Matching Items (8)

128649-Thumbnail Image.png

Toxic Oligomeric Alpha-Synuclein Variants Present in Human Parkinson’s Disease Brains Are Differentially Generated in Mammalian Cell Models

Description

Misfolding and aggregation of α-synuclein into toxic soluble oligomeric α-synuclein aggregates has been strongly correlated with the pathogenesis of Parkinson’s disease (PD). Here, we show that two different morphologically distinct

Misfolding and aggregation of α-synuclein into toxic soluble oligomeric α-synuclein aggregates has been strongly correlated with the pathogenesis of Parkinson’s disease (PD). Here, we show that two different morphologically distinct oligomeric α-synuclein aggregates are present in human post-mortem PD brain tissue and are responsible for the bulk of α-synuclein induced toxicity in brain homogenates from PD samples. Two antibody fragments that selectively bind the different oligomeric α-synuclein variants block this α-synuclein induced toxicity and are useful tools to probe how various cell models replicate the α-synuclein aggregation pattern of human PD brain. Using these reagents, we show that mammalian cell type strongly influences α-synuclein aggregation, where neuronal cells best replicate the PD brain α-synuclein aggregation profile. Overexpression of α-synuclein in the different cell lines increased protein aggregation but did not alter the morphology of the oligomeric aggregates generated. Differentiation of the neuronal cells into a cholinergic-like or dopaminergic-like phenotype increased the levels of oligomeric α-synuclein where the aggregates were localized in cell neurites and cell bodies.

Contributors

Agent

Created

Date Created
  • 2015-07-22

128456-Thumbnail Image.png

α-synuclein conformational antibodies fused to penetratin are effective in models of Lewy body disease

Description

Objective
Progressive accumulation of α-synuclein (α-syn) has been associated with Parkinson's disease (PD) and Dementia with Lewy body (DLB). The mechanisms through which α-syn leads to neurodegeneration are not completely

Objective
Progressive accumulation of α-synuclein (α-syn) has been associated with Parkinson's disease (PD) and Dementia with Lewy body (DLB). The mechanisms through which α-syn leads to neurodegeneration are not completely clear; however, the formation of various oligomeric species have been proposed to play a role. Antibody therapy has shown effectiveness at reducing α-syn accumulation in the central nervous system (CNS); however, most of these studies have been conducted utilizing antibodies that recognize both monomeric and higher molecular weight α-syn. In this context, the main objective of this study was to investigate the efficacy of immunotherapy with single-chain antibodies (scFVs) against specific conformational forms of α-syn fused to a novel brain penetrating sequence.
Method
We screened various scFVs against α-syn expressed from lentiviral vectors by intracerebral injections in an α-syn tg model. The most effective scFVs were fused to the cell-penetrating peptide penetratin to enhance transport across the blood–brain barrier, and lentiviral vectors were constructed and tested for efficacy following systemic delivery intraperitoneal into α-syn tg mice.
Result
Two scFVs (D5 and 10H) selectively targeted different α-syn oligomers and reduced the accumulation of α-syn and ameliorated functional deficits when delivered late in disease development; however, only one of the antibodies (D5) was also effective when delivered early in disease development. These scFVs were also utilized in an enzyme-linked immunosorbent assay (ELISA) assay to monitor the effects of immunotherapy on α-syn oligomers in brain and plasma.
Interpretation
The design and targeting of antibodies for specific species of α-syn oligomers is crucial for therapeutic immunotherapy and might be of relevance for the treatment of Lewy body disease.

Contributors

Agent

Created

Date Created
  • 2016-06-16

136483-Thumbnail Image.png

Optimizing Dialysis Conditions for A4 Stability

Description

The research objective is to maintain the A4 nanobody stability during dialysis. Various dialysis buffers were tested and compared, including PBS with varying amounts of the detergent, Tween: low, high,

The research objective is to maintain the A4 nanobody stability during dialysis. Various dialysis buffers were tested and compared, including PBS with varying amounts of the detergent, Tween: low, high, none. Furthermore, PBS, Tris, and HEPES, were tested and compared. PBS without Tween was the worst for preserving A4 stability. PBS was determined to be a better dialysis buffer than Tris or HEPES. To find the optimum buffer, other buffers will be tested and compared with PBS; methods such as gravity filtration and lyophilization will be considered as alternatives to dialysis.

Contributors

Agent

Created

Date Created
  • 2015-05

137774-Thumbnail Image.png

Detecting Oligomeric Forms of Alpha-Synuclein in Cell and Mouse Tissue

Description

Misfolding and aggregation of alpha-synuclein (a-syn) has been strongly correlated with the pathogenesis of Parkinson's disease (PD). Reagents such as single chain antibody fragments (scFv) that can interact with specific

Misfolding and aggregation of alpha-synuclein (a-syn) has been strongly correlated with the pathogenesis of Parkinson's disease (PD). Reagents such as single chain antibody fragments (scFv) that can interact with specific aggregate forms of a-syn can be very useful to study how different aggregate forms affect cells. Here we utilize two scFvs, D5 and 10H, that recognize two distinct oligomeric forms of a-syn to characterize the presence of different a-syn aggregates in animal models of PD.

Contributors

Agent

Created

Date Created
  • 2013-05

128955-Thumbnail Image.png

TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis

Description

Background
TDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. Using an atomic force microscopy based biopanning protocol

Background
TDP-43 aggregates accumulate in individuals affected by amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases, representing potential diagnostic and therapeutic targets. Using an atomic force microscopy based biopanning protocol developed in our lab, we previously isolated 23 TDP-43 reactive antibody fragments with preference for human ALS brain tissue relative to frontotemporal dementia, a related neurodegeneration, and healthy samples from phage-displayed single chain antibody fragment (scFv) libraries. Here we further characterize the binding specificity of these different scFvs and identify which ones have promise for detecting ALS biomarkers in human brain tissue and plasma samples.
Results
We developed a sensitive capture ELISA for detection of different disease related TDP-43 variants using the scFvs identified from the ALS biopanning. We show that a wide variety of disease selective TDP-43 variants are present in ALS as the scFvs show different reactivity profiles amongst the ALS cases. When assaying individual human brain tissue cases, three scFvs (ALS-TDP6, ALS-TDP10 and ALS-TDP14) reacted with all the ALS cases and 12 others reacted with the majority of the ALS cases, and none of the scFvs reacted with any control samples. When assaying individual human plasma samples, 9 different scFvs reacted with all the sporadic ALS samples and again none of them reacted with any control samples. These 9 different scFvs had different patterns of reactivity with plasma samples obtained from chromosome 9 open reading frame 72 (c9orf72) cases indicating that these familial ALS genetic variants may display different TDP-43 pathology than sporadic ALS cases.
Conclusions
These results indicated that a range of disease specific TDP-43 variants are generated in ALS patients with different variants being generated in sporadic and familial cases. We show that a small panel of scFvs recognizing different TDP-43 variants can generate a neuropathological and plasma biomarker profile with potential to distinguish different TDP-43 pathologies.

Contributors

Agent

Created

Date Created
  • 2017-01-25

Diagnosis and early detection of CNS-SLE in MRL/Ipr mice using peptide microarrays

Description

Background
An accurate method that can diagnose and predict lupus and its neuropsychiatric manifestations is essential since currently there are no reliable methods. Autoantibodies to a varied panel of antigens

Background
An accurate method that can diagnose and predict lupus and its neuropsychiatric manifestations is essential since currently there are no reliable methods. Autoantibodies to a varied panel of antigens in the body are characteristic of lupus. In this study we investigated whether serum autoantibody binding patterns on random-sequence peptide microarrays (immunosignaturing) can be used for diagnosing and predicting the onset of lupus and its central nervous system (CNS) manifestations. We also tested the techniques for identifying potentially pathogenic autoantibodies in CNS-Lupus. We used the well-characterized MRL/lpr lupus animal model in two studies as a first step to develop and evaluate future studies in humans.

Results
In study one we identified possible diagnostic peptides for both lupus and altered behavior in the forced swim test. When comparing the results of study one to that of study two (carried out in a similar manner), we further identified potential peptides that may be diagnostic and predictive of both lupus and altered behavior in the forced swim test. We also characterized five potentially pathogenic brain-reactive autoantibodies, as well as suggested possible brain targets.

Conclusions
These results indicate that immunosignaturing could predict and diagnose lupus and its CNS manifestations. It can also be used to characterize pathogenic autoantibodies, which may help to better understand the underlying mechanisms of CNS-Lupus.

Contributors

Agent

Created

Date Created
  • 2014-06-07

151602-Thumbnail Image.png

Detecting oligomeric beta-amyloid for the diagnosis of Alzheimer's disease

Description

Alzheimer's disease (AD) is the leading neurodegenerative disease, affecting roughly 8% of people 65 years of age or older. There exists an imperative need to develop a non-invasive test for

Alzheimer's disease (AD) is the leading neurodegenerative disease, affecting roughly 8% of people 65 years of age or older. There exists an imperative need to develop a non-invasive test for the earlier detection of AD. The use of biomarkers is a promising option that examines the toxic mechanisms and metabolic pathways that cause Alzheimer's disease, eventually leading to an early diagnostic method. This thesis presents the use of oligomeric beta-amyloid as a biomarker to detect Alzheimer's disease via a specialized enzyme-linked protein assay. Specifically, this paper details the optimization and development of a novel phage capture enzyme-linked immunosorbent assay (ELISA) that can detect the relative quantity of beta-amyloid oligomers in samples from a mouse model of AD. The objective of this thesis was to optimize a phage capture ELISA using the A4 single-chain variable fragment (scFv) to quantify the amount of beta-amyloid oligomers in various mice samples. A4 selectively recognizes a toxic oligomeric form of beta-amyloid. The level of A4-reactive oligomeric beta-amyloid was measured in triplicate in homogenized mouse brain tissue samples from eight transgenic (TG) and eight nontransgenic (NTG) animals aged five, nine, and thirteen months. There was a significant difference (p < 0.0005) between the five month TG and NTG mice. A decrease in beta-amyloid levels with the aging of the TG mice suggested that the beta-amyloid oligomers may be aggregating to form beta-amyloid fibrils. Conversely, the quantity of beta-amyloid increased with the aging of the NTG mice. This indicated that beta-amyloid oligomers may develop with normal aging.

Contributors

Agent

Created

Date Created
  • 2013

150063-Thumbnail Image.png

Understanding the role of predictive, diagnostic and pathogenic autoantibodies in systemic lupus erythematosus and its central nervous system (CNS) involvement

Description

Systemic lupus erytematosus (SLE) is an autoimmune disease where the immune system is reactive to self antigens resulting in manifestations like glomerulonephritis and arthritis. The immune system also affects

Systemic lupus erytematosus (SLE) is an autoimmune disease where the immune system is reactive to self antigens resulting in manifestations like glomerulonephritis and arthritis. The immune system also affects the central nervous system (known as CNS-SLE) leading to neuropsychiatric manifestations such as depression, cognitive impairment, psychosis and seizures. A subset of pathogenic brain-reactive autoantibodies (BRAA) is hypothesized to bind to integral membrane brain proteins, affecting their function, leading to CNS-SLE. I have tested this BRAA hypothesis, using our lupus-mouse model the MRL/lpr mice, and have found it to be a reasonable explanation for some of the manifestations of CNS-SLE. Even when the MRL/lpr had a reduced autoimmune phenotype, their low BRAA sera levels correlated with CNS involvement. The correlation existed between BRAA levels to integral membrane protein and depressive-like behavior. These results were the first to show a correlation between behavioral changes and BRAA levels from brain membrane antigen as oppose to cultured neuronal cells. More accurate means of predicting and diagnosing lupus and CNS-SLE is necessary. Using microarray technology I was able to determine peptide sets that could be predictive and diagnostic of lupus and each specific CNS manifestation. To knowledge no test currently exists that can effectively diagnose lupus and distinguish between each CNS manifestations. Using the peptide sets, I was able to determine possible natural protein biomarkers for each set as well as for five monoclonal BRAA from one MRL/lpr. These biomarkers can provide specific targets for therapy depending on the manifestation. It was necessary to investigate how these BRAA enter the brain. I hypothesized that substance P plays a role in altering the blood-brain barrier (BBB) allowing these BRAA to enter and affect brain function, when bound to its neurokinin-1 receptor (NK-1R). Western blotting results revealed an increase in the levels of NK-1R in the brain of the MRL/lpr compared to the MRL/mp. These MRL/lpr with increased levels of both NK-1R and BRAA displayed CNS dysfunction. Together, these results demonstrate that NK-1R may play a role in CNS manifestations. Overall, the research conducted here, add to the role that BRAA are playing in CNS-lupus.

Contributors

Agent

Created

Date Created
  • 2011