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Industrial yeasts, including a sake yeast strain Kyokai no. 7 (K7), are generally unable to sporulate. Previously, we have reported that in K7 (Saccharomyces cerevisiae) cells, deletion of the G1 cyclin gene CLN3, a key activator of the cell cycle, allows the cells to induce IME1 transcription and sporulate under sporulation conditions. Here we show that treatment with the immunosuppressive drug rapamycin also restores sporulation competence in K7 cells. Moreover, sporulation was observed after rapamycin treatment in other industrial yeasts, namely bottom fermenting yeast strains and a wine yeast strain, which are not able to sporulate under normal sporulation conditions. These findings suggest that activation of TORC1 under sporulation conditions leads to sporulation incompetence in these yeasts. Thus, rapamycin treatment will be useful to restore sporulation competence in industrial yeasts. Show less

Juvenile neuronal ceroid lipofuscinoses (JNCL) is the most common type of the neuronal ceroid lipofuscinoses (NCLs), a group of pediatric neurodegenerative diseases. In this chapter the genetic and biochemical basis, pathogenesis, clinical features, histopathological features, diagnosis and therapeutic strategies of the JNCL are reviewed. The premature death of the patients and subnormal life quality are inevitable due to the lack of understanding of pathogenesis and limitation in treatment. Hence we are still a long way to conquer the disease. Show less

The neuronal ceroid lipofuscinosis are classified based on age at onset into four main clinical forms in child-hood: infantile, late infantile, juvenile and congenital (CLN1, CLN2, CLN3 and CLN10). The variant late infantile forms (CLN5, CLN6, CLN7 and CLN8) are characterized by a wide variability of the clinical phenotypes and the most patients are originated from Finland and Turkey (Finnish, CLN5, and Turkish, CLN7 variants). We describe three unrelated patients with Finnish variant and another patient with Turkish variant. We describe an algorithm to facility the diagnosis of these low prevalence diseases. Patients with Finnish variant started with behaviour disorder between 2.6 and 4.6 years of age followed by learning difficulties and visual failure at an age of 6 years. Generalised tonic-clonic and myoclonic seizures were observed at 7 years of age with myoclonic jerks later on. Patients developed ataxia and blindness within 9 years and increasingly disability at 11 years of age. The patient with Turkish variant started with refractory epilepsy at age of 2, followed by a severe neurodegeneration manifested by ataxia, loss of walking ability within 2-3 years and vegetative state at 11 years of age. The clinical spectrum of the variant late infantile forms shows a wide geographical distribution. We report three novel mutations in the CLN5 gene and a diagnostic algorithm to facility the correlation genotype-phenotype studies. Show less

Loss-of-function mutations in CLN3 are responsible for juvenile-onset neuronal ceroid lipofuscinosis (JNCL), or Batten disease, which is an incurable lysosomal disease that manifests with vision loss, followed by seizures and progressive neurodegeneration, robbing children of motor skills, speech and cognition, and eventually leading to death in the second or third decade of life. Emerging clinical evidence points to JNCL pathology outside of the CNS, including the cardiovascular system. The CLN3 gene encodes an unusual transmembrane protein, CLN3 or battenin, whose elusive function has been the subject of intense study for more than 10 years. Owing to the detailed characterization of a large number of disease models, our knowledge of CLN3 protein function is finally coming into focus. This review will describe the most current understanding of CLN3 structure, function and dysfunction in JNCL. Show less

Neuronal ceroid lipofuscinosis (NCL) is a neurodegenerative disease caused by a number of different genes. A mutational analysis of the feline CLN3 gene was performed in a cat with NCL that had vacuolated lymphocytes, which is a feature of human NCL caused by defects of the CLN3 gene. To determine the candidate gene(s) responsible for this case, NCL-specific ultrastructures of storage materials were analysed. A sequence analysis indicated that the CLN3 gene was not likely to be responsible for this case of feline NCL because no deleterious mutation was detected. An ultrastructural analysis did not reveal any candidate gene because of inconsistency with any pattern found in human NCL. These findings suggest that the diagnostic criteria for human NCL are not directly applicable to feline NCL. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL; CLN3 disease; Batten disease) is an autosomal recessive neurodegenerative disease of childhood. Symptoms typically present at school age with vision loss followed by progressive cognitive decline, motor dysfunction, seizures, and behavior problems. Studies on sex differences in JNCL have yielded mixed results, but parent anecdotes suggest that females experience a more precipitous disease course. Therefore, we sought to determine if sex-based differences exist in JNCL. We used data from the Unified Batten Disease Rating Scale (UBDRS), the Batten Disease Support and Research Association (BDSRA) database, and the PedsQL quality of life (QoL) survey to evaluate sex-based differences in functional independence and time from symptom onset to death. On average, females had JNCL symptom onset one year later and death one year earlier than did males. Despite a later age at onset, females had lower functional capability, earlier loss of independent function, and lower physical QoL. Future research in sex differences in JNCL may help to further understand the biological mechanisms underpinning the disease course and may point to targeted therapies. Show less

Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Show less

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development. Show less

Neuronal ceroid lipofuscinosis (NCL) is a genetically heterogeneous group of lysosomal diseases that collectively compose the most common Mendelian form of childhood-onset neurodegeneration. It is estimated that ∼8% of individuals diagnosed with NCL by conservative clinical and histopathologic criteria have been ruled out for mutations in the nine known NCL-associated genes, suggesting that additional genes remain unidentified. To further understand the genetic underpinnings of the NCLs, we performed whole-exome sequencing on DNA samples from a Mexican family affected by a molecularly undefined form of NCL characterized by infantile-onset progressive myoclonic epilepsy (PME), vision loss, cognitive and motor regression, premature death, and prominent NCL-type storage material. Using a recessive model to filter the identified variants, we found a single homozygous variant, c.550C>T in KCTD7, that causes a p.Arg184Cys missense change in potassium channel tetramerization domain-containing protein 7 (KCTD7) in the affected individuals. The mutation was predicted to be deleterious and was absent in over 6,000 controls. The identified variant altered the localization pattern of KCTD7 and abrogated interaction with cullin-3, a ubiquitin-ligase component and known KCTD7 interactor. Intriguingly, murine cerebellar cells derived from a juvenile NCL model (CLN3) showed enrichment of endogenous KCTD7. Whereas KCTD7 mutations have previously been linked to PME without lysosomal storage, this study clearly demonstrates that KCTD7 mutations also cause a rare, infantile-onset NCL subtype designated as CLN14. Show less

A dynamic balance between targeted transport and endocytosis is critical for polarized cell growth. However, how actin-mediated endocytosis is regulated in different growth modes remains unclear. Here we report differential regulation of cortical actin patch dynamics between the yeast and hyphal growth in Candida albicans. The mechanism involves phosphoregulation of the endocytic protein Sla1 by the cyclin-dependent kinase (CDK) Cdc28-Cln3 and the actin-regulating kinase Prk1. Mutational studies of the CDK phosphorylation sites of Sla1 revealed that Cdc28-Cln3 phosphorylation of Sla1 enhances its further phosphorylation by Prk1, weakening Sla1 association with Pan1, an activator of the actin-nucleating Arp2/3 complex. Sla1 is rapidly dephosphorylated upon hyphal induction and remains so throughout hyphal growth. Consistently, cells expressing a phosphomimetic version of Sla1 exhibited markedly reduced actin patch dynamics, impaired endocytosis, and defective hyphal development, whereas a nonphosphorylatable Sla1 had the opposite effect. Taken together, our findings establish a molecular link between CDK and a key component of the endocytic machinery, revealing a novel mechanism by which endocytosis contributes to cell morphogenesis. Show less

Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4) and SCF(Grr1), redundantly target Cln3 for degradation. While the F-box proteins (FBPs) Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression. Show less

Cell size homeostasis is a conserved attribute in many eukaryotic species involving a tight regulation between the processes of growth and proliferation. In budding yeast S. cerevisiae, growth to a "critical cell size" must be achieved before a cell can progress past START and commit to cell division. Numerous studies have shown that progression past START is actively regulated by cell size control genes, many of which have implications in cell cycle control and cancer. Two initial screens identified genes that strongly modulate cell size in yeast. Since a second generation yeast gene knockout collection has been generated, we screened an additional 779 yeast knockouts containing 435 new ORFs (~7% of the yeast genome) to supplement previous cell size screens. Upon completion, 10 new strong size mutants were identified: nine in log-phase cells and one in saturation-phase cells, and 97% of the yeast genome has now been screened for cell size mutations. The majority of the logarithmic phase size mutants have functions associated with translation further implicating the central role of growth control in the cell division process. Genetic analyses suggest ECM9 is directly associated with the START transition. Further, the small (whi) mutants mrpl49Δ and cbs1Δ are dependent on CLN3 for cell size effects. In depth analyses of new size mutants may facilitate a better understanding of the processes that govern cell size homeostasis. Show less

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity. Show less

In Saccharomyces cerevisiae, the G1 cyclin/cyclin-dependent kinase (CDK) complexes Cln1,-2,-3/Cdk1 promote S phase entry during the mitotic cell cycle but do not function during meiosis. It has been proposed that the meiosis-specific protein kinase Ime2, which is required for normal timing of pre-meiotic DNA replication, is equivalent to Cln1,-2/Cdk1. These two CDK complexes directly catalyze phosphorylation of the B-type cyclin/CDK inhibitor Sic1 during the cell cycle to enable its destruction. As a result, Clb5,-6/Cdk1 become activated and facilitate initiation of DNA replication. While Ime2 is required for Sic1 destruction during meiosis, evidence now suggests that Ime2 does not directly catalyze Sic1 phosphorylation to target it for destabilization as Cln1,-2/Cdk1 do during the cell cycle. We demonstrated that Sic1 is eventually degraded in meiotic cells lacking the IME2 gene (ime2Δ), supporting an indirect role of Ime2 in Sic1 destruction. We further examined global RNA expression comparing wild type and ime2Δ cells. Analysis of these expression data has provided evidence that Ime2 is required early in meiosis for normal transcription of many genes that are also periodically expressed during late G1 of the cell cycle. Our results place Ime2 at a position in the early meiotic pathway that lies upstream of the position occupied by Cln1,-2/Cdk1 in the analogous cell cycle pathway. Thus, Ime2 may functionally resemble Cln3/Cdk1 in promoting S phase entry, or it could play a role even further upstream in the corresponding meiotic cascade. Show less

CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3∆ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport. Show less

The FAR1 gene encodes an 830 residue bifunctional protein, whose major function is inhibition of cyclin-dependent kinase complexes involved in the G1/S transition. FAR1 transcription is maximal between mitosis and early G1 phase. Enhanced FAR1 transcription is necessary but not sufficient for the pheromone-induced G1 arrest, since FAR1 overexpression itself does not trigger cell cycle arrest. Besides its well established role in the response to pheromone, recent evidences suggest that Far1 may also regulate the mitotic cell cycle progression: in particular, it has been proposed that Far1, together with the G1 cyclin Cln3, may be part of a cell sizer mechanism that controls the entry into S phase. Far1 is an unstable protein throughout the cell cycle except during G1 phase. Far1 levels peak in newborn cells as a consequence of a burst of synthetic activity at the end of the previous cycle, and the amounts per cell remain roughly constant during the G1 phase. Phosphorylation (at serine 87) by Cdk1-Cln complexes primes Far1 for ubiquitin-mediated proteolysis. By coupling a genome-wide transcriptional analysis of FAR1-overexpressing and far1Δ cells grown in ethanol- or glucose-supplemented minimal media with a range of phenotypic analysis, we show that FAR1 overexpression not only coordinately increases RNA and protein accumulation, but induces strong transcriptional remodeling, metabolism being the most affected cellular property, suggesting that the Far1/Cln3 sizer regulates cell growth either directly or indirectly by affecting metabolism and pathways known to modulate ribosome biogenesis. A crucial role in mediating the effect of Far1 overexpression is played by the Sfp1 protein, a key transcriptional regulator of ribosome biogenesis, whose presence is mandatory to allow a coordinated increase in both RNA and protein levels in ethanol-grown cells. Show less

BTN1, the yeast homolog to human CLN3 (which is defective in Batten disease), has been implicated in the regulation of vacuolar pH, potentially by modulating vacuolar-type H(+)-ATPase (V-ATPase) activity. However, we report that Btn1p and the V-ATPase complex do not physically interact, suggesting that any influence that Btn1p has on V-ATPase is indirect. Because membrane lipid environment plays a crucial role in the activity and function of membrane proteins, we investigated whether cells lacking BTN1 have altered membrane phospholipid content. Deletion of BTN1 (btn1-Δ) led to a decreased level of phosphatidylethanolamine (PtdEtn) in both mitochondrial and vacuolar membranes. In yeast there are two phosphatidylserine (PtdSer) decarboxylases, Psd1p and Psd2p, and these proteins are responsible for the synthesis of PtdEtn in mitochondria and Golgi-endosome, respectively. Deletion of both BTN1 and PSD1 (btn1-Δ psd1-Δ) led to a further decrease in levels of PtdEtn in ER membranes associated to mitochondria (MAMs), with a parallel increase in PtdSer. Fluorescent-labeled PtdSer (NBD-PtdSer) transport assays demonstrated that transport of NBD-PtdSer from the ER to both mitochondria and endosomes and/or vacuole is affected in btn1-Δ cells. Moreover, btn1-Δ affects the synthesis of PtdEtn by the Kennedy pathway and impairs the ability of psd1-Δ cells to restore PtdEtn to normal levels in mitochondria and vacuoles by ethanolamine addition. In summary, lack of Btn1p alters phospholipid levels and might play a role in regulating their subcellular distribution. Show less

The neuronal ceroid lipofuscinoses (NCLs) are clinically and genetically heterogeneous neurodegenerative disorders. Most are autosomal recessively inherited. Clinical features include a variable age of onset, motor and mental decline, epilepsy, visual loss, and premature death. Mutations in eight genes (PPT1/CLN1, TPP1/CLN2, CLN3, CLN5, CLN6, MFSD8/CLN7, CLN8) have been identified and several more are predicted to exist, including two provisionally named CLN4 and CLN9. Despite excessive in vitro and in vivo studies, the precise functions of the NCL proteins and the disease mechanisms remain elusive. To date 365 NCL-causing mutations are known, with 91 novel disease-causing mutations reported. These are reviewed with an emphasis on their complex correlation to phenotypes. Different mutations within the NCL spectrum can cause variable disease severity. The NCLs exemplify both phenotypic convergence or mimicry and phenotypic divergence. For example, mutations in CLN5, CLN6, MFSD8, or CLN8 can underlie the clinically similar late infantile variant NCL disease. Phenotypic divergence is exemplified by different CLN8 mutations giving rise to two very different diseases, the mild CLN8 disease, EPMR (progressive epilepsy with mental retardation), and the more severe CLN8 disease, late infantile variant. The increase in the genetic understanding of the NCLs has led to improved diagnostic approaches, and the recent proposal of a new nomenclature. Show less

Neuronal ceroid lipofuscinosis is a severe neurodegenerative lysosomal storage disorder. Gamma-aminobutyric acid and glutamate deficiency have been reported with CLN1, CLN3, and CLN6. Isolated biopterin/neopterin without dopamine deficiency has been reported in 1 patient with a CLN2 mutation. This report describes a patient with a CLN2 mutation with symptomatic biopterin and dopamine deficiency. A 4-year-old boy presented with intractable epilepsy and developmental regression starting 1 year previously. His exam showed retinopathy, scanning speech, dysmetria, and ataxic fenestrating gait with stooped posture. Electroencephalogram showed generalized spikes with occipital spikes on slow photic stimulation. Brain magnetic resonance images 1 year apart showed significant diffuse atrophy. CLN2 gene sequencing showed pathogenic compound heterozygous mutations. Cerebrospinal fluid neurotransmitters showed low homovanillic acid and tetrahydrobiopterin. Levodopa-carbidopa resulted in dramatic improvement of gait. Dopamine/biopterin deficiency is a possible secondary manifestation of CLN2 mutations. Levodopa and dopamine agonists might be useful in treating these secondary abnormalities and improving quality of life in these patients. Show less

Currently there is no treatment for juvenile Batten disease, a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. The Cln3-knockout (Cln3(Δex1-6)) mouse model recapitulates several features of the human disorder. Cln3(Δex1-6) mice, similarly to juvenile Batten disease patients, have a motor coordination deficit detectable as early as postnatal day 14. Previous studies demonstrated that acute attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity by the non-competitive AMPA antagonist, EGIS-8332, in both 1- and 6-7-month-old Cln3(Δex1-6) mice results in improvement in motor coordination. Here we show that acute inhibition of N-methyl-D-aspartate (NMDA)-type glutamate receptors by memantine (1 and 5 mg/kg i.p.) had no effect on the impaired motor coordination of one-month-old Cln3(Δex1-6) mice. At a later stage of the disease, in 6-7-month-old Cln3(Δex1-6) mice, memantine induced a delayed but extended (8 days) improvement of motor skills similarly to that observed previously with EGIS-8332 treatment. An age-dependent therapeutic effect of memantine implies that the pathomechanism in juvenile Batten disease changes during disease progression. In contrast to acute treatment, repeated administration of memantine or EGIS-8332 (1 mg/kg, once a week for 4 weeks) to 6-month-old Cln3(Δex1-6) mice had no beneficial effect on motor coordination. Moreover, repeated treatments did not impact microglial activation or the survival of vulnerable neuron populations. Memantine did not affect astrocytosis in the cortex. EGIS-8332, however, decreased astrocytic activation in the somatosensory barrelfield cortex. Acute inhibition of NMDA receptors can induce a prolonged therapeutic effect, identifying NMDA receptors as a new therapeutic target for juvenile Batten disease. Show less

Autophagy is a fundamental cellular process promoting survival under various environmental stress conditions. Selective types of autophagy have gained much interest recently as they are involved in specific quality control mechanisms removing, for example, aggregated proteins or dysfunctional mitochondria. This is considered to counteract the development of a number of neurodegenerative disorders and aging. Here we review the role of mitophagy and mitochondrial dynamics in ensuring quality control of mitochondria. In particular, we provide possible explanations why mitophagy in yeast, in contrast with the situation in mammals, was found to be independent of mitochondrial fission. We further discuss recent findings linking these processes to nutrient sensing pathways and the general stress response in yeast. In particular, we propose a model for how the stress response protein Whi2 and the Ras/PKA (protein kinase A) signalling pathway are possibly linked and thereby regulate mitophagy. Show less

Cellular and subcellular morphology reflects the physiological state of a cell. To determine the physiological nature of sake yeast with superior fermentation properties, we quantitatively analyzed the morphology of sake yeast cells by using the CalMorph system. All the sake strains examined here exhibited common morphological traits that are typically observed in the well-characterized whiskey (whi) mutants that show accelerated G(1)/S transition. In agreement with this finding, the sake strain showed less efficient G(0)/G(1) arrest and elevated expression of the G(1) cyclin gene CLN3 throughout the fermentation period. Furthermore, deletion of CLN3 remarkably impaired the fermentation rate in both sake and laboratory strains. Disruption of the SWI6 gene, a transcriptional coactivator responsible for Cln3p-mediated G(1)/S transition, also resulted in a decreased fermentation rate, whereas whi mutants exhibited significant improvement in the fermentation rate, demonstrating positive roles of Cln3p and its downstream signalling pathway in facilitating ethanol fermentation. The combined results indicate that enhanced induction of CLN3 contributes to the high fermentation rate of sake yeast, which are natural whi mutants. Show less

Cells have evolved complex regulatory networks that reorganize gene expression patterns in response to changing environmental conditions. These changes often involve redundant mechanisms that affect various levels of gene expression. Here, we examine the consequences of enhanced mRNA degradation in the galactose utilization network of Saccharomyces cerevisiae. We observe that glucose-induced degradation of GAL1 transcripts provides a transient growth advantage to cells upon addition of glucose. We show that the advantage arises from relief of translational competition between GAL1 transcripts and those of cyclin CLN3, a translationally regulated initiator of cell division. This competition creates a translational bottleneck that balances the production of Gal1p and Cln3p and represents a posttranscriptional control mechanism that enhances the cell's ability to adapt to changes in carbon source. We present evidence that the spatial regulation of GAL1 and CLN3 transcripts is what allows growth to be maintained during fluctuations of glucose availability. Our results provide unique insights into how cells optimize energy use during growth in a dynamic environment. Show less

Mutations in the CLN3 gene lead to juvenile neuronal ceroid lipofuscinosis, a pediatric neurodegenerative disorder characterized by visual loss, epilepsy and psychomotor deterioration. Although most CLN3 patients carry the same 1-kb deletion in the CLN3 gene, their disease phenotype can be variable. The aims of this study were to (i) study the clinical phenotype in CLN3 patients with identical genotype, (ii) identify genes that are dysregulated in CLN3 disease regardless of the clinical course that could be useful as biomarkers, and (iii) find modifier genes that affect the progression rate of the disease. A total of 25 CLN3 patients homozygous for the 1-kb deletion were classified into groups with rapid, average or slow disease progression using an established clinical scoring system. Genome-wide expression profiling was performed in eight CLN3 patients with different disease progression and matched controls. The study showed high phenotype variability in CLN3 patients. Five genes were dysregulated in all CLN3 patients and present candidate biomarkers of the disease. Of those, dual specificity phosphatase 2 (DUSP2) was also validated in acutely CLN3-depleted cell models and in CbCln3(Δex7/8) cerebellar precursor cells. A total of 13 genes were upregulated in patients with rapid disease progression and downregulated in patients with slow disease progression; one gene showed dysregulation in the opposite way. Among these potential modifier genes, guanine nucleotide exchange factor 1 for small GTPases of the Ras family (RAPGEF1) and transcription factor Spi-B (SPIB) were validated in an acutely CLN3-depleted cell model. These findings indicate that differential perturbations of distinct signaling pathways might alter disease progression and provide insight into the molecular alterations underlying neuronal dysfunction in CLN3 disease and neurodegeneration in general. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL, NCL3, Batten disease) is usually caused by a 1.02-kb deletion in the CLN3 gene. Mutations in the CLN1 gene may be associated with a variant form of JNCL (vJNCL). We report the clinical course and molecular studies in 24 patients with JNCL collected from 1975 to 2010 with the aim of assessing the natural history of the disorder and phenotype/genotype correlations. Patients were classified into the groups of vJNCL with mutations in the CLN1 gene and/or granular osmiophilic deposit (GROD) inclusion bodies (n = 11) and classic JNCL (cJNCL) with mutations in the CLN3 gene and/or fingerprint (FP) profiles (n = 13). Psychomotor impairment included regression of acquired skills, cognitive decline, and clinical manifestations of the disease. We used Kaplan-Meier analyses to estimate the age of onset of psychomotor impairment. Patients with vJNCL showed learning delay at an earlier age (median 4 years, 95% confidence interval [CI] 3.1-4.8) than those in the cJNCL group (median 8 years, 95% CI 6.2-9.7) (P = 0.001) and regression of acquired skills at a younger age. Patients with vJNCL showed a more severe and progressive clinical course than those with cJNCL. There may be a Gypsy ancestry for V181L missense mutation in the CLN1 gene. The rate of disease progression may be useful to diagnose vJNCL or cJNCL, which should be confirmed by molecular studies in CLN1/CLN3 genes. Further studies of genotype/phenotype correlation will be helpful for understanding the pathogenesis of this disease. Show less

The human Batten disease gene CLN3 and yeast orthologue BTN1 encode proteins of unclear function. We show that the loss of BTN1 phenocopies that of BTN2, which encodes a retromer accessory protein involved in the retrieval of specific cargo from late endosomes (LEs) to the Golgi. However, Btn1 localizes to Golgi and regulates soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) function to control retrograde transport. Specifically, BTN1 overexpression and deletion have opposing effects on phosphorylation of the Sed5 target membrane SNARE, on Golgi SNARE assembly, and on Golgi integrity. Although Btn1 does not interact physically with SNAREs, it regulates Sed5 phosphorylation by modulating Yck3, a palmitoylated endosomal kinase. This may involve modification of the Yck3 lipid anchor, as substitution with a transmembrane domain suppresses the deletion of BTN1 and restores trafficking. Correspondingly, deletion of YCK3 mimics that of BTN1 or BTN2 with respect to LE-Golgi retrieval. Thus, Btn1 controls retrograde sorting by regulating SNARE phosphorylation and assembly, a process that may be adversely affected in Batten Disease patients. Show less

Batten disease (BD) is the most common form of a group of disorders called neuronal ceroid lipofuscinosis, which are caused by a CLN3 gene mutation. A variety of pathogenic lysosomal storage disorder mechanisms have been suggested such as oxidative stress, endoplasmic reticulum (ER) stress, and altered protein trafficking. Resveratrol, a stilbenoid found in red grape skin, is a potent antioxidant chemical. Recent studies have suggested that resveratrol may have a curative effect in many neurodegenerative diseases. Therefore, we investigated the activities of resveratrol at the levels of oxidative and ER stress and apoptosis factors using normal and BD lymphoblast cells. We report that the BD lymphoblast cells contained low-levels of superoxide dismutase-1 (SOD-1) due to the long-term stress of reactive oxygen species. However, when we treated the cells with resveratrol, SOD-1 increased to levels observed in normal cells. Furthermore, we investigated the expression of glucose-regulated protein 78 as an ER stress marker. BD cells underwent ER stress, but resveratrol treatment resolved the ER stress in a dose-dependent manner. We further demonstrated that the levels of apoptosis markers such as apoptosis induce factor, cytochrome c, and cleavage of poly (ADP)-ribose polymerase decreased following resveratrol treatment. Thus, we propose that resveratrol may have beneficial effects in patients with BD. Show less

To use the Unified Batten Disease Rating Scale (UBDRS) to measure the rate of decline in physical and functional capability domains in patients with juvenile neuronal ceroid lipofuscinosis (JNCL) or Batten disease, a neurodegenerative lysosomal storage disorder. We have evaluated the UBDRS in subjects with JNCL since 2002; during that time, the scale has been refined to improve reliability and validity. Now that therapies are being proposed to prevent, slow, or reverse the course of JNCL, the UBDRS will play an important role in quantitatively assessing clinical outcomes in research trials. We administered the UBDRS to 82 subjects with JNCL genetically confirmed by CLN3 mutational analysis. Forty-four subjects were seen for more than one annual visit. From these data, the rate of physical impairment over time was quantified using multivariate linear regression and repeated-measures analysis. The UBDRS Physical Impairment subscale shows worsening over time that proceeds at a quantifiable linear rate in the years following initial onset of clinical symptoms. This deterioration correlates with functional capability and is not influenced by CLN3 genotype. The UBDRS is a reliable and valid instrument that measures clinical progression in JNCL. Our data support the use of the UBDRS to quantify the rate of progression of physical impairment in subjects with JNCL in clinical trials. Show less

Juvenile neuronal ceroid lipofuscinosis (JNCL; CLN3 disease; Batten disease) is an autosomal recessive neurodegenerative disease of childhood that typically presents at school age with vision loss followed by progressive cognitive decline, motor dysfunction, seizures, and behavior problems. No therapy has been shown to slow the progression of disease in JNCL patients, and all current treatments are symptomatic. Flupirtine has been shown in vitro to reduce apoptosis in CLN3 lymphocytes. Based on that preclinical study, several children with JNCL were given flupirtine by their parents. The purpose of this study was to determine if there was evidence of attenuated disease progression in any JNCL symptom domain. We administered a survey to parents of JNCL children to qualitatively assess flupirtine efficacy. We used the Unified Batten Disease Rating Scale (UBDRS) to determine specific aspects of disease progression and investigated three age-related factors: loss of independent ambulation, loss of intelligible speech, and loss of ability to perform independent activities of daily living. The median scores for the UBDRS physical, behavior, and capability subscales were determined in flupirtine-exposed subjects and compared to age-, sex-, and genotype-matched subjects who had never taken flupirtine. Twenty-one percent of survey responders reported administering flupirtine to their JNCL child, and 56% of these families perceived beneficial changes that they attributed to flupirtine. However, our quantitative, prospectively obtained data did not show any change in JNCL disease progression that could be attributed to flupirtine. This study highlights the need for prospective experimental therapeutic research. Show less

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of fatal inherited neurodegenerative diseases in humans distinguished by a common clinical pathology, characterized by the accumulation of storage body material in cells and gross brain atrophy. In this study, metabolic changes in three NCL mouse models were examined looking for pathways correlated with neurodegeneration. Two mouse models; motor neuron degeneration (mnd) mouse and a variant model of late infantile NCL, termed the neuronal ceroid lipofuscinosis (nclf) mouse were investigated experimentally. Both models exhibit a characteristic accumulation of autofluorescent lipopigment in neuronal and non neuronal cells. The NMR profiles derived from extracts of the cortex and cerebellum from mnd and nclf mice were distinguished according to disease/wildtype status. In particular, a perturbation in glutamine and glutamate metabolism, and a decrease in γ-amino butyric acid (GABA) in the cerebellum and cortices of mnd (adolescent mice) and nclf mice relative to wildtype at all ages were detected. Our results were compared to the Cln3 mouse model of NCL. The metabolism of mnd mice resembled older (6 month) Cln3 mice, where the disease is relatively advanced, while the metabolism of nclf mice was more akin to younger (1-2 months) Cln3 mice, where the disease is in its early stages of progression. Overall, our results allowed the identification of metabolic traits common to all NCL subtypes for the three animal models. Show less