- Email: [email protected]
P4-186 Regulation of lysosomal enzymes expression in fibroblasts from Alzheimer's disease patients
Poster Session P4: Molecular Mechanisms of Neurodegeneration - Enzyme Activities
$528
was to determine if there is a similar upregulation in calcipressin protein levels in AD to parallel the previously demonstrated upregulation in mRNA expression. Methods: Using Western blotting and immunohistochemistry techniques, we compared temporal lobe calcipressin protein expression in moderate to severe AD cases versus aging, as well as age-matched controls. Results: Although we found no change in AD (n = 3) compared to age-matched controls (n = 3) in either total calcipressin isoform-1 or -4 protein levels by Westem blotting (p > 0.05), immunohistochemistry did demonstrate an age-dependent (35 to 75 yrs; n = 8) increase in cytosolic calcipressin expression in the pyramidal neurons in cell layers III and V, which are cells that are selectively vulnerable to NFT formation. Preliminary analysis also suggests that there is an increased translocation of calcipressin isoform-1 and the calcineurin A subunit from the cytosolic to the nuclear compartment in AD (n = 10; aged-matched controls n = 8). We are currently investigating the neuronal co-expression of calcipressin with PHF-1 labeling of neurofibrillary tangles in AD. Conclusions: These data suggest that one of the mechanisms for a decrease in calcineurin activity and increased protein phosphorylation in AD could be altered cellular regulation and/or compartmentalization of calcipressin. (Supported in part by NIH grant
NS38162 to JML)
•
ANALYSIS OF BACE PROTEIN AND ACTIVITY IN HUMAN CEREBROSPINAL FLUID
R.M. Damian Holsinger*, Catriona A. McLean, Colin L. Masters, Genevieve Evin. The University of Melbourne, Parkville, Victoria,
Australia. Contact e-mail: [email protected] Background: BACE (B-site amyloid precursor cleaving enzyme) initiates A~ amyloid biogenesis by cleaving at the [~-secretase site of the amyloid precursor protein (APP). We and others have demonstrated that BACE protein and activity are elevated in AD brain in cortical regions susceptible to degeneration. Much emphasis has been placed on this enzyme as a probable target for therapeutic intervention. A key element in treatment of a disease is early detection. Unfortunately for diseases such as Alzhiemer's, Parkinson's and other neurodegenerative disorders the etiology is less clear and more than 90% are of sporadic origin making early detection and treatment significantly more difficult. Objective: As part of our analysis of BACE in AD we evaluated the possibility of detecting the protein in biological fluids. Methods: Analysis of crude human CSF by Western blotting revealed the presence of a BACE immunoreactive species migrating with a similar mobility (~70 kDa) to that observed for BACE in human brain. To determine whether BACE was present as an active species in CSF we used an in vitro fluurogenic assay based on the cleavage of a decapeptide substrate designed to encompass the [3-secretase cleavage site of the Swedish variant APP molecule. Results: Analysis of AD and control CSF samples consistently showed increased BACE activity in the AD group. Comparison of activity levels between the two groups revealed a statistically significant 3-fold increase of BACE activity in the AD group compared to controls. We are currently evaluating the feasibility of this assay in human plasma as well as its potential as a diagnostic marker of AD.
•86-]
REGULATION OF LYSOSOMAL ENZYMES
EXPRESSION IN FIBROBLASTS FROM ALZHEIMER'S DISEASE PATIENTS
Aldo Orlacchio* 1, Lorena Urbaneffi 1, Simona Mencarelli 1 Antonio Orlacchio 2'3 , Giuliana Pelicci 4, Sandro Sorbi 5 , Andrej Hasilik 6 , Giurgio Bemardi 2,3 , Carla Emiliani 1.1 Universit?~ di Perugia, Perugia,
Italy; 21RCCS Santa Lucia, Roma, Italy; 3policlinico Tor Vergata, Roma, Italy; 4European Institute of Oncology, Milan, Italy; 5Universit?t di Firenze, Firenze, Italy; 6University of Marburg, Marburg, Germany. Contact e-mail: orly@ unipg.it Background: An increased glycohydrolases activity and extracellular enzyme deposition in brain plaques has been described as an early marker of metabolic dysfunction potentially related to primary etiological events in Alzheimer's disease (AD). Moreover, many neurodegenerative diseases
arise from lysosomal dysfunction and a large part of these are associated with neurological features and characterized by intracelinlar deposition and protein aggregation, events also found in age-related neurodegenerative disorders such as AD. Objective(s): To characterize lysosomal system at peripheral level, we investigated the expression of lysosomal glycohydrolases c~-mannosidase, ~-hexosaminidase, ~-galactosidase and of lysosomal protease cathepsin D in skin fibroblasts from AD patients affected either by sporadic or familial forms of the disease, including pre-symptomatic individuals. In addition, we investigated the role of ras in the expression of lysosomal enzymes. Methods: Lysosomal enzymes expression was evaluated by enzymatic assays and RT-PCR. Ras, p441p42 and p38 MAPKs activation and PS1 were detected by Western blotting. Results: We observed an up-regulation of all three acidic glycohydrolases and a down-regulation of cathepsin D in AD patients as a consequence to regulation at transcriptional level. A parallel increase of ras transcript and ras protein, without an increase of p44/p42 MAPK activation, was also revealed in AD fibroblasts. An activation of p38 MAPK already described to occur in AD was also found. Infection with a retrovirus encoding a constitutively active ras mutant (rasV12), known to induce premature senescence, increased the expression of lysosomal glycohydrolases c~-mannosidase, ~-hexosaminidase, and ~-galactosidase. On the other hand, the same infected fibroblasts showed low levels of the protease cathepsin D. In addition, we also observed in these samples that expression of PS 1 was up-regulated, thus indicating that APP processing is modulated by ras activation. Conclusions: These results provide evidence that lysosomal dysfunction is not confined to CNS. In addition, enzyme changes, ras activation and p38MAPK activation might be related to the pathogenesis of the disease and also be considered a potential peripheral diagnostic marker. Promoter elements responsive to ras activation, the effect on APP processing and on lysosomal enzymes expression of different ras mutants and MAPKs inhibitors are currently under investigation.
•
DEVELOPMENT OF BACE 1 INHIBITORS
Stephen Brady 1, James Bruce 2, Satendra Singh 3 , Ming-Chih Crouthamel 1, Katharine M. Holoway 1, Craig Coburn 1, Joseph P. Vacca 1, Jules A. Shafer 1, Daria Hazuda 1, Ming-Tain Lai* I. 1Merck and
Co, West Point, PA, USA; 2Washington State University, Pullman, WA, USA; 3Bachem Bioscience Inc, King of Prussia, PA, USA. Contact e-mail: mingtain_Lai @merck,com Abstract: The 13-site amyloid precursor protein cleaving enzyme (BACE 1) has been demonstrated to be the major t3-secretase involved in amyloid precursor protein (APP) processing to generate AIM0/42 peptides. These peptides are the major components of amyloid plaques found in AD patients. Here we present the development of the an optimum substrate sequence for BACE 1 using combinatorial peptide libraries. The relative rate of peptide cleavage by BACE 1 was monitored by LC/MS. A distinct substrate sequence was identified, which is processed by BACE 1 much more efficiently than the Swedish sequence. A structural similarity search was employed to identify BACE 1 inhibitors in the Merck collection with molecular weights less than 600 amu. Based on an initial lead obtained from this similarity search, selective BACE 1 inhibitors were designed and synthesized by capitalizing on unique features of the S1'/$3' pocket of BACE 1 in comparison to the same pocket in cathepsin D and renin.
~ - ]
D E G R A D A T I O N O F BACE BY THE UBIQUITIN-PROTEASOME PATHWAY
Hong Qing, Weihui Zhou, Michelle A. Christensen, Xiulian Sun, Yigang Tong, Weihong Song*. The University of British Columbia,
Vancouver, BC, Canada. Contact e-mail: [email protected] The amyloid [3 protein (A[~) is derived from [3-amyloid precursor protein (APP). Cleavage of APP by [~-secretase generates a C-terminal fragment (APPCTF[5 or C99), which is subsequently cleaved by y-secretase to produce AlL BACE (or BACE1), the major [5-secretase involved in cleaving APE has been identified as a type 1 membrane-associated aspartyl protease.
$528
was to determine if there is a similar upregulation in calcipressin protein levels in AD to parallel the previously demonstrated upregulation in mRNA expression. Methods: Using Western blotting and immunohistochemistry techniques, we compared temporal lobe calcipressin protein expression in moderate to severe AD cases versus aging, as well as age-matched controls. Results: Although we found no change in AD (n = 3) compared to age-matched controls (n = 3) in either total calcipressin isoform-1 or -4 protein levels by Westem blotting (p > 0.05), immunohistochemistry did demonstrate an age-dependent (35 to 75 yrs; n = 8) increase in cytosolic calcipressin expression in the pyramidal neurons in cell layers III and V, which are cells that are selectively vulnerable to NFT formation. Preliminary analysis also suggests that there is an increased translocation of calcipressin isoform-1 and the calcineurin A subunit from the cytosolic to the nuclear compartment in AD (n = 10; aged-matched controls n = 8). We are currently investigating the neuronal co-expression of calcipressin with PHF-1 labeling of neurofibrillary tangles in AD. Conclusions: These data suggest that one of the mechanisms for a decrease in calcineurin activity and increased protein phosphorylation in AD could be altered cellular regulation and/or compartmentalization of calcipressin. (Supported in part by NIH grant
NS38162 to JML)
•
ANALYSIS OF BACE PROTEIN AND ACTIVITY IN HUMAN CEREBROSPINAL FLUID
R.M. Damian Holsinger*, Catriona A. McLean, Colin L. Masters, Genevieve Evin. The University of Melbourne, Parkville, Victoria,
Australia. Contact e-mail: [email protected] Background: BACE (B-site amyloid precursor cleaving enzyme) initiates A~ amyloid biogenesis by cleaving at the [~-secretase site of the amyloid precursor protein (APP). We and others have demonstrated that BACE protein and activity are elevated in AD brain in cortical regions susceptible to degeneration. Much emphasis has been placed on this enzyme as a probable target for therapeutic intervention. A key element in treatment of a disease is early detection. Unfortunately for diseases such as Alzhiemer's, Parkinson's and other neurodegenerative disorders the etiology is less clear and more than 90% are of sporadic origin making early detection and treatment significantly more difficult. Objective: As part of our analysis of BACE in AD we evaluated the possibility of detecting the protein in biological fluids. Methods: Analysis of crude human CSF by Western blotting revealed the presence of a BACE immunoreactive species migrating with a similar mobility (~70 kDa) to that observed for BACE in human brain. To determine whether BACE was present as an active species in CSF we used an in vitro fluurogenic assay based on the cleavage of a decapeptide substrate designed to encompass the [3-secretase cleavage site of the Swedish variant APP molecule. Results: Analysis of AD and control CSF samples consistently showed increased BACE activity in the AD group. Comparison of activity levels between the two groups revealed a statistically significant 3-fold increase of BACE activity in the AD group compared to controls. We are currently evaluating the feasibility of this assay in human plasma as well as its potential as a diagnostic marker of AD.
•86-]
REGULATION OF LYSOSOMAL ENZYMES
EXPRESSION IN FIBROBLASTS FROM ALZHEIMER'S DISEASE PATIENTS
Aldo Orlacchio* 1, Lorena Urbaneffi 1, Simona Mencarelli 1 Antonio Orlacchio 2'3 , Giuliana Pelicci 4, Sandro Sorbi 5 , Andrej Hasilik 6 , Giurgio Bemardi 2,3 , Carla Emiliani 1.1 Universit?~ di Perugia, Perugia,
Italy; 21RCCS Santa Lucia, Roma, Italy; 3policlinico Tor Vergata, Roma, Italy; 4European Institute of Oncology, Milan, Italy; 5Universit?t di Firenze, Firenze, Italy; 6University of Marburg, Marburg, Germany. Contact e-mail: orly@ unipg.it Background: An increased glycohydrolases activity and extracellular enzyme deposition in brain plaques has been described as an early marker of metabolic dysfunction potentially related to primary etiological events in Alzheimer's disease (AD). Moreover, many neurodegenerative diseases
arise from lysosomal dysfunction and a large part of these are associated with neurological features and characterized by intracelinlar deposition and protein aggregation, events also found in age-related neurodegenerative disorders such as AD. Objective(s): To characterize lysosomal system at peripheral level, we investigated the expression of lysosomal glycohydrolases c~-mannosidase, ~-hexosaminidase, ~-galactosidase and of lysosomal protease cathepsin D in skin fibroblasts from AD patients affected either by sporadic or familial forms of the disease, including pre-symptomatic individuals. In addition, we investigated the role of ras in the expression of lysosomal enzymes. Methods: Lysosomal enzymes expression was evaluated by enzymatic assays and RT-PCR. Ras, p441p42 and p38 MAPKs activation and PS1 were detected by Western blotting. Results: We observed an up-regulation of all three acidic glycohydrolases and a down-regulation of cathepsin D in AD patients as a consequence to regulation at transcriptional level. A parallel increase of ras transcript and ras protein, without an increase of p44/p42 MAPK activation, was also revealed in AD fibroblasts. An activation of p38 MAPK already described to occur in AD was also found. Infection with a retrovirus encoding a constitutively active ras mutant (rasV12), known to induce premature senescence, increased the expression of lysosomal glycohydrolases c~-mannosidase, ~-hexosaminidase, and ~-galactosidase. On the other hand, the same infected fibroblasts showed low levels of the protease cathepsin D. In addition, we also observed in these samples that expression of PS 1 was up-regulated, thus indicating that APP processing is modulated by ras activation. Conclusions: These results provide evidence that lysosomal dysfunction is not confined to CNS. In addition, enzyme changes, ras activation and p38MAPK activation might be related to the pathogenesis of the disease and also be considered a potential peripheral diagnostic marker. Promoter elements responsive to ras activation, the effect on APP processing and on lysosomal enzymes expression of different ras mutants and MAPKs inhibitors are currently under investigation.
•
DEVELOPMENT OF BACE 1 INHIBITORS
Stephen Brady 1, James Bruce 2, Satendra Singh 3 , Ming-Chih Crouthamel 1, Katharine M. Holoway 1, Craig Coburn 1, Joseph P. Vacca 1, Jules A. Shafer 1, Daria Hazuda 1, Ming-Tain Lai* I. 1Merck and
Co, West Point, PA, USA; 2Washington State University, Pullman, WA, USA; 3Bachem Bioscience Inc, King of Prussia, PA, USA. Contact e-mail: mingtain_Lai @merck,com Abstract: The 13-site amyloid precursor protein cleaving enzyme (BACE 1) has been demonstrated to be the major t3-secretase involved in amyloid precursor protein (APP) processing to generate AIM0/42 peptides. These peptides are the major components of amyloid plaques found in AD patients. Here we present the development of the an optimum substrate sequence for BACE 1 using combinatorial peptide libraries. The relative rate of peptide cleavage by BACE 1 was monitored by LC/MS. A distinct substrate sequence was identified, which is processed by BACE 1 much more efficiently than the Swedish sequence. A structural similarity search was employed to identify BACE 1 inhibitors in the Merck collection with molecular weights less than 600 amu. Based on an initial lead obtained from this similarity search, selective BACE 1 inhibitors were designed and synthesized by capitalizing on unique features of the S1'/$3' pocket of BACE 1 in comparison to the same pocket in cathepsin D and renin.
~ - ]
D E G R A D A T I O N O F BACE BY THE UBIQUITIN-PROTEASOME PATHWAY
Hong Qing, Weihui Zhou, Michelle A. Christensen, Xiulian Sun, Yigang Tong, Weihong Song*. The University of British Columbia,
Vancouver, BC, Canada. Contact e-mail: [email protected] The amyloid [3 protein (A[~) is derived from [3-amyloid precursor protein (APP). Cleavage of APP by [~-secretase generates a C-terminal fragment (APPCTF[5 or C99), which is subsequently cleaved by y-secretase to produce AlL BACE (or BACE1), the major [5-secretase involved in cleaving APE has been identified as a type 1 membrane-associated aspartyl protease.