scholarly journals Interaction with Btn2p Is Required for Localization of Rsg1p: Btn2p-Mediated Changes in Arginine Uptake in Saccharomyces cerevisiae

2002 ◽  
Vol 1 (4) ◽  
pp. 606-612 ◽  
Author(s):  
Subrata Chattopadhyay ◽  
David A. Pearce
Keyword(s):  
Regulatory Protein ◽  
Coiled Coil ◽  
Batten Disease ◽  
Cell Periphery ◽  
Lysosomal Storage ◽  
Distinct Structure ◽  
Arginine Uptake ◽  
Lysosomal Membrane Protein ◽  
Elevated Rate ◽  
Arginine Permease

ABSTRACT Btn2p, a novel coiled-coil protein, is up-regulated in btn1Δ yeast strains, and this up-regulation is thought to contribute to maintaining a stable vacuolar pH in btn1Δ 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 Rsg1p, a down-regulator of the Can1p arginine and lysine permease. Rsg1p localizes to a distinct structure toward the cell periphery, and strains lacking Btn2p (btn2Δ strains) fail to correctly localize Rsg1p. btn2Δ strains, like rsg1Δ strains, are sensitive for growth in the presence of the arginine analog canavanine. Furthermore, btn2Δ strains, like rsg1Δ 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, Rsg1p. Our original identification of Btn2p was that it is up-regulated in the btn1Δ strain which serves as a model for the lysosomal storage disorder Batten disease. Btn1p is a vacuolar/lysosomal membrane protein, and btn1Δ suppresses both the canavanine sensitivity and the elevated rate of uptake of arginine displayed by btn2Δ rsg1Δ strains. We conclude that Btn2p interacts with Rsg1p and modulates arginine uptake. Up-regulation of BTN2 expression in btn1Δ strains may facilitate either a direct or indirect effect on intracellular arginine levels.

scholarly journals Extracellular Vesicles as Drug Carriers for Enzyme Replacement Therapy to Treat CLN2 Batten Disease: Optimization of Drug Administration Routes

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1273 ◽  
Author(s):  
Matthew J. Haney ◽  
Yuling Zhao ◽  
Yeon S. Jin ◽  
Elena V. Batrakova
Keyword(s):  
Mouse Model ◽  
Extracellular Vesicles ◽  
Broad Class ◽  
Batten Disease ◽  
Drug Carriers ◽  
Lysosomal Storage ◽  
Storage Material ◽  
Enzyme Replacement ◽  
Daunting Task ◽  
The Brain

CLN2 Batten disease (BD) is one of a broad class of lysosomal storage disorders that is characterized by the deficiency of lysosomal enzyme, TPP1, resulting in a build-up of toxic intracellular storage material in all organs and subsequent damage. A major challenge for BD therapeutics is delivery of enzymatically active TPP1 to the brain to attenuate progressive loss of neurological functions. To accomplish this daunting task, we propose the harnessing of naturally occurring nanoparticles, extracellular vesicles (EVs). Herein, we incorporated TPP1 into EVs released by immune cells, macrophages, and examined biodistribution and therapeutic efficacy of EV-TPP1 in BD mouse model, using various routes of administration. Administration through intrathecal and intranasal routes resulted in high TPP1 accumulation in the brain, decreased neurodegeneration and neuroinflammation, and reduced aggregation of lysosomal storage material in BD mouse model, CLN2 knock-out mice. Parenteral intravenous and intraperitoneal administrations led to TPP1 delivery to peripheral organs: liver, kidney, spleen, and lungs. A combination of intrathecal and intraperitoneal EV-TPP1 injections significantly prolonged lifespan in BD mice. Overall, the optimization of treatment strategies is crucial for successful applications of EVs-based therapeutics for BD.

scholarly journals Interaction among Btn1p, Btn2p, and Ist2p Reveals Potential Interplay among the Vacuole, Amino Acid Levels, and Ion Homeostasis in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (2) ◽  
pp. 281-288 ◽  
Author(s):  
Yoojin Kim ◽  
Subrata Chattopadhyay ◽  
Sarahjane Locke ◽  
David A. Pearce
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Membrane Protein ◽  
Optimal Transport ◽  
Ion Homeostasis ◽  
Coiled Coil ◽  
Batten Disease ◽  
Yeast Saccharomyces Cerevisiae ◽  
Arginine Transport ◽  
Amino Acid Levels

ABSTRACT Btn2p, a novel cytosolic coiled-coil protein in Saccharomyces cerevisiae, was previously shown to interact with and to be necessary for the correct localization of Rhb1p, a regulator of arginine uptake, and Yif1p, a Golgi protein. We now report the biochemical and physical interactions of Btn2p with Ist2p, a plasma membrane protein that is thought to have a function in salt tolerance. A deletion in Btn2p (btn2Δ strains) results in a failure to correctly localize Ist2p, and strains lacking Btn2p and Ist2p (btn2Δ ist2Δ strains) are unable to grow in the presence of 0.5 or 1.0 M NaCl. Btn2p was originally identified as being up-regulated in a btn1Δ strain, which lacks the vacuolar-lysosomal membrane protein, Btn1p, and serves as a model for Batten disease. This up-regulation of Btn2p was shown to contribute to the maintenance of a stable vacuolar pH in the btn1Δ strain. Btn1p was subsequently shown to be required for the optimal transport of arginine into the vacuole. Interestingly, btn1Δ ist2Δ strains are also unable to grow in the presence of 0.5 or 1.0 M NaCl, and ist2Δ suppresses the vacuolar arginine transport defect in btn1Δ strains. Although further investigation is required, we speculate that altered vacuolar arginine transport in btn1Δ strains represents a mechanism for maintaining or balancing cellular ion homeostasis. Btn2p interacts with at least three proteins that are seemingly involved in different biological functions in different subcellular locations. Due to these multiple interactions, we conclude that Btn2p may play a regulatory role across the cell in response to alterations in the intracellular environment that may be caused by changes in amino acid levels or pH, a disruption in protein trafficking, or imbalances in ion homeostasis resulting from either genetic or environmental manipulation.

scholarly journals Neonatal brain-directed gene therapy rescues a mouse model of neurodegenerative CLN6 Batten disease

2019 ◽  
Vol 28 (23) ◽  
pp. 3867-3879 ◽  
Author(s):  
Sophia-Martha kleine Holthaus ◽  
Saul Herranz-Martin ◽  
Giulia Massaro ◽  
Mikel Aristorena ◽  
Justin Hoke ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mouse Model ◽  
Motor Coordination ◽  
Transmembrane Protein ◽  
Batten Disease ◽  
Premature Death ◽  
Neuronal Ceroid Lipofuscinoses ◽  
Therapeutic Effects ◽  
Lysosomal Storage ◽  
Enzyme Replacement

Abstract The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.

scholarly journals Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2

2020 ◽  
Vol 12 (572) ◽  
pp. eabb5413
Author(s):  
Dolan Sondhi ◽  
Stephen M. Kaminsky ◽  
Neil R. Hackett ◽  
Odelya E. Pagovich ◽  
Jonathan B. Rosenberg ◽  
...  
Keyword(s):  
Natural History ◽  
Gray Matter Volume ◽  
Batten Disease ◽  
Fold Increase ◽  
Lysosomal Storage ◽  
Gene Encoding ◽  
Adeno Associated Virus ◽  
Progression Of Disease ◽  
Brain Parenchymal ◽  
Burr Holes

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

scholarly journals Primary Mesenchymal Cells Isolated from SPARC-null Mice Exhibit Altered Morphology and Rates of Proliferation

1999 ◽  
Vol 10 (5) ◽  
pp. 1569-1579 ◽  
Author(s):  
Amy D. Bradshaw ◽  
Aleksandar Francki ◽  
Kouros Motamed ◽  
Chin Howe ◽  
E. Helene Sage
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Mesangial Cells ◽  
Regulatory Protein ◽  
Focal Adhesions ◽  
Muscle Cells ◽  
Matricellular Protein ◽  
Cell Periphery ◽  
Wild Type

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.

scholarly journals A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

2012 ◽  
Vol 23 (18) ◽  
pp. 3532-3541 ◽  
Author(s):  
Changfu Yao ◽  
Uttama Rath ◽  
Helder Maiato ◽  
David Sharp ◽  
Jack Girton ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Coiled Coil ◽  
Complete Understanding ◽  
Spindle Matrix ◽  
Lamin B ◽  
Amino Terminal ◽  
Distinct Structure ◽  
Spindle Apparatus ◽  
Imaging Approach ◽  
Amino Terminal Domain

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this “internal” matrix is a distinct structure from the microtubule spindle and from a lamin B–containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as “struts” stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.

scholarly journals Quantifying unusual neurological movement phenotypes in collective movement phenotypes

2021 ◽  
Author(s):  
Kehinde Owoeye ◽  
Mirco Musolesi ◽  
Stephen Hailes
Keyword(s):  
Full Range ◽  
Sampling Rate ◽  
Batten Disease ◽  
Genetic Mutation ◽  
Therapeutic Interventions ◽  
Control Group ◽  
Lysosomal Storage ◽  
Positional Accuracy ◽  
Building Models ◽  
Preclinical Trials

Building models of anomalous behaviour in animals is important for monitoring animal welfare as well as assessing the efficacy of therapeutic interventions in preclinical trials. In this paper, we describe methods that allow for the automatic discrimination of sheep with a genetic mutation that causes Batten disease from an age-matched control group, using GPS movement traces as input. Batten disease is an autosomal recessive lysosomal storage abnormality with symptoms that are likely to affect the way that those with it move and socialise, including loss of vision and dementia. The sheep in this study displayed a full range of symptoms and during the experiment, the sheep were mixed with a large group of younger animals. We used data obtained from bespoke raw data GPS sensors carried by all animals, with a sampling rate of 1 sample/second and a positional accuracy of around 30cm. The distance covered in each ten minute period and, more specifically, outliers in each period, were used as the basis for estimating the abnormal behaviour. Our results show that, despite the variability in the sample, the bulk of the outliers during the period of observation across six days came from the sheep with Batten disease. Our results point towards the potential of using relatively simple movement metrics in identifying the onset of a phenotype in symptomatically similar conditions.

scholarly journals Aggregation chimeras provide evidence of in vivo intercellular correction in ovine CLN6 neuronal ceroid lipofuscinosis (Batten disease)

2021 ◽  
Author(s):  
Lucy A. Barry ◽  
Graham W. Kay ◽  
Nadia L. Mitchell ◽  
Samantha J. Murray ◽  
Nigel P. Jay ◽  
...  
Keyword(s):  
Vision Loss ◽  
Inflammatory Responses ◽  
Neuronal Ceroid Lipofuscinosis ◽  
Batten Disease ◽  
Brain Regions ◽  
Glial Activation ◽  
Neuronal Ceroid Lipofuscinoses ◽  
Lysosomal Storage ◽  
Storage Body

AbstractThe neuronal ceroid lipofuscinoses (NCLs; Batten disease) are fatal, mainly childhood, inherited neurodegenerative lysosomal storage diseases. Sheep affected with a CLN6 form display progressive regionally defined glial activation and subsequent neurodegeneration, indicating that neuroinflammation may be causative of pathogenesis. In this study, aggregation chimeras were generated from homozygous unaffected normal and CLN6 affected sheep embryos, resulting in seven chimeric animals with varied proportions of normal to affected cells. These sheep were classified as affected-like, recovering-like or normal-like, based on their cell-genotype ratios and their clinical and neuropathological profiles.Neuropathological examination of the affected-like animals revealed intense glial activation, prominent storage body accumulation and severe neurodegeneration within all cortical brain regions, along with vision loss and decreasing intracranial volumes and cortical thicknesses consistent with ovine CLN6 disease. In contrast, intercellular communication affecting pathology was evident at both the gross and histological level in the normal-like and recovering-like chimeras, resulting in a lack of glial activation and rare storage body accumulation in only a few cells. Initial intracranial volumes of the recovering-like chimeras were below normal but progressively recovered to about normal by two years of age. All had normal cortical thicknesses, and none went blind. Extended neurogenesis was evident in the brains of all the chimeras.This study indicates that although CLN6 is a membrane bound protein, the consequent defect is not cell intrinsic. The lack of glial activation and inflammatory responses in the normal-like and recovering-like chimeras indicate that newly generated cells are borne into a microenvironment conducive to maturation and survival.

β1PIX, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles

2001 ◽  
Vol 114 (23) ◽  
pp. 4239-4251 ◽  
Author(s):  
Cheng-Gee Koh ◽  
Ed Manser ◽  
Zhou-Shen Zhao ◽  
Chee-Peng Ng ◽  
Louis Lim
Keyword(s):  
Splice Variants ◽  
Coiled Coil ◽  
Cell Periphery ◽  
Nucleotide Exchange ◽  
Dimerization Domain ◽  
Intracellular Location ◽  
Exchange Factor ◽  
C Terminus ◽  
Nucleotide Exchange Factor ◽  
Coiled Coil Region

PIX is a Rho-family guanine nucleotide exchange factor that binds PAK. We previously described two isoforms of PIX that differ in their N termini. Here, we report the identification of a new splice variant of βPIX, designated β2PIX, that is the dominant species in brain and that lacks the region of ∼120 residues with predicted coiled-coil structure at the C terminus of β1PIX. Instead, β2PIX contains a serine-rich C terminus. To determine whether these splice variants differ in their cellular function, we studied the effect of expressing these proteins in HeLa cells. We found that the coiled-coil region plays a key role in the localization of β1PIX to the cell periphery and is also responsible for PIX dimerization. Overexpression of β1, but not β2PIX, drives formation of membrane ruffles and microvillus-like structures (via activation of Rac1 and Cdc42, respectively), indicating that its function requires localized activation of these GTPases. Thus, β1PIX, like other RhoGEFs, exerts specific morphological functions that are dependent on its intracellular location and are mediated by its C-terminal dimerization domain.

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