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AMELIORATION OF AMYLOID BURDEN IN ADVANCED HUMAN AND MOUSE ALZHEIMER'S DISEASE BRAINS BY ORAL DELIVERY OF MYELIN BASIC PROTEIN BIOENCAPSULATED IN PLANT CELLS
Title
AMELIORATION OF AMYLOID BURDEN IN ADVANCED HUMAN AND MOUSE ALZHEIMER'S DISEASE BRAINS BY ORAL DELIVERY OF MYELIN BASIC PROTEIN BIOENCAPSULATED IN PLANT CELLS.
Creator
Kohli, Neha, Daniell, Henry, Kim, Yoon-Seong, Cheng, Zixi, University of Central Florida
Abstract / Description
One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-? (A?) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1...
Show moreOne of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-? (A?) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, 12-14 months old triple transgenic AD mice were fed with CTB-MBP bioencapsulated in the plant cells for 3 months. A reduction of 67.3% and 33.3% amyloid levels in hippocampal and cortical regions, respectively were observed by immunostaining of brain sections with anti- A? antibody. Similarly, 70% decrease in plaque number and 40% reduction of plaque intensity was observed through thioflavin S (ThS) staining that specifically stains amyloid in the AD brain. Furthermore, ex vivo 3xTg AD mice brain sections showed up to 45% reduction of ThS stained amyloid levels when incubated with enriched CTB-MBP in a concentration dependent manner. Similarly, incubation of enriched CTB-MBP with ex vivo postmortem human brain tissue sections with advanced stage of AD resulted up to 47% decrease of ThS stained amyloid plaque intensity. Lastly, lyophilization of plant material facilitates dehydration and long term storage of capsules at room temperature, in addition to increasing CTB-MBP concentration by 17 fold. These observations offer a low cost solution for treatment of even advanced stages of the AD by facilitating delivery of therapeutic proteins to central nervous system to address other neurodegenerative disease.
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Date Issued
2012
Identifier
CFE0004564, ucf:49237
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004564
EXPRESSION AND CHARACTERIZATION OF ANTIMICROBIAL PEPTIDES RETROCYCLIN-101 AND PROTEGRIN-1 IN CHLOROPLASTS TO CONTROL VIRAL AND BACTERIAL INFECTIONS
Title
EXPRESSION AND CHARACTERIZATION OF ANTIMICROBIAL PEPTIDES RETROCYCLIN-101 AND PROTEGRIN-1 IN CHLOROPLASTS TO CONTROL VIRAL AND BACTERIAL INFECTIONS.
Creator
Li, Baichuan, Daniell, Henry, University of Central Florida
Abstract / Description
Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101...
Show moreRetrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually-transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC-101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC-101 and PG1 accumulated up to 32-38% and 17~26% of the total soluble protein. Both RC-101 and PG1 were cleaved from GFP by corresponding proteases in vitro and Factor Xa like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6 fold higher yield of RC 101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to TMV infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further pre-clinical studies.
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Date Issued
2010
Identifier
CFE0003199, ucf:48571
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003199