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The CLN3 gene, which encodes the protein whose absence is responsible for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995. The function of the protein, Cln3p, still remains elusive. We previously cloned the Saccharomyces cerevisiae homolog to the human CLN3 gene, designated BTN1, whose product is 39% identical and 59% similar to Cln3p. We report that yeast strains lacking Btn1p, btn1-Delta deletion yeast strains, are more resistant to d-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP), in a pH-dependent manner. This phenotype is complemented in yeast by the human CLN3 gene. In addition, point mutations characterized in CLN3 from individuals with less severe forms of Batten disease, when introduced into BTN1, altered the degree of ANP resistance. Severity of Batten disease due to mutations in CLN3 and the degree of ANP resistance in yeast are related when the equivalent amino acid replacements in Cln3p and Btn1p are compared. These results indicate that yeast can be used as a model for the study of Batten disease. Show less

Juvenile neuronal ceroid lipofuscinosis or Batten disease (JNCL) is a neurodegenerative disorder characterized by blindness, seizures, cognitive decline and early death. Brain atrophy and retinitis pigmentosa ensue because of neuronal and photoreceptor apoptosis. The CLN3 gene defective in JNCL encodes a novel 438 amino acid protein. Most affected genes harbor a deletion resulting in a truncated protein. CLN3 overexpression in NT2 cells enhances growth, reverses growth inhibition induced by serum starvation and protects from apoptosis induced by vincristine, staurosporine, and etoposide but not from death caused by ceramide. CLN3 modulates endogenous and vincristine-activated ceramide, and therefore suppresses apoptosis by impacting generation of ceramide. Show less

Juvenile neuronal ceroid lipofuscinosis (Batten disease) is a progressive neurologic disorder which results from mutations in the CLN3 gene, which normally produces a 48-kDa polypeptide of unknown function. To help characterize the CLN3 protein, we have studied its tissue distribution and subcellular localization in human tissues using three epitope-specific polyclonal antibodies to human CLN3 by immunoblot, immunocytochemical, and immunoelectron microscopic analysis. The most abundant CLN3 protein expression was in the gray matter of the brain, where it was localized to astrocytes, capillary endothelium, and neurons. CLN3 was also evident in peripheral nerve, in pancreatic islet cells, and within the seminiferous tubules in the testis. Staining was generally diffuse within the cytoplasm with some nuclear reactivity. Subcellular localization identified the CLN3 protein within the nucleus and along cell membranes. These results were contrasted with the cellular distribution of palmitoyl-protein thioesterase (PPT), the enzyme whose deficiency is responsible for infantile neuronal ceroid lipofuscinosis (CLN1). PPT was most abundant in brain and visceral macrophages where it displayed a coarse granular staining pattern typical of lysosomal distribution. Immunoelectron microscopy confirmed that PPT immunoreactivity was limited to lysosomes. Show less

The CLN3 gene associated with Batten disease and encoding a novel protein of a predicted 438 amino acids was cloned in 1995 by the International Batten Disease Consortium. The function of CLN3 protein remains unknown. Computer-based analysis predicted that CLN3 may contain several posttranslational modifications. Thus, to study the posttranslational modification of CLN3 protein, we have expressed a full-length CLN3 protein as a C-terminal fusion with green fluorescent protein of the jellyfish Aequerea victoria in a Chinese hamster ovary cell line. Previously, we have shown that CLN3 is a glycosylated protein from lysosomal compartment, and now, by using in vivo labeling with 32P, detection with anti-phosphoamino acid antibodies, and phosphoamino acid analysis, we demonstrate that CLN3 is a phosphorylated protein. We demonstrate that CLN3 protein does not undergo mannose 6-phosphate modification and that it is a membrane protein. Furthermore, we show that the level of CLN3 protein phosphorylation may be modulated by several protein kinases and phosphatases activators or inhibitors. Show less

CLN3 gene, associated with juvenile neuronal ceroid lipofuscinosis, encodes a novel protein of a predicted 438 amino acid residues. We have expressed a full-length CLN3 protein and fragments thereof in fusion with green fluorescent protein in Chinese hamster ovary and human neuroblastoma cell lines to study its subcellular localization and intracellular trafficking pattern. By using laser scanning confocal microscopy, we demonstrate that the full-length CLN3 fusion protein is targeted to lysosomal compartments. Tunicamycin treatment did not alter the lysosomal targeting of the CLN3 protein, which indicates that extensive N-glycosylation of the full-length CLN3 fusion protein is not engaged in its lysosomal sorting. Monensin produced retention of CLN3 fusion protein in vesicular structure of the Golgi apparatus in the perinuclear space, suggesting that CLN3 fusion protein is transported to the lysosomal compartments through the trans-Golgi cisternae. Neither of the truncated CLN3 fusion proteins encompassing its 1-138, 1-322, and 138-438 amino acid residues was disclosed in lysosomal compartments. However, CLN3 fusion protein showing double-point mutations at amino acid residues 425 and 426, thus at its putative dileucine lysosomal signaling motif, was still targeted to lysosomes, suggesting that a dileucine motif alone is not sufficient for lysosomal sorting of the CLN3 fusion protein. Show less

The product of the CLN3 gene is a novel protein of unknown function. Simulations using amphiphacy algorithms have shown that structurally CLN3 may be another candidate for the family of membranous proteins. Signals controlling intracellular targeting of many membrane proteins are present as short sequences within their cytoplasmic domains. In fact, the sequence of CLN3 protein contains several such signaling sequences, which are conserved among mammals. First, at the N-terminus, potential N-myristoylation motif is present. Second, the C-terminal part of CLN3 protein contains both the dileucine motif, which is a potential lysosomal targeting signal, and the prenylation motif. There is scanty evidence of lysosomal and/or mitochondrial localization of CLN3 protein. However, the question of where the functional site of the cln3 protein exists in vivo remains unanswered. From theoretical calculations, we hypothesized that CLN3 should be an integral part of the membranous micro-environment. First, to test this hypothesis, we initiated detergent-partitioning experiments, localizing CLN3 predominantly in a pool of membranous protein. Further studies have shown that CLN3 protein integrates spontaneously with cellular membranes. Second, based on the prenylation results of CLN3 protein in vitro, we discussed the possible topological consequences of C-terminal fragment of CLN3 protein. Show less

Juvenile neuronal ceroid lipofuscinosis is a lysosomal storage disease that causes visual impairment, progressive mental deterioration, and eventually death. A predominant 1.02-kb deletion as well as other mutations have been described in the CLN3 gene. Lacking significant identity with proteins of known function and no overt targeting signals within the primary amino acid sequence, accurate predictions of the intracellular location and function could not be made. Further, recent conflicting reports identified CLN3 as either a lysosomal or a mitochondrial protein. Transfection experiments using native and epitope-tagged fusion proteins were evaluated to help delineate CLN3 localization. We confirmed by immunohistochemistry and brefeldin A treatment that NH2-terminal green fluorescence protein (GFP)-CLN3 fusion proteins were retained in the Golgi apparatus, with no colocalization with mitochondrial markers. Anti-CLN3 antibodies directed against amino acids 67-90 of CLN3 were generated and shown to be specific for a 50-kDa protein in HEK 293 cells and GFP-CLN3 in transfected cells. However, cells transfected with nontagged CLN3 or carboxyl-terminal-tagged CLN3 were not immunoreactive with anti-CLN3 antibodies, suggesting that normally, the amino terminus interacts with other molecules. Thus, tags on the NH2-terminus probably inhibited these interactions and movement of CLN3 from the Golgi to more distal compartments. Also, CLN3 tagged at the COOH-terminus with either GFP or FLAG epitopes were retained in the ER, indicating a role for the COOH-terminus in trafficking. Taken together, these data confirm that CLN3 traffics through the ER and Golgi. Show less

This study describes the phenotype/genotype analyses of 56 probands with a juvenile onset, some of which had atypical features of neuronal ceroid lipofuscinosis, collected at the New York State Institute for Basic Research (IBR). In this group, we found probands with abundant curvilinear profiles in lysosomal storage material, deficiency of pepstatin-insensitive peptidase, and mutations in the CLN2 gene, as well as patients with a predominance of granular osmiophilic deposits in the lysosomal storage material, deficiency of palmitoyl-protein thioesterase, and mutations in the CLN1 gene. We have divided the probands into two categories: typical (or classic) and atypical. Most of the typical and atypical probands had onset of symptoms about or after 4 years of age. Interfamiliar and intrafamiliar variations were found, especially in the speed of becoming practically blind. Thus, our study indicates that some mutations in the CLN1, CLN2, and CLN3 genes may be associated with late onset of the disease process, may have a more benign clinical course, and clinic overlap with other forms of neuronal ceroid lipofuscinosis. Show less

The infantile form of neuronal ceroid lipofuscinosis (NCL) has been well studied in Finland, where there is a high carrier frequency (1:70) for a single mutation in the causative gene, CLN1, or PPT. We have recently studied a group of 29 NCL subjects in the United States with palmitoyl-protein thioesterase (PPT) deficiency and described 19 different CLN1/PPT mutations in our population. In this report, we present a review of our previous findings, including a more detailed analysis of phenotype-genotype correlations, and present previously unpublished data concerning the clinical manifestations of the disorder in children of families with multiple affected members. Our studies indicate that about half of PPT-deficient patients in the United States are very similar to Finnish infants with INCL, but that a different mutation (R151X) accounts for 40% of U.S. alleles. The Finnish mutation (R122W) is rare in the United States. The other half of U.S. PPT-deficient patients develop symptoms after the age of 2 years, much later than Finnish patients. One common mutation (the "Scottish" allele, T75P) accounts for 13% of alleles and results in a juvenile-onset phenotype that is clinically indistinguishable from JNCL with CLN3 mutations. Other rare mutations were also associated with JNCL phenotypes, such as D79G and G250V. A preliminary expression study of two of these mutant enzymes supports the conclusion that juvenile-onset NCL (JNCL with GROD) is caused by missense mutations in the PPT gene that result in mutated enzymes with residual PPT enzyme activity. Show less

In Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition. Show less

Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition. Show less

Batten disease (juvenile neuronal ceroid lipofuscinosis) is a recessive neurodegenerative disorder of childhood. The gene, CLN3, was recently identified and found to encode a novel 438 amino acid protein of unknown function. In order to gain insight into the function of the Batten disease protein (CLN3p), we investigated its subcellular localization. Protein constructs incorporating CLN3p fused to the green fluorescence protein or an eight amino acid peptide tag were transiently expressed in fibroblasts, HeLa and COS-7 cells. A juxtanuclear, asymmetric localization pattern was observed that correlated with the Golgi apparatus in all three cell types. However, a proportion of transiently transfected cells exhibited a punctate vesicular distribution throughout the cytoplasm in addition to or without the Golgi localization. In order to account for localization patterns arising from intracellular protein transport disruption due to exaggerated overexpression in transiently transfected cells, we isolated a stably transfected cell line expressing only one copy of the CLN3 -GFP DNA construct. Fluorescence and biochemical analyses using this cell line demonstrated that CLN3p is an integral membrane protein that localizes primarily in the Golgi apparatus. The functional implications of this finding are discussed. Show less

To correlate the phenotypes with the genotypes of 10 Finnish juvenile neuronal ceroid lipofuscinosis (JNCL; late-onset Batten disease) patients who all are compound heterozygotes for the major 1.02-kb deletion in the CLN3 gene. The mutations on the non-1.02-kb deletion chromosomes were screened in 6 patients; in the other 4 patients the mutations were known (one affecting a splice site, two missense mutations, and one deletion of exons 10 through 13). Clinical features were examined, and MRI, MRS, somatosensory evoked magnetic field (SEF), and overnight polysomnography (PSG) studies were performed. A novel deletion of exons 10 through 13 was found in 6 patients belonging to three families. In the patients carrying the deletions of exons 10 through 13 the clinical course of the disease was fairly similar. Variation was greatest in the time course to blindness. In these patients the mental and motor decline was slower than in classic JNCL, but more severe than in the two patients with missense mutations in exons 11 and 13. MRI showed brain atrophy in 4 patients. One patient had hyperintense periventricular white matter, otherwise brain signal intensities were normal. SEFs were enhanced in patients older than 14 years, whereas in PSG all but the youngest 6-year-old patient showed epileptiform activity in slow-wave sleep. JNCL can manifest as at least three different phenotypes: classic, delayed classic, and protracted JNCL with predominantly ocular symptoms. Finnish compound heterozygotes have the delayed classic or the protracted form of JNCL. Show less

In the absence of a successful mating, pheromone-arrested Saccharomyces cerevisiae cells reenter the mitotic cycle through a recovery process that involves downregulation of the mating mitogen-activated protein kinase (MAPK) cascade. We have isolated a novel gene, POG1, whose promotion of recovery parallels that of the MAPK phosphatase Msg5. POG1 confers alpha-factor resistance when overexpressed and enhances alpha-factor sensitivity when deleted in the background of an msg5 mutant. Overexpression of POG1 inhibits alpha-factor-induced G1 arrest and transcriptional repression of the CLN1 and CLN2 genes. The block in transcriptional repression occurs at SCB/MCB promoter elements by a mechanism that requires Bck1 but not Cln3. Genetic tests strongly argue that POG1 promotes recovery through upregulation of the CLN2 gene and that the resulting Cln2 protein promotes recovery primarily through an effect on Ste20, an activator of the mating MAPK cascade. A pog1 cln3 double mutant displays synthetic mutant phenotypes shared by cell-wall integrity and actin cytoskeleton mutants, with no synthetic defect in the expression of CLN1 or CLN2. These and other results suggest that POG1 may regulate additional genes during vegetative growth and recovery. Show less

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality. Show less

Diploid yeast cells switch from mitosis to meiosis when starved of essential nutrients. While G1 cyclins play a key role in initiating the mitotic cell cycle, entry into meiosis depends on Ime1, a transcriptional activator regulated by both nutritional and cell-type signals. We show here that G1 cyclins downregulate IME1 transcription and prevent the accumulation of the Ime1 protein within the nucleus, which results in repression of early-meiotic gene expression. As G1-cyclin deficient cells do not require nutrient starvation to undergo meiosis, G1 cyclin would exert its role by transmitting essential nutritional signals to Ime1 function. The existence of a negative cross-talk mechanism between mitosis and meiosis may help explain why these two developmental options are incompatible in budding yeast. Show less

1. In order to investigate the biological function of the human CLN3 gene that is defective in Batten disease, we created a yeast strain by PCR-targeted disruption of the yeast gene (YHC3), which is a homologue of the human CLN3 gene. 2. The phenotypic characterization revealed that the yhc3 delta mutants are more sensitive to combined heat and alkaline stress than the wild-type strains as determined by inhibition of cell proliferation. 3. This suggests that the yhc3 delta mutant is a good model to investigate the biological function of human CLN3 gene in mammalian cells and to understand the pathophysiology of juvenile Batten disease. Show less

Several lines of evidence suggest that the morphogenetic transition from the yeast form to pseudohyphae in Saccharomyces cerevisiae may be regulated by the cyclin-dependent kinase (Cdk). To examine this hypothesis, we mutated all of the G1 cyclin genes in strains competent to form pseudohyphae. Interestingly, mutation of each G1 cyclin results in a different filamentation phenotype, varying from a significant defect in cln1/cln1 strains to enhancement of filament production in cln3/cln3 strains. cln1 cln2 double mutants are more defective in pseudohyphal development and haploid invasive growth than cln1 strains. FLO11 transcription, which correlates with the level of invasive growth, is low in cln1 cln2 mutants and high in grr1 cells (defective in proteolysis of Cln1,2), suggesting that Cln1,2/Cdks regulate the pseudohyphal transcriptional program. Epistasis analysis reveals that Cln1,2/Cdk and the filamentation MAP kinase pathway function in parallel in regulating filamentous and invasive growth. Cln1 and Cln2, but not Ste20 or Ste12, are responsible for most of the elevated FLO11 transcription in grr1 strains. Furthermore, phenotypic comparison of various filamentation mutants illustrates that cell elongation and invasion/cell-cell adhesion during filamentation are separable processes controlled by the pseudohyphal transcriptional program. Potential targets for G1 cyclin/Cdks during filamentous growth are discussed. Show less

The transcription complexes SBF and MBF mediate the G(1)-S transition in the cell cycle of Saccharomyces cerevisiae. In late G(1), SBF and MBF induce a burst of transcription in a number of genes, including G(1)- and S-phase cyclins. Activation of SBF and MBF depends on the G(1) cyclin Cln3 and a largely uncharacterized protein called Bck2. We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF. Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28. Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF. We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression. One of these suppressors was the negative regulator of meiosis RME1. Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity. Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1. Show less

Batten disease, a degenerative neurological disorder with juvenile onset, is the most common form of the neuronal ceroid lipofuscinoses. Mutations in the CLN3 gene cause Batten disease. To facilitate studies of Batten disease pathogenesis and treatment, a murine model was created by targeted disruption of the Cln3 gene. Mice homozygous for the disrupted Cln3 allele had a neuronal storage disorder resembling that seen in Batten disease patients: there was widespread and progressive intracellular accumulation of autofluorescent material that by EM displayed a multilamellar rectilinear/fingerprint appearance. Inclusions contained subunit c of mitochondrial ATP synthase. Mutant animals also showed neuropathological abnormalities with loss of certain cortical interneurons and hypertrophy of many interneuron populations in the hippocampus. Finally, as is true in Batten disease patients, there was increased activity in the brain of the lysosomal protease Cln2/TPP-1. Our findings are evidence that the Cln3-deficient mouse provides a valuable model for studying Batten disease. Show less

Among the epilepsies, the progressive myoclonus epilepsies (PMEs) form a heterogeneous group of rare diseases characterized by myoclonus, epilepsy, and progressive neurologic deterioration, particularly dementia and ataxia. The success of the Human Genome Project and the fact that most PMEs are inherited through a mendelian or mitochondrial mode have resulted in important advances in the definition of the molecular basis of PME. The gene defects for the most common forms of PME (Unverricht-Lundborg disease, the neuronal ceroid lipofuscinoses, Lafora disease, type I sialidosis, and myoclonus epilepsy with ragged-red fibers) have been either identified or mapped to specific chromosome sites. Unverricht-Lundborg disease has been shown to be caused by mutations in the gene that codes for cystatin B, an inhibitor of cysteine protease. The most common mutation in Unverricht-Lundborg disease is an expansion of a dodecamer repeat located in a noncoding region upstream of the transcription start site of the cystatin B gene, making it the first human disease associated with instability of a dodecamer repeat. Juvenile neuronal ceroid lipofuscinosis is caused by mutations in the CLN3 gene, a gene of unknown function that encodes a 438-amino-acid protein of possible mitochondrial location. Other forms of neuronal ceroid lipofuscinosis that occur as PME and Lafora disease have been mapped by means of linkage analysis, but the corresponding gene defects remain unknown. Sialidosis has been shown to be caused by mutations in the sialidase gene, and myoclonus epilepsy with ragged-red fibers is well known to be caused by mutations in the mitochondrial gene that codes for tRNA(Lys). How the different PME gene defects described produce the various PME phenotypes, including epileptic seizures, remains unknown. The development of animal models that bear these mutations is needed to increase our knowledge of the basic mechanisms involved in the PMEs. This knowledge should lead to the development of new and effective forms of therapy, which are especially lacking for the PMEs. Show less

During brain development, excess neurons that are formed die by apoptosis. cln3 was recently identified as the gene defective in juvenile Batten disease, an inherited neurodegenerative disease of childhood. In this disease, neurons die by apoptosis. Overexpression of this gene increases survival of human NT2 neuronal precursor cells. We, therefore, hypothesized that cln3 may be present in developing neurons and may play an important role in regulating the developmental process. NT2 neuronal cells were induced to develop into mature neurons. We evaluated cln3 expression by reverse transcription PCR and immunohistochemistry over a 7-wk period of differentiation. Also, cln3 expression was characterized in neonatal rat brain during the first week of life (P-1, P0, P4, and P8) and at P30. cln3 was differentially expressed during neuronal development into nondividing post-mitotic neurons. The greatest expression was noted during wk 6 and then dropped to predifferentiation levels during wk 7. cln3 expression was detected in all the rat brain developmental stages evaluated. The greatest expression was seen at P0 and was double compared with the other stages. We conclude that cln3 is present during critical periods of neuronal cell differentiation and brain development. As cln3 is antiapoptotic, we hypothesize that cln3 plays an important role in regulating brain development. These findings may have implications for identifying strategies aimed at neuroprotection and neuronal survival during development. Show less

BTN1 of Saccharomyces cerevisiae encodes an ortholog of CLN3, the human Batten disease gene. We have reported previously that deletion of BTN1, btn1-Delta, resulted in a pH-dependent resistance to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP). This phenotype was caused by btn1-Delta strains having an elevated ability to acidify growth medium through an elevated activity of the plasma membrane H(+)-ATPase, resulting from a decreased vacuolar pH during early growth. We have determined that growing btn1-Delta strains in the presence of chloroquine reverses the resistance to ANP, decreases the rate of medium acidification, decreases the activity of plasma membrane H(+)-ATPase, and elevates vacuolar pH. However, an additional effect of this phenotypic reversal is that activity of plasma membrane H(+)-ATPase is decreased further and vacuolar pH is increased further as btn1-Delta strains continue to grow. This phenotypic reversal of btn1-Delta can be considered for developing a therapy for Batten disease. Show less

The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a group of neurodegenerative disorders characterised by the accumulation of an autofluorescent lipopigment in many cell types. Different NCL types are distinguished according to age of onset, clinical phenotype, ultrastructural characterisation of the storage material, and chromosomal location of the disease gene. At least eight genes underlie the NCLs, of which four have been isolated and mutations characterised: CLN1, CLN2, CLN3, CLN5. Two of these genes encode lysosomal enzymes, and two encode transmembrane proteins, at least one of which is likely to be in the lysosomal membrane. The basic defect in the NCLs appears to be associated with lysosomal function. Show less

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, stress resistance and proliferation, in particular in connection with the available nutrient conditions. Extensive information has been obtained on the core section of the pathway, i.e. Cdc25, Ras, adenylate cyclase, PKA, and on components interacting directly with this core section, such as the Ira proteins, Cap/Srv2 and the two cAMP phosphodiesterases. Recent work has now started to reveal upstream regulatory components and downstream targets of the pathway. A G-protein-coupled receptor system (Gpr1-Gpa2) acts upstream of adenylate cyclase and is required for glucose activation of cAMP synthesis in concert with a glucose phosphorylation-dependent mechanism. Although a genuine signalling role for the Ras proteins remains unclear, they appear to mediate at least part of the potent stimulation of cAMP synthesis by intracellular acidification. Recently, several new targets of the PKA pathway have been discovered. These include the Msn2 and Msn4 transcription factors mediating part of the induction of STRE-controlled genes by a variety of stress conditions, the Rim15 protein kinase involved in stationary phase induction of a similar set of genes and the Pde1 low-affinity cAMP phosphodiesterase, which specifically controls agonist-induced cAMP signalling. A major issue that remains to be resolved is the precise connection between the cAMP-PKA pathway and other nutrient-regulated components involved in the control of growth and of phenotypic characteristics correlated with growth, such as the Sch9 and Yak1 protein kinases. Cln3 appears to play a crucial role in the connection between the availability of certain nutrients and Cdc28 kinase activity, but it remains to be clarified which nutrient-controlled pathways control Cln3 levels. Show less

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterised by the accumulation of autofluorescent storage material in neurons and other cell types. The clinical features include visual impairment, progressive myoclonic epilepsy, and cognitive decline reflecting progressive neurodegeneration. The NCLs are subdivided into several subtypes according to age of onset, clinical course, and ultrastructure of the storage material. The molecular genetic basis of this group of disorders has recently been clarified. Mutations in the gene encoding a lysosomal enzyme, palmitoyl protein thioesterase (PPT), cause infantile NCL (locus CLN1 on chromosome 1p32) or Haltia-Santavuori disease. This Finnish disease is characterised ultrastructurally by granular osmiophilic deposits (GRODs). Juvenile-onset NCL with GRODs also is caused by mutations in PPT. Classic late-infantile NCL (Jansky-Bielschowsky disease) is caused by mutations in a gene encoding a pepstatin-insensitive lysosomal peptidase (CLN2 on chromosome 11p15), and juvenile-onset NCL (Batten disease) is caused by mutations in a gene encoding a 438-amino-acid membrane protein (CLN3 on chromosome 16p12) of unknown function. A locus for Finnish variant late-infantile NCL, CLN5, has been mapped to chromosome 13q22 and a locus for variant late-infantile NCL, CLN6, to chromosome 15q21-23. These and further advances will allow the molecular basis of the NCLs to be elucidated and may lead to new strategies for diagnosis and treatment. Show less

In the yeast Saccharomyces cerevisiae, heat shock stress induces a variety of cellular responses including a transient cell cycle arrest before G(1)/S transition. Previous studies have suggested that this G(1) delay is probably attributable to a reduced level of the G(1) cyclin gene (CLN1 and CLN2) transcripts. Here we report our finding that the G(1) cyclin Cln3 and the S cyclin Clb5 are the key factors required for recovery from heat shock-induced G(1) arrest. Heat shock treatment of G(1) cells lacking either CLN3 or CLB5/CLB6 functions leads to prolonged cell cycle arrest before the initiation of DNA synthesis, concomitant with a severe deficiency in bud formation. The inability of the clb5 clb6 mutant to resume normal budding after heat shock treatment is unanticipated, since the S phase cyclins are generally thought to be required mainly for initiation of DNA synthesis and have no significant roles in bud formation in the presence of functional G(1) cyclins. Further studies reveal that the accumulation of G(1) cyclin transcripts is markedly delayed in the clb5 clb6 mutant following heat shock treatment, indicating that the CLN gene expression may require Clb5/Clb6 to attain a threshold level for driving the cell cycle through G(1)/S transition. Consistent with this assumption, overproduction of Clb5 greatly enhances the transcription of at least two G(1) cyclin genes (CLN1 and CLN2) in heat-shocked G(1) cells. These results suggest that Clb5 may positively regulate the expression of G(1) cyclins during cellular recovery from heat shock-induced G(1) arrest. Additional evidence is presented to support a role for Clb5 in maintaining the synchrony between budding and DNA synthesis during normal cell division as well. Show less

The human hereditary ceroid-lipofuscinoses are a group of autosomal recessively inherited diseases characterized by massive accumulations of autofluorescent lysosomal storage bodies in the cells of many tissues and by neuronal degeneration throughout the central nervous system. There are a number of clinically and genetically distinct forms of ceroid-lipofuscinosis, the most common of which is the juvenile type, also known as Batten disease and CLN3. To study the mechanisms that lead to pathology in CLN3 and to evaluate potential therapies, a mouse model has been generated by targeted disruption of the mouse ortholog of the CLN3 gene (Cln3). As in affected humans, mice homozygous for the disrupted Cln3 allele show accumulation of autofluorescent storage material in neurons and other cell types. The storage material consists of membrane-bounded intracellular inclusions with ultrastructural features typical of the ceroid-lipofuscinoses. The accumulation of this storage material validates the Cln3 knockout mice as a model for the human disorder. Show less

When Saccharomyces cerevisiae cells are transferred from poor medium to fresh medium containing glucose, they rapidly increase the transcription of a large group of genes as they resume rapid growth and accelerate progress through the cell cycle. Among those genes induced by glucose is CLN3, encoding a G(1) cyclin that is thought to play a pivotal role in progression through Start. Deletion of CLN3 delays the increase in proliferation normally observed in response to glucose medium. ADA2 and ADA3/NGG1 are necessary for the rapid induction of CLN3 message levels in response to glucose. Loss of either ADA2 or ADA3/NGG1 also affects a large number of genes and inhibits the rapid global increase in transcription that occurs in response to glucose. Surprisingly, these effects are transitory, and expression of CLN3 and total poly(A)(+) RNA appear normal when ADA2 or ADA3/NGG1 deletion mutants are examined in log-phase growth. These results indicate a role for ADA2 and ADA3/NGG1 in allowing rapid transcriptional responses to environmental signals. Consistent with the role of the Ada proteins in positive regulation of CLN3, deletion of RPD3, encoding a histone deacetylase, prevented the down regulation of CLN3 mRNA in the absence of glucose. Show less

The first prenatal diagnosis of variant late infantile neuronal ceroid lipofuscinosis (vLINCL[Finnish]; CLN5) is reported. The disease belongs to the group of progressive encephalopathies in children with psycho-motor deterioration, visual failure and premature death. Neurons and several extraneural cells harbour lysosomal inclusions showing accumulation of material with histochemical characteristics of ceroid and lipofuscin. A Finnish woman with a daughter with vLINCL came for genetic counselling for her current pregnancy. Electron microscopy of a chorionic villus sample (CVS) at the 11th week of gestation did not reveal inclusions characteristic for NCL. DNA analysis showed that the fetus had inherited the major mutation, a 2 bp deletion of the CLN5 gene from the mother, and the same paternal (and maternal) haplotypes for COLAC1 and AC224 as the affected daughter. The pregnancy was terminated. Electron microscopy of the CVS of the aborted fetus at the 14th week of pregnancy showed lysosomal electron dense inclusions with straight and curved lamellar profiles consistent with vLINCL. Prenatal diagnosis of NCL-disorders (CLN1, CLN2, CLN3) can be made from CVS by demonstrating the mutations of the affected genes or by haplotype analysis using the closely linked markers in most cases. In various clinical settings the DNA diagnostics may not be possible. Demonstration of the characteristic inclusions of the placenta and fetal tissues remains a helpful adjunct in such cases. Show less