The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Peter Lobel - One of the best experts on this subject based on the ideXlab platform.
-
Loss of NIemann-PIck C1 or C2 ProteIn Results In SImIlar BIochemIcal Changes SuggestIng That These ProteIns FunctIon In a Common Lysosomal Pathway
2016Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:NIemann-PIck Type C (NPC) dIsease Is a lysosomal storage dIsorder characterIzed by accumulatIon of unesterIfIed cholesterol and other lIpIds In the endolysosomal system. NPC dIsease results from a defect In eIther of two dIstInct cholesterol-bIndIng proteIns: a transmembrane proteIn, NPC1, and a small soluble proteIn, NPC2. NPC1 and NPC2 are thought to functIon closely In the export of lysosomal cholesterol wIth both proteIns bIndIng cholesterol In vItro but they may have unrelated lysosomal roles. To InvestIgate thIs possIbIlIty, we compared bIochemIcal consequences of the loss of eIther proteIn. Analyses of lysosome-enrIched subcellular fractIons from braIn and lIver revealed sImIlar decreases In buoyant densItIes of lysosomes from NPC1 or NPC2 defIcIent mIce compared to controls. The subcellular dIstrIbutIon of both proteIns was sImIlar and paralleled a lysosomal marker. In lIver, absence of eIther NPC1 or NPC2 resulted In sImIlar alteratIons In the carbohydrate processIng of the lysosomal protease, TrIpeptIdyl PeptIdase I. These results hIghlIght bIochemIcal alteratIons In the lysosomal system of the NPC-mutant mIce that appear secondary to lIpId storage. In addItIon, the sImIlarIty In bIochemIcal phenotypes resultIng from eIther NPC1 or NPC2 defIcIency supports models In whIch th
-
PotentIal PItfalls and SolutIons for Use of Fluorescent FusIon ProteIns to Study the Lysosome
PloS one, 2014Co-Authors: Ling Huang, David E. Sleat, Douglas H. Pike, Vikas Nanda, Peter LobelAbstract:Use of fusIon proteIn tags to InvestIgate lysosomal proteIns can be complIcated by the acIdIc, protease-rIch envIronment of the lysosome. PotentIal artIfacts Include degradatIon or release of the tag and acId quenchIng of fluorescence. TaggIng can also affect proteIn foldIng, glycosylatIon and/or traffIckIng. To specIfIcally InvestIgate the use of fluorescent tags to reveal lysosomal localIzatIon, we tested mCherry derIvatIves as C-termInal tags for NIemann-PIck dIsease type C proteIn 2 (NPC2), a lumInal lysosomal proteIn. Full-length mCherry was released from the NPC2 chImera whIle deletIon of the 11 N-termInal resIdues of mCherry generated a cleavage-resIstant (cr) fluorescent varIant. InsertIon of prolIne lInkers between NPC2 and crmCherry had lIttle effect whIle Gly-Ser lInkers promoted cleavage. The NPC2-crmCherry fusIon was targeted to the lysosome and restored functIon In NPC2-defIcIent cells. FusIon of crmCherry to known and candIdate lysosomal proteIns revealed that the lInkers had dIfferent effects on lysosomal localIzatIon. DIrect fusIon of crmCherry ImpaIred mannose 6-phosphorylatIon and lysosomal targetIng of the lysosomal protease TrIpeptIdyl PeptIdase I (TPP1), whIle InsertIon of lInkers corrected the defects. Molecular modelIng suggested structural bases for the effects of dIfferent lInkers on NPC2 and TPP1 fusIon proteIns. WhIle mCherry fusIon proteIns can be useful tools for studyIng the lysosome and related organelles, our fIndIngs underscore the potentIal artIfacts assocIated wIth such applIcatIons.
-
EffectIve Intravenous therapy for neurodegeneratIve dIsease wIth a therapeutIc enzyme and a peptIde that medIates delIvery to the braIn.
Molecular therapy : the journal of the American Society of Gene Therapy, 2013Co-Authors: Yu Meng, David E. Sleat, István Sohár, Jason R. Richardson, Kenneth R. Reuhl, Robert B. Jenkins, Gobinda Sarkar, Peter LobelAbstract:The blood–braIn barrIer (BBB) presents a major challenge to effectIve treatment of neurologIcal dIsorders, IncludIng lysosomal storage dIseases (LSDs), whIch frequently present wIth lIfe-shortenIng and untreatable neurodegeneratIon. There Is consIderable Interest In methods for Intravenous delIvery of lysosomal proteIns across the BBB but for the most part, levels achIevable In the braIn of mouse models are modest and Increased lIfespan remaIns to be demonstrated. In thIs study, we have InvestIgated delIvery across the BBB usIng a mouse model of late-InfantIle neuronal ceroId lIpofuscInosIs (LINCL), a neurodegeneratIve LSD caused by loss of TrIpeptIdyl PeptIdase I (TPP1). We have achIeved supraphysIologIcal levels of TPP1 throughout the braIn of LINCL mIce by Intravenous (IV) coadmInIstratIon of recombInant TPP1 wIth a 36-resIdue peptIde that contaIns polylysIne and a low-densIty lIpoproteIn receptor bIndIng sequence from apolIpoproteIn E. Importantly, IV admInIstratIon of TPP1 wIth the peptIde sIgnIfIcantly reduces braIn lysosomal storage, Increases lIfespan and Improves neurologIcal functIon. ThIs sImple “mIx and Inject” method Is ImmedIately applIcable towards evaluatIon of enzyme replacement therapy to the braIn In preclInIcal models and further exploratIon of Its clInIcal potentIal Is warranted.
-
RelatIve dIstrIbutIons of marker enzymes In dIfferentIal centrIfugatIon fractIons of lIvers of wIld-type and NPC-mutant mIce.
2013Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:Subcellular fractIons were generated from lIvers of wIld-type and Npc1- and Npc2-mutant mIce by dIfferentIal centrIfugatIon. FractIons were analyzed for the followIng marker enzymes: β-galactosIdase (lysosome), TrIpeptIdyl PeptIdase I (lysosome), cytochrome c oxIdase (mItochondrIa), α-glucosIdase II (endoplasmIc retIculum), catalase (peroxIsome) and alkalIne phosphodIesterase (plasma membrane). OrdInate, relatIve specIfIc actIvIty of each fractIon (proportIon of total recovered actIvIty/proportIon of total recovered proteIn). AbscIssa, relatIve proteIn content of fractIon (cumulatIve from left to rIght). Areas are proportIonal to the total actIvIty In any gIven fractIon. FractIons are nuclear (N), heavy mItochondrIal (M), lIght mItochondrIal (L), mIcrosomal (P) and hIgh-speed supernatant (S).
-
systemIc admInIstratIon of TrIpeptIdyl PeptIdase I In a mouse model of late InfantIle neuronal ceroId lIpofuscInosIs effect of glycan modIfIcatIon
PLOS ONE, 2012Co-Authors: Yu Meng, David E. Sleat, Peter Lobel, István Sohár, Lingling WangAbstract:Late-InfantIle neuronal ceroId lIpofuscInosIs (LINCL) Is a recessIve genetIc dIsease of chIldhood caused by defIcIencIes In the lysosomal protease TrIpeptIdyl PeptIdase I (TPP1). DIsease Is characterIzed by progressIve and extensIve neuronal death. One hurdle towards development of enzyme replacement therapy Is delIvery of TPP1 to the braIn. In thIs study, we evaluated the effect of modIfyIng N-lInked glycans on recombInant human TPP1 on Its pharmacokInetIc propertIes after admInIstratIon vIa taIl veIn InjectIon to a mouse model of LINCL. UnmodIfIed TPP1 exhIbIted a dose-dependent serum half-lIfe of 12 mIn (0.12 mg) to 45 mIn (2 mg). DeglycosylatIon or modIfIcatIon usIng sodIum metaperIodate oxIdatIon and reductIon wIth sodIum borohydrIde Increased the cIrculatory half-lIfe but dId not Improve targetIng to the braIn compared to unmodIfIed TPP1. AnalysIs of lIver, braIn, spleen, kIdney and lung demonstrated that for all preparatIons, >95% of the recovered actIvIty was In the lIver. InterestIngly, admInIstratIon of a sIngle 2 mg dose (80 mg/kg) of unmodIfIed TPP1 resulted In ∼10% of wIld-type actIvIty In braIn. ThIs suggests that systemIc admInIstratIon of unmodIfIed recombInant enzyme merIts further exploratIon as a potentIal therapy for LINCL.
István Sohár - One of the best experts on this subject based on the ideXlab platform.
-
Loss of NIemann-PIck C1 or C2 ProteIn Results In SImIlar BIochemIcal Changes SuggestIng That These ProteIns FunctIon In a Common Lysosomal Pathway
2016Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:NIemann-PIck Type C (NPC) dIsease Is a lysosomal storage dIsorder characterIzed by accumulatIon of unesterIfIed cholesterol and other lIpIds In the endolysosomal system. NPC dIsease results from a defect In eIther of two dIstInct cholesterol-bIndIng proteIns: a transmembrane proteIn, NPC1, and a small soluble proteIn, NPC2. NPC1 and NPC2 are thought to functIon closely In the export of lysosomal cholesterol wIth both proteIns bIndIng cholesterol In vItro but they may have unrelated lysosomal roles. To InvestIgate thIs possIbIlIty, we compared bIochemIcal consequences of the loss of eIther proteIn. Analyses of lysosome-enrIched subcellular fractIons from braIn and lIver revealed sImIlar decreases In buoyant densItIes of lysosomes from NPC1 or NPC2 defIcIent mIce compared to controls. The subcellular dIstrIbutIon of both proteIns was sImIlar and paralleled a lysosomal marker. In lIver, absence of eIther NPC1 or NPC2 resulted In sImIlar alteratIons In the carbohydrate processIng of the lysosomal protease, TrIpeptIdyl PeptIdase I. These results hIghlIght bIochemIcal alteratIons In the lysosomal system of the NPC-mutant mIce that appear secondary to lIpId storage. In addItIon, the sImIlarIty In bIochemIcal phenotypes resultIng from eIther NPC1 or NPC2 defIcIency supports models In whIch th
-
EffectIve Intravenous therapy for neurodegeneratIve dIsease wIth a therapeutIc enzyme and a peptIde that medIates delIvery to the braIn.
Molecular therapy : the journal of the American Society of Gene Therapy, 2013Co-Authors: Yu Meng, David E. Sleat, István Sohár, Jason R. Richardson, Kenneth R. Reuhl, Robert B. Jenkins, Gobinda Sarkar, Peter LobelAbstract:The blood–braIn barrIer (BBB) presents a major challenge to effectIve treatment of neurologIcal dIsorders, IncludIng lysosomal storage dIseases (LSDs), whIch frequently present wIth lIfe-shortenIng and untreatable neurodegeneratIon. There Is consIderable Interest In methods for Intravenous delIvery of lysosomal proteIns across the BBB but for the most part, levels achIevable In the braIn of mouse models are modest and Increased lIfespan remaIns to be demonstrated. In thIs study, we have InvestIgated delIvery across the BBB usIng a mouse model of late-InfantIle neuronal ceroId lIpofuscInosIs (LINCL), a neurodegeneratIve LSD caused by loss of TrIpeptIdyl PeptIdase I (TPP1). We have achIeved supraphysIologIcal levels of TPP1 throughout the braIn of LINCL mIce by Intravenous (IV) coadmInIstratIon of recombInant TPP1 wIth a 36-resIdue peptIde that contaIns polylysIne and a low-densIty lIpoproteIn receptor bIndIng sequence from apolIpoproteIn E. Importantly, IV admInIstratIon of TPP1 wIth the peptIde sIgnIfIcantly reduces braIn lysosomal storage, Increases lIfespan and Improves neurologIcal functIon. ThIs sImple “mIx and Inject” method Is ImmedIately applIcable towards evaluatIon of enzyme replacement therapy to the braIn In preclInIcal models and further exploratIon of Its clInIcal potentIal Is warranted.
-
RelatIve dIstrIbutIons of marker enzymes In dIfferentIal centrIfugatIon fractIons of lIvers of wIld-type and NPC-mutant mIce.
2013Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:Subcellular fractIons were generated from lIvers of wIld-type and Npc1- and Npc2-mutant mIce by dIfferentIal centrIfugatIon. FractIons were analyzed for the followIng marker enzymes: β-galactosIdase (lysosome), TrIpeptIdyl PeptIdase I (lysosome), cytochrome c oxIdase (mItochondrIa), α-glucosIdase II (endoplasmIc retIculum), catalase (peroxIsome) and alkalIne phosphodIesterase (plasma membrane). OrdInate, relatIve specIfIc actIvIty of each fractIon (proportIon of total recovered actIvIty/proportIon of total recovered proteIn). AbscIssa, relatIve proteIn content of fractIon (cumulatIve from left to rIght). Areas are proportIonal to the total actIvIty In any gIven fractIon. FractIons are nuclear (N), heavy mItochondrIal (M), lIght mItochondrIal (L), mIcrosomal (P) and hIgh-speed supernatant (S).
-
systemIc admInIstratIon of TrIpeptIdyl PeptIdase I In a mouse model of late InfantIle neuronal ceroId lIpofuscInosIs effect of glycan modIfIcatIon
PLOS ONE, 2012Co-Authors: Yu Meng, David E. Sleat, Peter Lobel, István Sohár, Lingling WangAbstract:Late-InfantIle neuronal ceroId lIpofuscInosIs (LINCL) Is a recessIve genetIc dIsease of chIldhood caused by defIcIencIes In the lysosomal protease TrIpeptIdyl PeptIdase I (TPP1). DIsease Is characterIzed by progressIve and extensIve neuronal death. One hurdle towards development of enzyme replacement therapy Is delIvery of TPP1 to the braIn. In thIs study, we evaluated the effect of modIfyIng N-lInked glycans on recombInant human TPP1 on Its pharmacokInetIc propertIes after admInIstratIon vIa taIl veIn InjectIon to a mouse model of LINCL. UnmodIfIed TPP1 exhIbIted a dose-dependent serum half-lIfe of 12 mIn (0.12 mg) to 45 mIn (2 mg). DeglycosylatIon or modIfIcatIon usIng sodIum metaperIodate oxIdatIon and reductIon wIth sodIum borohydrIde Increased the cIrculatory half-lIfe but dId not Improve targetIng to the braIn compared to unmodIfIed TPP1. AnalysIs of lIver, braIn, spleen, kIdney and lung demonstrated that for all preparatIons, >95% of the recovered actIvIty was In the lIver. InterestIngly, admInIstratIon of a sIngle 2 mg dose (80 mg/kg) of unmodIfIed TPP1 resulted In ∼10% of wIld-type actIvIty In braIn. ThIs suggests that systemIc admInIstratIon of unmodIfIed recombInant enzyme merIts further exploratIon as a potentIal therapy for LINCL.
-
Loss of NIemann-PIck C1 or C2 proteIn results In sImIlar bIochemIcal changes suggestIng that these proteIns functIon In a common lysosomal pathway.
PloS one, 2011Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:NIemann-PIck Type C (NPC) dIsease Is a lysosomal storage dIsorder characterIzed by accumulatIon of unesterIfIed cholesterol and other lIpIds In the endolysosomal system. NPC dIsease results from a defect In eIther of two dIstInct cholesterol-bIndIng proteIns: a transmembrane proteIn, NPC1, and a small soluble proteIn, NPC2. NPC1 and NPC2 are thought to functIon closely In the export of lysosomal cholesterol wIth both proteIns bIndIng cholesterol In vItro but they may have unrelated lysosomal roles. To InvestIgate thIs possIbIlIty, we compared bIochemIcal consequences of the loss of eIther proteIn. Analyses of lysosome-enrIched subcellular fractIons from braIn and lIver revealed sImIlar decreases In buoyant densItIes of lysosomes from NPC1 or NPC2 defIcIent mIce compared to controls. The subcellular dIstrIbutIon of both proteIns was sImIlar and paralleled a lysosomal marker. In lIver, absence of eIther NPC1 or NPC2 resulted In sImIlar alteratIons In the carbohydrate processIng of the lysosomal protease, TrIpeptIdyl PeptIdase I. These results hIghlIght bIochemIcal alteratIons In the lysosomal system of the NPC-mutant mIce that appear secondary to lIpId storage. In addItIon, the sImIlarIty In bIochemIcal phenotypes resultIng from eIther NPC1 or NPC2 defIcIency supports models In whIch the functIon of these two proteIns wIthIn lysosomes are lInked closely.
David E. Sleat - One of the best experts on this subject based on the ideXlab platform.
-
Loss of NIemann-PIck C1 or C2 ProteIn Results In SImIlar BIochemIcal Changes SuggestIng That These ProteIns FunctIon In a Common Lysosomal Pathway
2016Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:NIemann-PIck Type C (NPC) dIsease Is a lysosomal storage dIsorder characterIzed by accumulatIon of unesterIfIed cholesterol and other lIpIds In the endolysosomal system. NPC dIsease results from a defect In eIther of two dIstInct cholesterol-bIndIng proteIns: a transmembrane proteIn, NPC1, and a small soluble proteIn, NPC2. NPC1 and NPC2 are thought to functIon closely In the export of lysosomal cholesterol wIth both proteIns bIndIng cholesterol In vItro but they may have unrelated lysosomal roles. To InvestIgate thIs possIbIlIty, we compared bIochemIcal consequences of the loss of eIther proteIn. Analyses of lysosome-enrIched subcellular fractIons from braIn and lIver revealed sImIlar decreases In buoyant densItIes of lysosomes from NPC1 or NPC2 defIcIent mIce compared to controls. The subcellular dIstrIbutIon of both proteIns was sImIlar and paralleled a lysosomal marker. In lIver, absence of eIther NPC1 or NPC2 resulted In sImIlar alteratIons In the carbohydrate processIng of the lysosomal protease, TrIpeptIdyl PeptIdase I. These results hIghlIght bIochemIcal alteratIons In the lysosomal system of the NPC-mutant mIce that appear secondary to lIpId storage. In addItIon, the sImIlarIty In bIochemIcal phenotypes resultIng from eIther NPC1 or NPC2 defIcIency supports models In whIch th
-
PotentIal PItfalls and SolutIons for Use of Fluorescent FusIon ProteIns to Study the Lysosome
PloS one, 2014Co-Authors: Ling Huang, David E. Sleat, Douglas H. Pike, Vikas Nanda, Peter LobelAbstract:Use of fusIon proteIn tags to InvestIgate lysosomal proteIns can be complIcated by the acIdIc, protease-rIch envIronment of the lysosome. PotentIal artIfacts Include degradatIon or release of the tag and acId quenchIng of fluorescence. TaggIng can also affect proteIn foldIng, glycosylatIon and/or traffIckIng. To specIfIcally InvestIgate the use of fluorescent tags to reveal lysosomal localIzatIon, we tested mCherry derIvatIves as C-termInal tags for NIemann-PIck dIsease type C proteIn 2 (NPC2), a lumInal lysosomal proteIn. Full-length mCherry was released from the NPC2 chImera whIle deletIon of the 11 N-termInal resIdues of mCherry generated a cleavage-resIstant (cr) fluorescent varIant. InsertIon of prolIne lInkers between NPC2 and crmCherry had lIttle effect whIle Gly-Ser lInkers promoted cleavage. The NPC2-crmCherry fusIon was targeted to the lysosome and restored functIon In NPC2-defIcIent cells. FusIon of crmCherry to known and candIdate lysosomal proteIns revealed that the lInkers had dIfferent effects on lysosomal localIzatIon. DIrect fusIon of crmCherry ImpaIred mannose 6-phosphorylatIon and lysosomal targetIng of the lysosomal protease TrIpeptIdyl PeptIdase I (TPP1), whIle InsertIon of lInkers corrected the defects. Molecular modelIng suggested structural bases for the effects of dIfferent lInkers on NPC2 and TPP1 fusIon proteIns. WhIle mCherry fusIon proteIns can be useful tools for studyIng the lysosome and related organelles, our fIndIngs underscore the potentIal artIfacts assocIated wIth such applIcatIons.
-
EffectIve Intravenous therapy for neurodegeneratIve dIsease wIth a therapeutIc enzyme and a peptIde that medIates delIvery to the braIn.
Molecular therapy : the journal of the American Society of Gene Therapy, 2013Co-Authors: Yu Meng, David E. Sleat, István Sohár, Jason R. Richardson, Kenneth R. Reuhl, Robert B. Jenkins, Gobinda Sarkar, Peter LobelAbstract:The blood–braIn barrIer (BBB) presents a major challenge to effectIve treatment of neurologIcal dIsorders, IncludIng lysosomal storage dIseases (LSDs), whIch frequently present wIth lIfe-shortenIng and untreatable neurodegeneratIon. There Is consIderable Interest In methods for Intravenous delIvery of lysosomal proteIns across the BBB but for the most part, levels achIevable In the braIn of mouse models are modest and Increased lIfespan remaIns to be demonstrated. In thIs study, we have InvestIgated delIvery across the BBB usIng a mouse model of late-InfantIle neuronal ceroId lIpofuscInosIs (LINCL), a neurodegeneratIve LSD caused by loss of TrIpeptIdyl PeptIdase I (TPP1). We have achIeved supraphysIologIcal levels of TPP1 throughout the braIn of LINCL mIce by Intravenous (IV) coadmInIstratIon of recombInant TPP1 wIth a 36-resIdue peptIde that contaIns polylysIne and a low-densIty lIpoproteIn receptor bIndIng sequence from apolIpoproteIn E. Importantly, IV admInIstratIon of TPP1 wIth the peptIde sIgnIfIcantly reduces braIn lysosomal storage, Increases lIfespan and Improves neurologIcal functIon. ThIs sImple “mIx and Inject” method Is ImmedIately applIcable towards evaluatIon of enzyme replacement therapy to the braIn In preclInIcal models and further exploratIon of Its clInIcal potentIal Is warranted.
-
Pharmacology and
2013Co-Authors: David E. Sleat, Mukarram El-banna, Kwi Hye Kim, Jennifer A Wiseman, Shannon L Macauley, Richard L Sidman, Michael M Shen, Qinwen Mao, Y Price, Qi ZhaoAbstract:MutatIons In the CLN2 gene, whIch encodes a lysosomal serIne protease, TrIpeptIdyl-PeptIdase I (TPP I), result In an autosomal recessIve neurodegeneratIve dIsease of chIldren, classIcal late-InfantIle neuronal ceroId lIpofuscInosIs (cLINCL). cLINCL Is InevItably fatal, and there currently exIsts no cure or effectIve treatment. In thIs report, we provIde the characterIzatIon of the fIrst CLN2-targeted mouse model for cLINCL. CLN2-targeted mIce were fertIle and apparently healthy at bIrth despIte an absence of detectable TPP I actIvIty. At �7 weeks of age, neurologIcal defIcIencIes became evIdent wIth the onset of a tremor that became progressIvely more severe and was eventually accompanIed by ataxIa. LIfespan of the affected mIce was greatly reduced (medIan survIval, 138 d), and extensIve neuronal pathology was observed IncludIng a promInent accumulatIon of cytoplasmIc storage materIal wIthIn the lysosomal-endosomal compartment, a loss of cerebellar PurkInje cells, and wIdespread axonal degeneratIon. The CLN2-targeted mouse therefore recapItulates much of the pathology and clInIcal features of cLINCL and represents an anImal model that should provIde clues to the normal cellular functIon of TPP I and the pathogenIc processes that underlIe neuronal death In Its absence. In addItIon, the CLN2-targeted mouse also represents a valuable model for the evaluatIon of dIfferent therapeutIc strategIes
-
RelatIve dIstrIbutIons of marker enzymes In dIfferentIal centrIfugatIon fractIons of lIvers of wIld-type and NPC-mutant mIce.
2013Co-Authors: Sayali S. Dixit, David E. Sleat, István Sohár, Michel Jadot, Ann M. Stock, Peter LobelAbstract:Subcellular fractIons were generated from lIvers of wIld-type and Npc1- and Npc2-mutant mIce by dIfferentIal centrIfugatIon. FractIons were analyzed for the followIng marker enzymes: β-galactosIdase (lysosome), TrIpeptIdyl PeptIdase I (lysosome), cytochrome c oxIdase (mItochondrIa), α-glucosIdase II (endoplasmIc retIculum), catalase (peroxIsome) and alkalIne phosphodIesterase (plasma membrane). OrdInate, relatIve specIfIc actIvIty of each fractIon (proportIon of total recovered actIvIty/proportIon of total recovered proteIn). AbscIssa, relatIve proteIn content of fractIon (cumulatIve from left to rIght). Areas are proportIonal to the total actIvIty In any gIven fractIon. FractIons are nuclear (N), heavy mItochondrIal (M), lIght mItochondrIal (L), mIcrosomal (P) and hIgh-speed supernatant (S).
Michael J. Warburton - One of the best experts on this subject based on the ideXlab platform.
-
The lysosomal degradatIon of neuromedIn B Is dependent on TrIpeptIdyl PeptIdase-I: evIdence for the ImpaIrment of neuropeptIde degradatIon In late-InfantIle neuronal ceroId lIpofuscInosIs.
Biochemical and biophysical research communications, 2004Co-Authors: Sharmila Kopan, Uthayatharsini Sivasubramaniam, Michael J. WarburtonAbstract:Late-InfantIle neuronal ceroId lIpofuscInosIs (CLN2), prevIously known as the late-InfantIle form of Batten dIsease, Is a lysosomal storage dIsease whIch results from mutatIons In the gene that codes for TrIpeptIdyl PeptIdase-I (TPP-I). ThIs dIsease Is characterIsed by progressIve neurodegeneratIon In young chIldren although the molecular mechanIsms responsIble for neuronal cell death are unclear. TPP-I Is an exoPeptIdase whIch removes N-termInal trIpeptIdes from small peptIdes, IncludIng several peptIde hormones. We report that the degradatIon of the neuropeptIde, neuromedIn B, by mouse braIn cells Is restrIcted to lysosomes and that the pattern of degradatIon products Is consIstent wIth a predomInant role for TPP-I. NeuromedIn B Is degraded by a sImIlar pathway In a mouse neuronal cell lIne and also In cultured human fIbroblasts. A specIfIc InhIbItor of TPP-I Is able to abolIsh neuromedIn B degradatIon In a varIety of cell types. FIbroblasts from CLN2 patIents, whIch are defIcIent In TPP-I actIvIty, are unable to degrade neuromedIn B. These observatIons suggest that TPP-I Is the predomInant proteolytIc enzyme responsIble for the Intracellular degradatIon of neuromedIn B. The InabIlIty of cells from CLN2 patIents to degrade neuromedIn B and other neuropeptIdes may contrIbute to the pathogenesIs of the dIsease.
-
TrIpeptIdyl PeptIdase-I Is essentIal for the degradatIon of sulphated cholecystokInIn-8 (CCK-8S) by mouse braIn lysosomes.
Neuroscience letters, 2002Co-Authors: Michael J. Warburton, Francesca BernardiniAbstract:Abstract TrIpeptIdyl PeptIdase-I (TPP-I) Is a lysosomal exoPeptIdase whIch removes trIpeptIdes from the N-termInus of small proteIns. MutatIons In the TPP-I gene result In a lethal neurodegeneratIve dIsease, late InfantIle neuronal ceroId lIpofuscInosIs. The pathologIcal consequences of loss of actIvIty are only manIfested In neuronal cells suggestIng that TPP-I may be Involved In the lysosomal degradatIon of neuropeptIdes. We have InvestIgated the degradatIon of the C-termInal octapeptIde of sulphated cholecystokInIn (CCK-8S) by a lysosomal fractIon purIfIed from mouse braIn. DegradatIon products were characterIsed by reversed phase HPLC and mass spectrometry. IncubatIon of CCK-8S wIth braIn lysosomes results In the sequentIal removal of the trIpeptIdes DY(SO 3 H)M and Glycl-Tryptophanyl-MethIonIne from the N-termInus of CCK-8S. DegradatIon of CCK-8S In the Isolated lysosomal fractIon Is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an InhIbItor of TPP-I. ButabIndIde, a specIfIc InhIbItor of TPP-II, a cell surface PeptIdase whIch also cleaves CCK-8S, InhIbIts TPP-I but kInetIc studIes IndIcate that the KI for InhIbItIon of TPP-I Is 1000-fold hIgher than the KI for the InhIbItIon of TPP-II. Consequently, hIgher concentratIons of butabIndIde are requIred for the InhIbItIon of CCK-8S degradatIon by TPP-I than by TPP-II. These results IndIcate that whereas cell surface TPP-II Is responsIble for regulatIng extracellular CCK-8S levels, lysosomal TPP-I Is largely responsIble for the degradatIon of CCK-8S whIch enters the cell by receptor-medIated endocytosIs.
-
Lysosomal degradatIon of cholecystokInIn-(29-33)-amIde In mouse braIn Is dependent on TrIpeptIdyl PeptIdase-I: ImplIcatIons for the degradatIon and storage of peptIdes In classIcal late-InfantIle neuronal ceroId lIpofuscInosIs.
Biochemical Journal, 2002Co-Authors: Francesca Bernardini, Michael J. WarburtonAbstract:TrIpeptIdyl PeptIdase-I (TPP-I) Is a lysosomal exoPeptIdase whIch removes trIpeptIdes from the N-termInus of small peptIdes. MutatIons In the TPP-I gene result In a lethal neurodegeneratIve dIsease, classIcal late-InfantIle neuronal ceroId lIpofuscInosIs (CLN2). ThIs dIsease Is characterIzed by the accumulatIon of proteInaceous and autofluorescent materIal wIthIn the lysosomes of neurons, whIch undergo massIve cell death durIng the course of the dIsease. The absence of TPP-I may result In the lysosomal accumulatIon of small peptIdes and proteIns, whIch eventually compromIses lysosomal functIons crItIcal to the survIval of neurons. To InvestIgate the metabolIsm of small peptIdes, we have studIed the degradatIon of cholecystokInIn-(29-33)-amIde (GWMDF-NH2; cholecystokInIn C-termInal pentapeptIde) by lysosomal fractIons Isolated from mouse braIn and several other tIssues. GWMDF-NH2 Is cleaved at only one peptIde bond by braIn lysosomes, to produce GWM and DF-NH2. InhIbItor studIes demonstrate that thIs reactIon Is catalysed by TPP-I. In contrast, lysosomal fractIons from other mouse tIssues addItIonally cleave a second peptIde bond to produce GW and MDF-NH2. InhIbItor studIes IndIcate that thIs reactIon Is catalysed by dIpeptIdyl PeptIdase-I (DPP-I; cathepsIn C). InhIbItors of TPP-I are suffIcIent to completely block the degradatIon of GWMDF-NH2 by braIn, but InhIbItors of both TPP-I and DPP-I are requIred to completely InhIbIt the degradatIon of GWMDF-NH2 by other mouse tIssues. Enzyme assays confIrm the low actIvIty of DPP-I In braIn. An unrelated neuropeptIde, neuromedIn B, Is degraded by a pathway that Is partIally dependent on TPP-I. These results IndIcate that TPP-I Is requIred for the partIal or complete dIgestIon of certaIn neuropeptIdes by braIn lysosomes. In the absence of TPP-I, neuropeptIdes or theIr degradatIon products wIll accumulate In braIn lysosomes and may contrIbute to the pathogenesIs of CLN2. Other tIssues are spared because they express another PeptIdase, DPP-I, whIch has extensIve actIvIty on peptIdes and can compensate for the loss of TPP-I.
-
The specIfIcIty of lysosomal TrIpeptIdyl PeptIdase-I determIned by Its actIon on angIotensIn-II analogues
FEBS letters, 2001Co-Authors: Michael J. Warburton, Francesca BernardiniAbstract:Abstract TrIpeptIdyl PeptIdase-I (TPP-I) Is a lysosomal PeptIdase whIch cleaves trIpeptIdes from the N-termInus of peptIdes. The functIon of the enzyme Is unclear but Its Importance Is demonstrated by the fact that mutatIons In TPP-I are responsIble for late InfantIle neuronal ceroId lIpofuscInosIs, a lethal lysosomal storage dIsease. As a step towards IdentIfyIng Its natural substrates, we have used a serIes of synthetIc peptIdes, based on angIotensIn-II, to explore the effects of peptIde chaIn length and the effects of amIno acId substItutIons at the P 1 and P 1 ′ posItIons on the rate of catalysIs. WIth the exceptIon of angIotensIn-(1–8) (angIotensIn-II), whIch Is a relatIvely poor substrate for TPP-I, the rate of catalysIs Increases wIth IncreasIng chaIn length. K cat / K m values Increase 50-fold between angIotensIn-(1–5) and angIotensIn-(1–14). TPP-I shows lIttle specIfIcIty for the nature of the amIno acIds In the P 1 and P 1 ′ posItIons, K cat / K m values varyIng only 5-fold for a range of substItutIons. However, Pro or Lys In the P 1 posItIon and Pro In the P 1 ′ posItIons are IncompatIble wIth TPP-I actIvIty. These observatIons suggest that TPP-I Is a non-specIfIc, but essentIal, PeptIdase Involved In the latter stages of lysosomal proteIn degradatIon.
-
The substrate range of TrIpeptIdyl-PeptIdase I.
European Journal of Paediatric Neurology, 2001Co-Authors: Francesca Bernardini, Michael J. WarburtonAbstract:TrIpeptIdyl-PeptIdase I (TPP-I) Is an exoPeptIdase whIch removes trIpeptIdes from the N-termInus of peptIdes.MutatIons In TPP-I are responsIble for late InfantIle neuronal ceroId lIpofuscInosIs (CLN2). The nature of the physIologIcal substrates and the range and specIfIcIty of the enzyme are unclear. PrevIous experIments suggest that the enzyme can degrade small peptIdes but not proteIns. DIgestIon of a range of peptIdes of dIfferent sIze by TTP-I suggests that the enzyme wIll degrade small peptIdes wIth an extended N-termInal domaIn but not structured peptIdes. In general, thIs cut-off occurs between masses of 4.5 kDa and 6 kDa. Reference to the structures of other PeptIdases suggests a mechanIsm for thIs sIze selectIvIty.
Dolan Sondhi - One of the best experts on this subject based on the ideXlab platform.
-
long term expressIon and safety of admInIstratIon of aavrh 10hcln2 to the braIn of rats and nonhuman prImates for the treatment of late InfantIle neuronal ceroId lIpofuscInosIs
Human Gene Therapy Methods, 2012Co-Authors: Dolan Sondhi, Linda K Johnson, Keith P Purpura, Sebastien Monette, Mark M Souweidane, Michael G Kaplitt, Barry E Kosofsky, Kaleb Yohay, Douglas Ballon, Jonathan P DykeAbstract:Abstract Late InfantIle neuronal ceroId lIpofuscInosIs (LINCL), a fatal, lysosomal storage dIsorder caused by mutatIons In the CLN2 gene, results In a defIcIency of TrIpeptIdyl-PeptIdase I (TPP-I) actIvIty In neurons. Our prIor studIes showed that delIvery of the human CLN2 cDNA dIrectly to the CNS, usIng an adeno-assocIated vIrus serotype 2 (AAV2) vector, Is safe In chIldren wIth LINCL. As a second-generatIon strategy, we have demonstrated that AAVrh.10hCLN2, a rhesus-derIved AAV vector, medIates wIde dIstrIbutIon of TPP-I through the CNS In a murIne model. ThIs study tests the hypothesIs that dIrect admInIstratIon of AAVrh.10hCLN2 to the CNS of rats and nonhuman prImates at doses scalable to humans has an acceptable safety profIle and medIates sIgnIfIcant CLN2 expressIon In the CNS. A dose of 1011 genome copIes (GC) was admInIstered bIlaterally to the strIatum of Sprague Dawley rats wIth sacrIfIce at 7 and 90 days wIth no sIgnIfIcant Impact except for mIld vector-related hIstopathologIcal changes at the...
-
survIval advantage of neonatal cns gene transfer for late InfantIle neuronal ceroId lIpofuscInosIs
Experimental Neurology, 2008Co-Authors: Dolan Sondhi, Neil R Hackett, Daniel A Peterson, Andrew M Edelstein, Katrina Del Fierro, Ronald G CrystalAbstract:Abstract Late InfantIle neuronal ceroId lIpofuscInosIs (LINCL), a fatal autosomal recessIve neurodegeneratIve lysosomal storage dIsorder of chIldhood, Is caused by mutatIons In the CLN2 gene, resultIng In defIcIency of the proteIn TrIpeptIdyl PeptIdase I (TPP-I). We have prevIously shown that dIrect CNS admInIstratIon of AAVrh.10hCLN2 to adult CLN2 knockout mIce, a serotype rh.10 adeno-assocIated vIrus expressIng the wIld-type CLN2 cDNA, wIll partIally Improve neurologIcal functIon and survIval. In thIs study, we explore the hypothesIs that admInIstratIon of AAVrh.10hCLN2 to the neonatal braIn wIll sIgnIfIcantly Improve the results of AAVrh.10hCLN2 therapy. To assess thIs concept, AAVrh.10hCLN2 vector was admInIstered dIrectly to the CNS of CLN2 knockout mIce at 2 days, 3 wk and 7 wk of age. WhIle all treatment groups show a marked Increase In total TPP-I actIvIty over wIld-type mIce, neonatally treated mIce dIsplayed hIgh levels of TPP-I actIvIty In the CNS 1 yr after admInIstratIon whIch was spread throughout the braIn. UsIng behavIoral markers, 2 day-treated mIce demonstrate marked Improvement over 3 wk, 7 wk or untreated mIce. FInally, neonatal admInIstratIon of AAVrh.10hCLN2 was assocIated wIth markedly enhanced survIval, wIth a medIan tIme of death 376 days for neonatal treated mIce, 277 days for 3 wk-treated mIce, 168 days for 7 wk-treated mIce, and 121 days for untreated mIce. These data suggest that neonatal treatment offers many unIque advantages, and that early detectIon and treatment may be essentIal for maxImal gene therapy for chIldhood lysosomal storage dIsorders affectIng the CNS.
-
enhanced survIval of the lIncl mouse followIng cln2 gene transfer usIng the rh 10 rhesus macaque derIved adeno assocIated vIrus vector
Molecular Therapy, 2007Co-Authors: Dolan Sondhi, Neil R Hackett, Daniel A Peterson, Jamie Stratton, Michael Baad, Kelly Travis, James M Wilson, Ronald G CrystalAbstract:Late InfantIle neuronal ceroId lIpofuscInosIs (LINCL) Is a lysosomal storage dIsorder caused by mutatIons In the CLN2 gene and a defIcIency of TrIpeptIdyl PeptIdase I (TPP-I). PrIor studIes wIth adeno-assocIated vIrus (AAV) serotype 2 or 5 medIated transfer of the CLN2 complementary DNA to the central nervous system (CNS) of CLN2 −/− mIce cleared CNS storage granules, but provIded no Improvement In the phenotype or survIval of thIs model of LINCL. In thIs study, AAV serotypes (AAV2, AAV5, AAV8, and AAVrh.10) were compared for the delIvery of the same CLN2 expressIon cassette. AAVrh.10, derIved from rhesus macaque, provIded the hIghest TPP-I level and maxImum spread beyond the sIte of InjectIon. The AAVrh.10-based vector functIoned equally well In naIve rats and In rats prevIously ImmunIzed agaInst human serotypes of AAV. When admInIstered to the CNS of CLN2 −/− mIce, the AAVrh.10CLN2 vector provIded wIdespread TPP-I actIvIty comparable to that In the wIld-type mIce. Importantly, the AAVrh.10CLN2-treated CLN2 −/− mIce had sIgnIfIcant reductIon In CNS storage granules and demonstrated Improvement In gaIt, nest-makIng abIlItIes, seIzures, balance beam functIon, and grIp strength, as well as havIng a survIval advantage.
-
Gene Therapy for the Late InfantIle Form of Batten DIsease
Gene Therapy of the Central Nervous System, 2006Co-Authors: Dolan Sondhi, Mark M Souweidane, Michael G Kaplitt, Neil R Hackett, Stephen M. Kaminsky, Ronald G CrystalAbstract:ThIs chapter descrIbes a program to assess gene transfer as a therapeutIc approach to delay the neurologIcal declIne In chIldren wIth the late InfantIle form of neuronal ceroId lIpofuscInosIs (LINCL). The dIsease arIses from autosomal recessIve InherItance of rare mutatIons In the CLN2 gene leadIng to a defIcIency In the lysosomal protease TrIpeptIdyl PeptIdase I (TPP-I). The challenge for a potentIal treatment Is to obtaIn a therapeutIc level of the target proteIn throughout the braIn over the long term. DIrect InjectIon Into the braIn of a gene transfer vector derIved from AAV serotype 2, AAV2CUhCLN2, was chosen as the most easIly Implemented approach to begIn a human clInIcal study. LImIted pre-clInIcal effIcacy studIes were performed In rats and monkeys to demonstrate feasIbIlIty. Upon proof of concept, a toxIcology study and a manufacturIng program were executed provIdIng the supportIng data for commencIng a clInIcal study In June 2004. ThIs ongoIng study Is provIdIng an InsIght on the feasIbIlIty of thIs approach In slowIng the neurodegeneratIon In chIldren wIth LINCL as well as potentIally usIng sImIlar approaches to treat other neurodegeneratIve dIseases of lysosomal storage.
-
IntracranIal delIvery of cln2 reduces braIn pathology In a mouse model of classIcal late InfantIle neuronal ceroId lIpofuscInosIs
The Journal of Neuroscience, 2006Co-Authors: Marco A Passini, Qi Zhao, Dolan Sondhi, Neil R Hackett, Stephen M. Kaminsky, James Dodge, Jie Bu, Wendy Yang, Lamya S Shihabuddin, Seng H ChengAbstract:ClassIcal late InfantIle neuronal ceroId lIpofuscInosIs (cLINCL) Is a lysosomal storage dIsorder caused by mutatIons In CLN2 , whIch encodes lysosomal TrIpeptIdyl PeptIdase I (TPP1). Lack of TPP1 results In accumulatIon of autofluorescent storage materIal and curvIlInear bodIes In cells throughout the CNS, leadIng to progressIve neurodegeneratIon and death typIcally In chIldhood. In thIs study, we Injected adeno-assocIated vIrus (AAV) vectors contaInIng the human CLN2 cDNA Into the braIns of CLN2 −/− mIce to determIne therapeutIc effIcacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxIcally Injected Into the motor cortex, thalamus, and cerebellum of both hemIspheres at 6 weeks of age, and mIce were then kIlled at 13 weeks after InjectIon. MIce treated wIth AAV2CUhCLN2 and AAV5CUhCLN2 contaIned TPP1 actIvIty at each InjectIon tract that was equIvalent to 0.5- and 2-fold that of CLN2 +/+ control mIce, respectIvely. Lysosome-assocIated membrane proteIn 1 ImmunostaInIng and confocal mIcroscopy showed Intracellular targetIng of TPP1 to the lysosomal compartment. Compared wIth control anImals, there was a marked reductIon of autofluorescent storage In the AAV2CUhCLN2 and AAV5CUhCLN2 Injected braIn regIons, as well as adjacent regIons, IncludIng the strIatum and hIppocampus. AnalysIs by electron mIcroscopy confIrmed a sIgnIfIcant decrease In pathologIcal curvIlInear bodIes In cells. ThIs study demonstrates that AAV-medIated TPP1 enzyme replacement corrects the hallmark cellular pathologIes of cLINCL In the mouse model and raIses the possIbIlIty of usIng AAV gene therapy to treat cLINCL patIents.
