein, is up-regulated in btn1delta yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1delta strains (D. A. Pearce, T. Ferea, S. A. Nosel, B. Das, and F. Sherman, Nat. Genet. 22:55-58, 1999). We now report that Btn2p interacts biochemically and functionally with Rsglp, a down-regulator of the Can1p arginine and lysine permease. Rsglp localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2delta strains) fail to correctly localize Rsg1p. btn2delta strains, like rsg1delta strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2delta strains, like rsg1delta strains, demonstrate an elevated rate of uptake of [14C]arginine, which leads to increased intracellular levels of arginine. Overexpression of BTN2 results in a decreased rate of arginine uptake. Collectively, these results indicate that altered levels of Btn2p can modulate arginine uptake through localization of the Can1p-arginine permease regulatory protein, Rsglp. Our original identification of Btn2p was that it is up-regulated in the btn1delta strain which serves as a model for the lysosomal storage disorder Batten disease. Btnlp is a vacuolar/lysosomal membrane protein, and btn1delta suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2delta rsg1delta strains. We conclude that Btn2p interacts with Rsglp and modulates arginine uptake. Up-regulation of BTN2 expression in btn1delta strains may facilitate either a direct or indirect effect on intracellular arginine levels. Show less

Two homeodomain proteins, Yox1 and Yhp1, act as repressors at early cell cycle boxes (ECBs) to restrict their activity to the M/G1 phase of the cell cycle in budding yeast. These proteins bind to Mcm1 and to a typical homeodomain binding site. The expression of Yox1 is periodic and directly correlated with its binding to, and repression of, ECB activity. The absence of Yox1 and Yhp1 or the constitutive expression of Yox1 leads to the loss of cell-cycle regulation of ECB activity. Therefore, the cell-cycle-regulated expression of these repressors defines the interval of ECB-dependent transcription. Twenty-eight genes, including MCM2-7, CDC6, SWI4, CLN3, and a number of genes required during late M phase have been identified that are coordinately regulated by this pathway. Show less

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. Show less

Eukaryotic cells commit in G1 to a new mitotic cycle or to diverse differentiation processes. Here we show that Whi3 is a negative regulator of Cln3, a G1 cyclin that promotes transcription of many genes to trigger the G1/S transition in budding yeast. Whi3 contains an RNA-recognition motif that specifically binds the CLN3 mRNA, with no obvious effects on Cln3 levels, and localizes the CLN3 mRNA into discrete cytoplasmic foci. This is the first indication that G1 events may be regulated by locally restricting the synthesis of a cyclin. Moreover, Whi3 is also required for restraining Cln3 function in meiosis, filamentation, and mating, thus playing a key role in cell fate determination in budding yeast. Show less

In the past decade there have been significant advances in our understanding of the molecular genetic basis of the neuronal ceroid lipofuscinoses, a clinically and genetically heterogeneous group of childhood neurodegenerative storage disorders. Recent research progress is reviewed here, to summarize new disease gene identification, diagnostics, treatment, protein functional studies and investigations into the underlying molecular pathogenesis of these devastating disorders. Show less

A transactivation motif has been identified in the neurodegenerative disease protein, CLN3. The C-terminal domain (residues 394-438) of CLN3 can function as a transcriptional activator when fused to the DNA binding domain, LexA. A series of deletion and substitution constructs have been generated to identify the essential region for transactivation. A similar motif is also present in the POU domain transcription factor, nubbin. However, this domain alone does not activate transcription, allowing further localisation of the critical residues in CLN3 required for activity. Show less

The Saccharomyces cerevisiae cyclin-dependent kinase Cdc28 forms complexes with nine different cyclins to promote cell division. These nine cyclin-Cdc28 complexes have different roles, but share the same catalytic subunit; thus, it is not clear how substrate specificity is achieved. One possible mechanism is specific sub-cellular localization of specific complexes. We investigated the location of two G1 cyclins using fractionation and microscopy. In addition, we developed 'forced localization' cassettes, which direct proteins to particular locations, to test the importance of localization. Cln2 was found in both nucleus and cytoplasm. A substrate of Cln2, Sic1, was also in both compartments. Cytoplasmic Cln2 was concentrated at sites of polarized growth. Forced localization showed that some functions of Cln2 required a cytoplasmic location, while other functions required a nuclear location. In addition, one function apparently required shuttling between the two compartments. The G1 cyclin Cln3 required nuclear localization. An autonomous, nuclear localization sequence was found near the C-terminus of Cln3. Our data supports the hypothesis that Cln2 and Cln3 have distinct functions and locations, and the specificity of cyclin-dependent kinases is mediated in part by subcellular location. Show less

Decay-accelerating factor (DAF) is a membrane regulator of C3 activation that protects self cells from autologous complement attack. In humans, DAF is uniformly expressed as a glycosylphosphatidylinositol (GPI)-anchored molecule. In mice, both GPI-anchored and transmembrane-anchored DAF proteins are produced, each of which can be derived from two different genes (Daf1 and Daf2). In this report, we describe a Daf1 gene knock-out mouse arising as the first product of a strategy for targeting one or both Daf genes. As part of the work, we characterize recently described monoclonal antibodies against murine DAF protein using deletion mutants synthesized in yeast, and then employ the monoclonal antibodies in conjunction with wild-type and the Daf1 knock-out mice to determine the tissue distribution of the mouse Daf1 and Daf2 gene products. To enhance the immunohistochemical detection of murine DAF protein, we utilized the sensitive tyramide fluorescence method. In wild-type mice, we found strong DAF labelling of glomeruli, airway and gut epithelium, the spleen, vascular endothelium throughout all tissues, and seminiferous tubules of the testis. In Daf1 knock-out mice, DAF labelling was ablated in most tissues, but strong labelling of the testis and splenic dendritic cells remained. In both sites, reverse transcription-polymerase chain reaction analyses identified both GPI and transmembrane forms of Daf2 gene-derived protein. The results have relevance for studies of in vivo murine DAF function and of murine DAF structure. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a severe autosomal recessive neurodegenerative disorder resulting from mutations in the CLN3 gene. The gene product is a 438-amino acid hydrophobic peptide of unknown function containing five transmembrane domains. In order to study the tissue distribution of the peptide, polyclonal antibodies were raised in rabbits to three epitopes and were affinity purified before use. All three antibodies were used together for immunocytochemical staining of human pancreas. This staining showed localization in pancreatic islet cells. Double labelling of the tissue indicated that cells staining for the CLN3 protein were also positive for somatostatin. Show less

The neuronal ceroid lipofuscinoses (NCLs) are the most common neurodegenerative disorders of childhood. The CLN1, CLN2 and CLN3 genes are associated to the infantile, late infantile and juvenile forms of NCL, respectively. We have subcloned the cDNAs encoding CLN1, CLN2 and BTN1, the yeast homologue of human CLN3, into plasmid vectors to evaluate whether these proteins interact with other proteins co-expressed from either a cDNA library derived from human cerebellum or from yeast, respectively, using the two-hybrid system. We concluded that CLN1 most likely does not interact with any other proteins in vivo. Furthermore, it is unlikely that CLN2 interacts with other proteins in vivo, although this study utilized a cDNA encoding the CLN2 precursor and it is possible that interacting partners may be excluded by the nature of this protein structure. Finally, we conclude that proteins that interact with Btn1p and therefore CLN3 cannot be identified using the whole proteins in a two-hybrid system, due to the hydrophobic nature of this protein. By understanding the topology of CLN3, specific regions of CLN3 need to be tested by two-hybrid to identify any interacting partners. Show less

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a childhood neurodegenerative disease that is caused by mutations in the CLN3 gene. The protein encoded by CLN3 has no homology with any proteins of known function and its cellular role remains elusive. In order to investigate the role played by the CLN3 protein we aimed to identify interacting proteins. Here, we describe the yeast two-hybrid system as the approach taken to investigate such protein-protein interactions. CLN3 was expressed as a fusion protein with a DNA-binding domain and used to screen a library of human fetal brain cDNAs fused to a transcriptional activation domain. Owing to low level expression of the full length CLN3 fusion protein, truncated regions corresponding to the predicted hydrophilic regions were also tested. No proteins that interact with CLN3 were detected, nor was there any evidence for CLN3-CLN3 interactions. Potential interaction of CLN3 with subunit c of mitochondrial ATP synthase, the major component of the storage material that accumulates in Batten disease patients, was also tested. No interaction was detected suggesting that the accumulation of subunit c does not result from loss of a process that requires a direct interaction with CLN3. We conclude that either CLN3 does not interact with other proteins or such interactions cannot be detected using the two-hybrid system. Show less

Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid-beta protein precursor and cathepsin D in vitro. The Leu101Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin. Show less

The classic late infantile form of neuronal ceroid lipofuscinosis (CLN2, cLINCL) is associated with mutations in the gene encoding tripeptidyl-peptidase I (TPP-I), a lysosomal aminopeptidase that cleaves off tripeptides from the free N-termini of oligopeptides. To date over 30 different mutations and 14 polymorphisms associated with CLN2 disease process have been identified. In the present study, we analysed the molecular basis of 15 different mutations of TPP-I by using immunocytochemistry, immunofluorescence, Western blotting, enzymatic assay and subcellular fractionation. In addition, we studied the expression of TPP-I in other lysosomal storage disorders such as CLN1, CLN3, muccopolysaccharidoses and GM1 and GM2 gangliosidoses. Our study shows that TPP-I is absent or appears in very small amounts not only in cLINCL subjects with mutations producing severely truncated protein, but also in individuals with missense point mutations, which correlates with loss of TPP-I activity. Of interest, small amounts of TPP-I were detected in lysosomal fraction from fibroblasts from cLINCL subject with protracted form. This observation suggests that the presence of small amounts of TPP-I in lysosomes is able to delay significantly CLN2 disease process. We also show that TPP-I immunoreactivity is increased in the brain tissue of CLN1 and CLN3 subjects, stronger in glial cells and macrophages than neurons. Less prominent increase of TPP-I staining was found in muccopolysaccharidoses and GM1 and GM2 gangliosidoses. These data suggest that TPP-I participates in lysosomal turnover of proteins in pathological conditions associated with cell/tissue injury. Show less

Thirty-eight mutations and seven polymorphisms have recently been reported in the genes underlying the neuronal ceroid lipofuscinoses (NCLs) including 11 new mutations described here. A total of 114 mutations and 28 polymorphisms have now been described in the five human genes identified which cause NCL. Thirty-eight mutations are recorded for CLN1/PPT; 40 for CLN2/TTP-1, 31 for CLN3, four for CLN5, one for CLN8. Two mutations have been described in animal genes (cln8/mnd, CTSD). All mutations in NCL genes are contained in the NCL Mutation Database (http://www.ucl.ac.uk/NCL). Show less

One variant form of late infantile neuronal ceroid lipofuscinosis (LINCL) is found predominantly within the Turkish population (CLN7). Exclusion mapping showed that CLN7 was not an allelic variant of known NCL loci (CLN1, CLN2, CLN3, CLN5 or CLN6). Using the method of homozygosity mapping, a genome-wide search was undertaken and a total of 358 microsatellite markers were typed at an average distance of about 10 cM. A region of shared homozygosity was identified on chromosome 8p23. This telomeric region contained the recently identified CLN8 gene. A missense mutation in CLN8 causes progressive epilepsy with mental retardation (EPMR) or Northern epilepsy, which has so far been reported only from Finland and is now classified as an NCL. The mouse model mnd has been shown to carry a 1 bp insertion in the orthologous Cln8 gene. Statistically significant evidence for linkage was obtained in this region, with LOD scores > 3, assuming either homogeneity or heterogeneity. Flanking recombinants defined a critical region of 14 cM between D8S504 and D8S1458 which encompasses CLN8. This suggests that Turkish variant LINCL, despite having an earlier onset and more severe phenotype, may be an allelic variant of Northern epilepsy. However mutation analysis has not so far identified a disease causing mutation within the coding or non-coding exons of CLN8 in the families. The Turkish variant LINCL disease-causing mutation remains to be delineated. Show less