- Email: [email protected]
P3-353 A protease resistant PRP isoform in plasma of hamsters infected with the 263K scrapie strain
Poster Session P3: Related Neurodegenerative Conditions - Other Prion Disease 3f3-~
L E V E L S OF CSF-TAU AND A M Y L O I D BETA(I-42) IN F R O N T O T E M P O R A L L O B A R DEGENERATION: C O R R E L A T I O N W I T H C L I N I C A L AND I M A G I N G CHARACTERISTICS
Yolande A. Pijnenburg*. VU University Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected]
Background: Pathologic findings of frontotemporal lobar degeneration (FTLD) are heterogeneous and most often non-specific. The usefulness of the CSF biomarkers tan and amyloid betao_~2 ) (A~42) in the diagnosis of FTLD is doubtful, because of the wide range of both CSF tau and A~42 levels, which partly overlap with levels in Alzheimer's disease. No relationship between the presence of intracerebral tan deposition and elevated levels of CSF tau was found in several tauopathies. Objective: To investigate the relationships between CSF levels of tan and A[342 and disease characteristics in FTLD. Methods: Included were 32 patients with FTLD (16 frontotemporal dementia, 11 semantic dementia, 5 progressive nonfluent aphasia; 16 males; median age 63, range 54-85).The diagnosis of FTLD was based on international clinical diagnostic criteria, supported by structural and/or functional neuroimaging (HMPAO-SPECT). CSF tan and A[342 were determined using sandwich ELISA's. MRI and CT scans were rated for regional atrophy using a 0-3 scale. Spearman's correlation coefficients between CSF markers, clinical and imaging parameters were computed. Multiple regression analysis was performed to predict CSF tan and CSF A1~42 as dependent variables with clinical and imaging parameters as independent variables. Results: Median CSF tan level was 350 pg/ml (range 49-1200) and median CSF A1342 level was 574 pg/ml (range 2451408). There was a positive correlation between the level of A~42 and the degree of asymmetry between the left and right frontotemporal cortices (r = 0.501, p < 0.01). No relationships between CSF tan and frontotemporal atrophy and/or dementia severity could be demonstrated. However, within the subgroup of temporal variant FTLD age, disease duration and asymmetry were independent predictors of CSF-tau. Conclusions: CSF biomarkers in FTLD seem to be related to a combination of clinical and imaging characteristics. Future studies are needed to investigate these relationships in more detail. I C A L STUDY ON P•-3-5J-] PI MH OMSUPNHOOHRIYSLTAOTCEHDE MSITES O F TAU P R O T E I N IN A M Y O T R O P H I C L A T E R A L SCLEROSIS/ P A R K I N S O N I S M - D E M E N T I A C O M P L E X OF THE KII PENINSULA O F JAPAN Yasumasa Kokubo*. Mie Univ. Schol. of Med., Tsu, Japan. Contact e-maih kokubo-y @clin.medic.mie-u, ac.jp
Background: There are abundant tan depositions in the brains of amyotrophic lateral sclerosis/parkinsonism-dementia complex of the Kii peninsula (Kii ALS/PDC) of Japan. The purpose of this study is to reveal phosphorylated sites of tan protein of Kii ALS/PDC brains immunohistochemically. Objective and Method: Formalin-fixed, paraffin-embedded sections of the medial temporal lobes derived from five patients with ALS/PDC, five patients with Alzheimer's disease (AD) and five normal subjects were submitted for the study. Eighteen different anti -au antibodies, including AT8, AT100 (Innogenetics), Tau5 (Neo Markers), Tan (Dr. Ihara), anti-PSI99, PS202, PT205, PS208, PT231/235, PS262, PS396, PS404, PS409, PS 412, PS413, PS422, taa-C and tan-N (Dr. Ishiguro), were used. Result: In Kii ALS/PDC, tan deposits were positive for all of anti -an antibodies except for anti-tan antibody specific to PS412, while in AD, they were positive for anti-tan antibodies. Conclusion: Immnnohistochemical properties of tau of Kii ALS/PDC were similar to that of AD except for PS412.
~
$455
THE M I D D E L H E I M FRONTALITY SCORE: A BEHAVIORAL ASSESSMENT S C A L E THAT DISCRIMINATES F R O N T O T E M P O R A L D E M E N T I A F R O M A L Z H E I M E R ' S DISEASE
Peter Patti De Deyn 1,2, Sebastiaan Engelborghs* 1,2, Jos Saerens 2, Johan Goeman 2, Peter Mari6n 2, Karen Maertens 1, Guy Nagels 1, Barbara A. Pickut 2. 1University of Antwerp, Antwerp, Belgium; 2Middelheim General Hospital, Antwerp, Belgium. Contact e-mail: Sebastiaan.Engelborghs @ua.ac, be
Background and Objective(s): Despite striking neuropsychological and behavioral differences between Alzheimer's disease (AD) and frontotemporal dementia (FYD), clinical diagnostic criteria and frequently used behavioral assessment scales failed to discriminate FTD from AD patients. We therefore developed the Middelheim Frontality Score (MFS), a disease-long clinical and behavioral assessment tool that measures frontal lobe features, and set up this prospective study to assess discriminatory power and intra- and inter-rater variability. Methods: Patients with probable AD (N = 400) and FTD (N = 62) were included. The MFS was obtained by summating the scores obtained in a standardized fashion on 10 items yielding a total maximal score of 10. Information was obtained through hetero-anamnesis of the caregiver, anamnesis of the patient, clinical files and behavioral observation. Results: Comparing mean total MFS scores, FTD patients (6.3 :tz 1.8) had significantly higher scores than AD patients (3.1 4- 1.8) (P < 0.001). Distribution of scores on individual MFS items was significantly different comparing both disease groups 0~2 = 76.2; P < 0.001). A moderately positive and highly significant correlation was shown between the total MFS score and diagnosis FTD (R = 0.478; P < 0.0001). Applying a total MFS score of 5 as discriminatory cut-off, a specificity of 89.0% and a sensitivity of 88.7% were achieved. Intra- and inter-rater variability was calculated in a different study population by means of retest correlation, revealing moderate to strong positive COlTelations of high statistical significance. Conclusions: The MFS is a clinical and behavioral assessment scale that measures frontal lobe features and that was shown to reliably discriminate FTD from AD patients.
Poster Session P3: Related N e u r o d e g e n e r a t i v e Conditions - Other Prion Disease ~
A
PROTEASE RESISTANT P R P I S O F O R M IN P L A S M A O F H A M S T E R S INFECTED W I T H THE 263K S C R A P I E STRAIN
Chuanhua Wang*, Jae-I1 Kim, Salomon Kuizon, Denis Barengoits, Cliff Meeker, Richard Kascsak, Richard Carp, Richard Rubenstein. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY,, USA. Contact e-maiL" Chuanhua. [email protected]
Background: Prion diseases are infectious and fatal neurodegenerative disorders which include Creutzfeldt-Jakob disease (CJD), scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Consumption of BSE contaminated meat has presumably been responsible for new variant CJD. (vCJD). Although the number of BSE cases have dramatically subsided following the feed bans, the question of whether the disease is endemic remains unanswered. More recently, BSE have been found in the US and Canada. The development of a diagnostic test would therefore be an important step in controlling the spread of infection and insuring public safety. However, a rapid, consistent, and relatively non-invasive diagnostic test has not been available for the detection of priori diseases. Neuropathological changes are caused by the accumulation in the central nervous system of PrP sc, a protease resistant abnormal isoform of PrP c, a host-coded glycoprotein. The detection of PrP sc is used as the diagnostic marker for priori diseases. Objective: In this study, a protease resistant PrP isoform was detected in the plasma from hamsters infected with the 263K scrapie strain, and not control hamster plasma. Method: Collect 10 mis. of normal or 263K-infected hamster blood in 3.8% sodium citrate by cardiac puncture and separate the plasma by centrifugation (2000g, 10rain).
$456
Poster Session P3: Related Neurodegenerative Conditions - Other Prion Disease
The plasma is then processed using dialysis, affinity chromatography and treatment with proteinase K followed by gel electrophoresis and western blotting. Results: Using biotinylated 3F4 monoclonal antibody, a prpSC-like isoform was found in 263K-infected hamster plasma. No corresponding form was found in normal hamster plasma. This suggests that a prpSC-like isoform is present in plasma and may prove to be a useful marker for the infectious agent. Conclusion: A plasma-derived prpSC-like protein, which is resistant to proteinase K digestion and immunoreactive with PrP-specific antibodies, may be a useful diagnostic marker for prion diseases. Further studies are currently being conducted.
•
MONOCLONAL ANTIBODIES FOR THE TREATMENT OF PRION INFECTION
Joanna Pankiewicz* 1, Frances Prelli 1, Henrieta Scholtzova 1, Marcin Sadowski 1, Einar M. Sigurdsson 1, Fernando Goni 1, Richard Kascsak 2, Regina Kascsak 2, Richard I. Carp z, Harry C. Meeker 2, Man-Sun Sy 3, Thomas Wisniewski 1 1New York University School of
Medicine, New York, NY, USA; 2New York State Institute for Basic Research in Development Disabilities, Staten Island, NY,, USA; 3Case Western Reserve University School of Medicine, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Prion diseases are fatal, highly transmissible conformational disorders with a prolonged incubation period followed by a brief clinically symptomatic stage. Conformational transformation of PrPc into a toxic and proteinase resistant conformer PrP sc can be induced by introduction of allogenic (iatrogenic Creutzfeldt-Jakob disease [CJD]) or in certain cases xenogenic PrP sc (variant CJD). Prion infection does not elicit an immune response but PrP sc replicates and accumulates within the lymphoreticular system. Our own studies and those of others have demonstrated that humoral immunity may significantly prolong the incubation period and even prevent symptoms. Objective(s): To investigate the mechanism of action of antiPrP monoclonal antibodies (mAbs) as a potential preventive therapy for prion infections. Methods: (1) The effect of a large single dose of mAb administered immediately following infection of CD-1 mice with 139A scrapie strain was investigated. (2) The neutralizing effect of different mAbs on scrapie inoculum infectivity was studied. 100 Ixl of 1 : 10 brain homogenate from terminally sick 139A infected CD-1 mice was incubated 1 : 1 with various mAbs and administered intraperitoneaily into CD-1 mice. (3) A cell culture model of prion infection was created to study the effect of mAbs on PrP sc replication. Results: (1) Administration of lmg 7D9 mAb (non-linear PrP epitope) 1 hour following inoculation resulted in a significant delay in onset of disease symptoms (p < 0.001). (2) mAbs significantly reduced the infectivity of brain homogenate. The longest delay in onset of symptoms was observed with mAb 8B4 (recognizing residues 37-45 of PrP; p < 0.001), followed by mAbs llG5 and 8H4 (residues 115-130 and 175-185, respectively; p < 0.05). No significant effect was observed for mAb 8F9 (residues 220-231). (3) A cell culture model of PrP sc replication has been created using neuroblastoma cell lines over-expressing human or mufine PrP C which were infected with sporadic CJD and various mouse adapted scrapie strains respectively. Large library of anti-PrP mAbs will be used to map epitopes of PrP crucial for prion replication. Conclusions: Anti-PrP mAbs effectively extend the incubation period in prion infection, representing a potential therapeutic approach. Ongoing studies are designed to map PrP sc epitopes which are the most crucial for its infectivity and ability to replicate.
~3f~
EXPRESSION AND POTENTIAL ROLES OF THE MEMBERS OF CALSENILIN FAMILY IN THE BRAINS OF SCRAPIE-INFECTED MICE
Eun-Kyoung Choi* 1, Jin-Kyu Choi 1 Yulian Jin 1, Jae-Kwang Jin 1, Ju-Hong Lee 1, Hyun-Pil Lee 1, Joseph D. Buxbaum 2, Wilma Wasco 3, Richard I. Carp 4, Yong-Sun Kim 1'5. lllsong Institute of Life Science,
Hallym University, Anyang, Kyonggi-do, Republic of Korea; 2Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, New York, NY, USA; 3Genetics and Aging Research Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA; 4New York State Institute for Basic Research In Developmental Disabilities, Staten Island, NY,, USA; 5Department of Microbiology, Chuncheon, Kangwon-do, Republic of Korea. Contact e-mail: [email protected] Background: Scrapie is the archetype of transmissible spongiform encephalopathies (TSEs) or prion diseases caused by misfolding of the prion protein (PrP). PrP so, abnormal isoforms of the normal cellular prion protein, can induce neuronal degeneration and astrogliosis. Calsenilin, which has also been termed DREAM and KChIP3, is a member of the neuronal calcium sensor (NCS) family of calcium-binding proteins that interacts with the presenilins. Calsenilin has been found to act as a Kv4 channel interactor and as a transcriptional repressor, and a number of calsenilin-like proteins (KChIP1, KChIP2 and CALP/KChIP4) have been identified. We have recently shown that caisenilin can be cleaved by caspase-3 and that its phosphorylation at Set 63 can regulate this caspase cleavage. Objective(s): To investigate possible correlations between the expression levels and the potential roles of the members of calsenilin family in prion diseases. To identify the specific cell types that express calsenilin/KChIPs. Methods: To determine the expression of ealsenilin/KChlPs, we generated appropriate primers of calsenilin/KChIPs and carried out RT-PCR and Western blot using the brains of control and scrapie-infected mice. To characterize the specific cell types that express calsenilin/KChlPs, we carried out in situ hybridization and immunohistochemistry analyses. To confirm the expression pattern and its functional role of calsenilirdKChlPs, we carried out the immunofluorescence using the brains of control and scrapie-infected mice. Results: We have found that calsenilin mRNA is mainly detected in neurons of cerebral cortex, hippocampus, striatum, and cerebellum in both control and scrapie-infected brains and that its expression levels are markedly increased by scrapie infection. Interestingly, the expression of calsenilin was significantly increased in the GFAP-positive reactive astrocytes of scrapie-infected brains than in the control brains. We also found the different expression pattern of calsenilin/KChIPs in scrapie-infected brains. Conclusions: The expression patterns of calsenilin/KChlPs and its specific expression in reactive astrocytes may be involved in the glial activation and the development of prion diseases.
~ - ]
USE OF EXPRESSION ARRAYS TO PRIORITIZE AND RNAI TO TEST PRION INCUBATION TIME MODIFIER GENE CANDIDATES
Rajeev Kumar .1 , Inyoul Lee 2, Anna Gibson 1, Leroy E. Hood 2, George A. Carlson 1. l McLaughIin Research Institute, Great Falls, MT], USA;
2Institute for Systems Biology, Seattle, WA, USA. Contact e-mail: gac @po.mri.montana. edu Background: Although the most dramatic control of prion incubation time in the mouse is exerted by alternative alleles of the prion protein (PrP) gene, other loci strongly influence the length of the interval between prion inoculation and onset of disease. Crosses between CAST/El and SJL/J mice, which share PrP gene coding sequences but differ in incubation time, and quantitative trait analysis were used to identify to regions on Chromosomes 9 and 11 that harbor prion incubation time modifier genes. Objective: The goal is to identify the genes within these chromosomal intervals that underlie differential prion susceptibility. Method: Differences in activity of aUelic gene products are responsible for the majority of quantitative trait loci and some activity differences may reflect level of mRNA expression. SJL strains congenic for either the Chromosome 9 or Chromosome 11 interval from long incubation time CAST mice were produced by backcrossing and marker-
L E V E L S OF CSF-TAU AND A M Y L O I D BETA(I-42) IN F R O N T O T E M P O R A L L O B A R DEGENERATION: C O R R E L A T I O N W I T H C L I N I C A L AND I M A G I N G CHARACTERISTICS
Yolande A. Pijnenburg*. VU University Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected]
Background: Pathologic findings of frontotemporal lobar degeneration (FTLD) are heterogeneous and most often non-specific. The usefulness of the CSF biomarkers tan and amyloid betao_~2 ) (A~42) in the diagnosis of FTLD is doubtful, because of the wide range of both CSF tau and A~42 levels, which partly overlap with levels in Alzheimer's disease. No relationship between the presence of intracerebral tan deposition and elevated levels of CSF tau was found in several tauopathies. Objective: To investigate the relationships between CSF levels of tan and A[342 and disease characteristics in FTLD. Methods: Included were 32 patients with FTLD (16 frontotemporal dementia, 11 semantic dementia, 5 progressive nonfluent aphasia; 16 males; median age 63, range 54-85).The diagnosis of FTLD was based on international clinical diagnostic criteria, supported by structural and/or functional neuroimaging (HMPAO-SPECT). CSF tan and A[342 were determined using sandwich ELISA's. MRI and CT scans were rated for regional atrophy using a 0-3 scale. Spearman's correlation coefficients between CSF markers, clinical and imaging parameters were computed. Multiple regression analysis was performed to predict CSF tan and CSF A1~42 as dependent variables with clinical and imaging parameters as independent variables. Results: Median CSF tan level was 350 pg/ml (range 49-1200) and median CSF A1342 level was 574 pg/ml (range 2451408). There was a positive correlation between the level of A~42 and the degree of asymmetry between the left and right frontotemporal cortices (r = 0.501, p < 0.01). No relationships between CSF tan and frontotemporal atrophy and/or dementia severity could be demonstrated. However, within the subgroup of temporal variant FTLD age, disease duration and asymmetry were independent predictors of CSF-tau. Conclusions: CSF biomarkers in FTLD seem to be related to a combination of clinical and imaging characteristics. Future studies are needed to investigate these relationships in more detail. I C A L STUDY ON P•-3-5J-] PI MH OMSUPNHOOHRIYSLTAOTCEHDE MSITES O F TAU P R O T E I N IN A M Y O T R O P H I C L A T E R A L SCLEROSIS/ P A R K I N S O N I S M - D E M E N T I A C O M P L E X OF THE KII PENINSULA O F JAPAN Yasumasa Kokubo*. Mie Univ. Schol. of Med., Tsu, Japan. Contact e-maih kokubo-y @clin.medic.mie-u, ac.jp
Background: There are abundant tan depositions in the brains of amyotrophic lateral sclerosis/parkinsonism-dementia complex of the Kii peninsula (Kii ALS/PDC) of Japan. The purpose of this study is to reveal phosphorylated sites of tan protein of Kii ALS/PDC brains immunohistochemically. Objective and Method: Formalin-fixed, paraffin-embedded sections of the medial temporal lobes derived from five patients with ALS/PDC, five patients with Alzheimer's disease (AD) and five normal subjects were submitted for the study. Eighteen different anti -au antibodies, including AT8, AT100 (Innogenetics), Tau5 (Neo Markers), Tan (Dr. Ihara), anti-PSI99, PS202, PT205, PS208, PT231/235, PS262, PS396, PS404, PS409, PS 412, PS413, PS422, taa-C and tan-N (Dr. Ishiguro), were used. Result: In Kii ALS/PDC, tan deposits were positive for all of anti -an antibodies except for anti-tan antibody specific to PS412, while in AD, they were positive for anti-tan antibodies. Conclusion: Immnnohistochemical properties of tau of Kii ALS/PDC were similar to that of AD except for PS412.
~
$455
THE M I D D E L H E I M FRONTALITY SCORE: A BEHAVIORAL ASSESSMENT S C A L E THAT DISCRIMINATES F R O N T O T E M P O R A L D E M E N T I A F R O M A L Z H E I M E R ' S DISEASE
Peter Patti De Deyn 1,2, Sebastiaan Engelborghs* 1,2, Jos Saerens 2, Johan Goeman 2, Peter Mari6n 2, Karen Maertens 1, Guy Nagels 1, Barbara A. Pickut 2. 1University of Antwerp, Antwerp, Belgium; 2Middelheim General Hospital, Antwerp, Belgium. Contact e-mail: Sebastiaan.Engelborghs @ua.ac, be
Background and Objective(s): Despite striking neuropsychological and behavioral differences between Alzheimer's disease (AD) and frontotemporal dementia (FYD), clinical diagnostic criteria and frequently used behavioral assessment scales failed to discriminate FTD from AD patients. We therefore developed the Middelheim Frontality Score (MFS), a disease-long clinical and behavioral assessment tool that measures frontal lobe features, and set up this prospective study to assess discriminatory power and intra- and inter-rater variability. Methods: Patients with probable AD (N = 400) and FTD (N = 62) were included. The MFS was obtained by summating the scores obtained in a standardized fashion on 10 items yielding a total maximal score of 10. Information was obtained through hetero-anamnesis of the caregiver, anamnesis of the patient, clinical files and behavioral observation. Results: Comparing mean total MFS scores, FTD patients (6.3 :tz 1.8) had significantly higher scores than AD patients (3.1 4- 1.8) (P < 0.001). Distribution of scores on individual MFS items was significantly different comparing both disease groups 0~2 = 76.2; P < 0.001). A moderately positive and highly significant correlation was shown between the total MFS score and diagnosis FTD (R = 0.478; P < 0.0001). Applying a total MFS score of 5 as discriminatory cut-off, a specificity of 89.0% and a sensitivity of 88.7% were achieved. Intra- and inter-rater variability was calculated in a different study population by means of retest correlation, revealing moderate to strong positive COlTelations of high statistical significance. Conclusions: The MFS is a clinical and behavioral assessment scale that measures frontal lobe features and that was shown to reliably discriminate FTD from AD patients.
Poster Session P3: Related N e u r o d e g e n e r a t i v e Conditions - Other Prion Disease ~
A
PROTEASE RESISTANT P R P I S O F O R M IN P L A S M A O F H A M S T E R S INFECTED W I T H THE 263K S C R A P I E STRAIN
Chuanhua Wang*, Jae-I1 Kim, Salomon Kuizon, Denis Barengoits, Cliff Meeker, Richard Kascsak, Richard Carp, Richard Rubenstein. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY,, USA. Contact e-maiL" Chuanhua. [email protected]
Background: Prion diseases are infectious and fatal neurodegenerative disorders which include Creutzfeldt-Jakob disease (CJD), scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. Consumption of BSE contaminated meat has presumably been responsible for new variant CJD. (vCJD). Although the number of BSE cases have dramatically subsided following the feed bans, the question of whether the disease is endemic remains unanswered. More recently, BSE have been found in the US and Canada. The development of a diagnostic test would therefore be an important step in controlling the spread of infection and insuring public safety. However, a rapid, consistent, and relatively non-invasive diagnostic test has not been available for the detection of priori diseases. Neuropathological changes are caused by the accumulation in the central nervous system of PrP sc, a protease resistant abnormal isoform of PrP c, a host-coded glycoprotein. The detection of PrP sc is used as the diagnostic marker for priori diseases. Objective: In this study, a protease resistant PrP isoform was detected in the plasma from hamsters infected with the 263K scrapie strain, and not control hamster plasma. Method: Collect 10 mis. of normal or 263K-infected hamster blood in 3.8% sodium citrate by cardiac puncture and separate the plasma by centrifugation (2000g, 10rain).
$456
Poster Session P3: Related Neurodegenerative Conditions - Other Prion Disease
The plasma is then processed using dialysis, affinity chromatography and treatment with proteinase K followed by gel electrophoresis and western blotting. Results: Using biotinylated 3F4 monoclonal antibody, a prpSC-like isoform was found in 263K-infected hamster plasma. No corresponding form was found in normal hamster plasma. This suggests that a prpSC-like isoform is present in plasma and may prove to be a useful marker for the infectious agent. Conclusion: A plasma-derived prpSC-like protein, which is resistant to proteinase K digestion and immunoreactive with PrP-specific antibodies, may be a useful diagnostic marker for prion diseases. Further studies are currently being conducted.
•
MONOCLONAL ANTIBODIES FOR THE TREATMENT OF PRION INFECTION
Joanna Pankiewicz* 1, Frances Prelli 1, Henrieta Scholtzova 1, Marcin Sadowski 1, Einar M. Sigurdsson 1, Fernando Goni 1, Richard Kascsak 2, Regina Kascsak 2, Richard I. Carp z, Harry C. Meeker 2, Man-Sun Sy 3, Thomas Wisniewski 1 1New York University School of
Medicine, New York, NY, USA; 2New York State Institute for Basic Research in Development Disabilities, Staten Island, NY,, USA; 3Case Western Reserve University School of Medicine, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Prion diseases are fatal, highly transmissible conformational disorders with a prolonged incubation period followed by a brief clinically symptomatic stage. Conformational transformation of PrPc into a toxic and proteinase resistant conformer PrP sc can be induced by introduction of allogenic (iatrogenic Creutzfeldt-Jakob disease [CJD]) or in certain cases xenogenic PrP sc (variant CJD). Prion infection does not elicit an immune response but PrP sc replicates and accumulates within the lymphoreticular system. Our own studies and those of others have demonstrated that humoral immunity may significantly prolong the incubation period and even prevent symptoms. Objective(s): To investigate the mechanism of action of antiPrP monoclonal antibodies (mAbs) as a potential preventive therapy for prion infections. Methods: (1) The effect of a large single dose of mAb administered immediately following infection of CD-1 mice with 139A scrapie strain was investigated. (2) The neutralizing effect of different mAbs on scrapie inoculum infectivity was studied. 100 Ixl of 1 : 10 brain homogenate from terminally sick 139A infected CD-1 mice was incubated 1 : 1 with various mAbs and administered intraperitoneaily into CD-1 mice. (3) A cell culture model of prion infection was created to study the effect of mAbs on PrP sc replication. Results: (1) Administration of lmg 7D9 mAb (non-linear PrP epitope) 1 hour following inoculation resulted in a significant delay in onset of disease symptoms (p < 0.001). (2) mAbs significantly reduced the infectivity of brain homogenate. The longest delay in onset of symptoms was observed with mAb 8B4 (recognizing residues 37-45 of PrP; p < 0.001), followed by mAbs llG5 and 8H4 (residues 115-130 and 175-185, respectively; p < 0.05). No significant effect was observed for mAb 8F9 (residues 220-231). (3) A cell culture model of PrP sc replication has been created using neuroblastoma cell lines over-expressing human or mufine PrP C which were infected with sporadic CJD and various mouse adapted scrapie strains respectively. Large library of anti-PrP mAbs will be used to map epitopes of PrP crucial for prion replication. Conclusions: Anti-PrP mAbs effectively extend the incubation period in prion infection, representing a potential therapeutic approach. Ongoing studies are designed to map PrP sc epitopes which are the most crucial for its infectivity and ability to replicate.
~3f~
EXPRESSION AND POTENTIAL ROLES OF THE MEMBERS OF CALSENILIN FAMILY IN THE BRAINS OF SCRAPIE-INFECTED MICE
Eun-Kyoung Choi* 1, Jin-Kyu Choi 1 Yulian Jin 1, Jae-Kwang Jin 1, Ju-Hong Lee 1, Hyun-Pil Lee 1, Joseph D. Buxbaum 2, Wilma Wasco 3, Richard I. Carp 4, Yong-Sun Kim 1'5. lllsong Institute of Life Science,
Hallym University, Anyang, Kyonggi-do, Republic of Korea; 2Laboratory of Molecular Neuropsychiatry, Mount Sinai School of Medicine, New York, NY, USA; 3Genetics and Aging Research Unit, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA; 4New York State Institute for Basic Research In Developmental Disabilities, Staten Island, NY,, USA; 5Department of Microbiology, Chuncheon, Kangwon-do, Republic of Korea. Contact e-mail: [email protected] Background: Scrapie is the archetype of transmissible spongiform encephalopathies (TSEs) or prion diseases caused by misfolding of the prion protein (PrP). PrP so, abnormal isoforms of the normal cellular prion protein, can induce neuronal degeneration and astrogliosis. Calsenilin, which has also been termed DREAM and KChIP3, is a member of the neuronal calcium sensor (NCS) family of calcium-binding proteins that interacts with the presenilins. Calsenilin has been found to act as a Kv4 channel interactor and as a transcriptional repressor, and a number of calsenilin-like proteins (KChIP1, KChIP2 and CALP/KChIP4) have been identified. We have recently shown that caisenilin can be cleaved by caspase-3 and that its phosphorylation at Set 63 can regulate this caspase cleavage. Objective(s): To investigate possible correlations between the expression levels and the potential roles of the members of calsenilin family in prion diseases. To identify the specific cell types that express calsenilin/KChIPs. Methods: To determine the expression of ealsenilin/KChlPs, we generated appropriate primers of calsenilin/KChIPs and carried out RT-PCR and Western blot using the brains of control and scrapie-infected mice. To characterize the specific cell types that express calsenilin/KChlPs, we carried out in situ hybridization and immunohistochemistry analyses. To confirm the expression pattern and its functional role of calsenilirdKChlPs, we carried out the immunofluorescence using the brains of control and scrapie-infected mice. Results: We have found that calsenilin mRNA is mainly detected in neurons of cerebral cortex, hippocampus, striatum, and cerebellum in both control and scrapie-infected brains and that its expression levels are markedly increased by scrapie infection. Interestingly, the expression of calsenilin was significantly increased in the GFAP-positive reactive astrocytes of scrapie-infected brains than in the control brains. We also found the different expression pattern of calsenilin/KChIPs in scrapie-infected brains. Conclusions: The expression patterns of calsenilin/KChlPs and its specific expression in reactive astrocytes may be involved in the glial activation and the development of prion diseases.
~ - ]
USE OF EXPRESSION ARRAYS TO PRIORITIZE AND RNAI TO TEST PRION INCUBATION TIME MODIFIER GENE CANDIDATES
Rajeev Kumar .1 , Inyoul Lee 2, Anna Gibson 1, Leroy E. Hood 2, George A. Carlson 1. l McLaughIin Research Institute, Great Falls, MT], USA;
2Institute for Systems Biology, Seattle, WA, USA. Contact e-mail: gac @po.mri.montana. edu Background: Although the most dramatic control of prion incubation time in the mouse is exerted by alternative alleles of the prion protein (PrP) gene, other loci strongly influence the length of the interval between prion inoculation and onset of disease. Crosses between CAST/El and SJL/J mice, which share PrP gene coding sequences but differ in incubation time, and quantitative trait analysis were used to identify to regions on Chromosomes 9 and 11 that harbor prion incubation time modifier genes. Objective: The goal is to identify the genes within these chromosomal intervals that underlie differential prion susceptibility. Method: Differences in activity of aUelic gene products are responsible for the majority of quantitative trait loci and some activity differences may reflect level of mRNA expression. SJL strains congenic for either the Chromosome 9 or Chromosome 11 interval from long incubation time CAST mice were produced by backcrossing and marker-