Traumatic Brain Injury in Mice Generates Early-Stage Alzheimer's Disease Related Protein Pathology that Correlates with Neurobehavioral Deficits.

Traumatic brain injury (TBI) increases the long-term risk of neurodegenerative diseases, including Alzheimer's disease (AD). Here, we demonstrate that protein variant pathology generated in brain tissue of an experimental TBI mouse model is similar to protein variant pathology observed during early stages of AD, and that subacute accumulation of AD associated variants of amyloid beta (Aß) and tau in the TBI mouse model correlated with behavioral deficits. Male C57BL/6 mice were subjected to midline fluid percussion injury or to sham injury, after which sensorimotor function (rotarod, neurological severity score), cognitive deficit (novel object recognition), and affective deficits (elevated plus maze, forced swim task) were assessed post-injury (DPI). Protein pathology at 7, 14, and 28 DPI was measured in multiple brain regions using an immunostain panel of reagents selectively targeting different neurodegenerative disease-related variants of Aß, tau, TDP-43, and alpha-synuclein. Overall, TBI resulted in sensorimotor deficits and accumulation of AD-related protein variant pathology near the impact site, both of which returned to sham levels by 14 DPI. Individual mice, however, showed persistent behavioral deficits and/or accumulation of toxic protein variants at 28 DPI. Behavioral outcomes of each mouse were correlated with levels of seven different protein variants in ten brain regions at specific DPI. Out of 21 significant correlations between protein variant levels and behavioral deficits, 18 were with variants of Aß or tau. Correlations at 28 DPI were all between a single Aß or tau variant, both of which are strongly associated with human AD cases. These data provide a direct mechanistic link between protein pathology resulting from TBI and the hallmarks of AD.

Traumatic brain injury in mice generates early-stage Alzheimer's disease related protein pathology that correlates with neurobehavioral deficits.

Traumatic brain injury (TBI) increases the long-term risk of neurodegenerative diseases, including Alzheimer's disease (AD). Here, we demonstrate that protein variant pathology generated in brain tissue of an experimental TBI mouse model is similar to protein variant pathology observed in human ADbrains, and that subacute accumulation of two AD associated variants of amyloid beta (Aß) and tau in the TBI mouse model correlated with behavioral deficits. Male C57BL/6 mice were subjected to midline fluid percussion injury or to sham injury, after which sensorimotor function (rotarod, neurological severity score), cognitive deficit (novel object recognition), and affective deficits (elevated plus maze, forced swim task) were assessed at different days post-injury (DPI). Protein pathology at 7, 14, and 28 DPI was measured in multiple brain regions using an immunostain panel of reagents selectively targeting different neurodegenerative disease-related variants of Aß, tau, TDP-43, and alpha-synuclein. Overall, TBI resulted in sensorimotor deficits and accumulation of AD-related protein variant pathology near the impact site, both of which returned to sham levels by 14 DPI. Individual mice, however, showed persistent behavioral deficits and/or accumulation of selected toxic protein variants at 28 DPI. Behavioral outcomes of each mouse were correlated with levels of seven different protein variants in ten brain regions at specific DPI. Out of 21 significant correlations between protein variant levels and behavioral deficits, 18 were with variants of Aß or tau. Correlations at 28 DPI were all between a single Aß or tau variant, both of which are strongly associated with human AD cases. These data provide a direct mechanistic link between protein pathology resulting from TBI and the hallmarks of AD.

Sex-Specific Multiparameter Blood Test for the Early Diagnosis of Alzheimer's Disease.

Blood-based biomarkers are needed for the early diagnosis of Alzheimer's disease (AD). We analyzed longitudinal human plasma samples from AD and control cases to identify biomarkers for the early diagnosis of AD. Plasma samples were grouped based on clinical diagnosis at the time of collection: AD, mild cognitive impairment (MCI), and pre-symptomatic (preMCI). Samples were analyzed by ELISA using a panel of reagents against nine different AD-related amyloid-ß (Aß), tau, or TDP-43 variants. Receiver operating characteristic (ROC) curves of different biomarker panels for different diagnostic sample groups were determined. Analysis of all of the samples gave a sensitivity of 92% and specificity of 76% for the diagnosis of AD. Early-stage diagnosis of AD, utilizing only the preMCI and MCI samples, identified 88% of AD cases. Using sex-biased biomarker panels, early diagnosis of AD cases improved to 96%. Using the sex-biased panels, we also identified 6 of the 25 control group cases as being at high risk of AD, which is consistent with what is expected given the advanced age of the control cases. Specific AD-associated protein variants are effective blood-based biomarkers for the early diagnosis of AD. Notably, significant differences were observed in biomarker profiles for the early detection of male and female AD cases.

Soluble α-synuclein-antibody complexes activate the NLRP3 inflammasome in hiPSC-derived microglia.

Parkinson's disease is characterized by accumulation of α-synuclein (αSyn). Release of oligomeric/fibrillar αSyn from damaged neurons may potentiate neuronal death in part via microglial activation. Heretofore, it remained unknown if oligomeric/fibrillar αSyn could activate the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome in human microglia and whether anti-αSyn antibodies could prevent this effect. Here, we show that αSyn activates the NLRP3 inflammasome in human induced pluripotent stem cell (hiPSC)-derived microglia (hiMG) via dual stimulation involving Toll-like receptor 2 (TLR2) engagement and mitochondrial damage. In vitro, hiMG can be activated by mutant (A53T) αSyn secreted from hiPSC-derived A9-dopaminergic neurons. Surprisingly, αSyn-antibody complexes enhanced rather than suppressed inflammasome-mediated interleukin-1ß (IL-1ß) secretion, indicating these complexes are neuroinflammatory in a human context. A further increase in inflammation was observed with addition of oligomerized amyloid-ß peptide (Aß) and its cognate antibody. In vivo, engraftment of hiMG with αSyn in humanized mouse brain resulted in caspase-1 activation and neurotoxicity, which was exacerbated by αSyn antibody. These findings may have important implications for antibody therapies aimed at depleting misfolded/aggregated proteins from the human brain, as they may paradoxically trigger inflammation in human microglia.

A conformation-specific antibody against oligomeric ß-amyloid restores neuronal integrity in a mouse model of Alzheimer's disease.

Conformationally distinct aggregates of the amyloid ß (Aß) peptide accumulate in brains of patients with Alzheimer's disease (AD), but the roles of the different aggregates in disease progression are not clear. We previously isolated two single-chain variable domain antibody fragments (scFvs), C6T and A4, that selectively bind different toxic conformational variants of oligomeric Aß. Here, we utilize these scFvs to localize the presence of these Aß variants in human AD brain and to demonstrate their potential as therapeutic agents for treating AD. Both A4 and C6T label oligomeric Aß in extracellular amyloid plaques, whereas C6T also labels intracellular oligomeric Aß in human AD brain tissue and in an AD mouse model. For therapeutic studies, the A4 and C6T scFvs were expressed in the AD mice by viral infection of liver cells. The scFvs were administered at 2 months of age, and mice sacrificed at 9 months. The scFvs contained a peptide tag to facilitate transport across the blood brain barrier. While treatment with C6T only slightly decreased Aß deposits and plaque-associated inflammation, it restored neuronal integrity to WT levels, significantly promoted growth of new neurons, and impressively rescued survival rates to WT levels. Treatment with A4 on the other hand significantly decreased Aß deposits but did not significantly decrease neuroinflammation or promote neuronal integrity, neurogenesis, or survival rate. These results suggest that the specific Aß conformation targeted in therapeutic applications greatly affects the outcome, and the location of the targeted Aß variants may also play a critical factor.

Isolation and characterization of antibody fragments selective for human FTD brain derived TDP-43 variants.

BACKGROUND: Frontotemporal dementia (FTD) is the second leading cause of early onset dementia following Alzheimer's disease. It involves atrophy of the frontal and temporal regions of the brain affecting language, memory, and behavior. Transactive response DNA-binding protein 43 (TDP-43) pathology is found in most FTD and ALS cases. It plays a role in transcription, translation and serves as a shuttle between the nucleus and cytoplasm. Prior to its aggregation, TDP-43 exists as polyubiquitinated, hyperphosphorylated C-terminal fragments that correlate well with FTD disease progression. Because of the importance of TDP-43 in these diseases, reagents that can selectively recognize specific toxic TDP variants associated with onset and progression of FTD can be effective diagnostic and therapeutic tools. RESULTS: We utilized a novel atomic force microscopy (AFM) based biopanning protocol to isolate single chain variable fragments (scFvs) from a phage display library that selectively bind TDP variants present in human FTD but not cognitively normal age matched brain tissue. We then used the scFvs (FTD-TDP1 through 5) to probe post-mortem brain tissue and sera samples for the presence of FTD related TDP variants. The scFvs readily selected the FTD tissue and sera samples over age matched controls. The scFvs were used in immunohistochemical analysis of FTD and control brain slices where the reagents showed strong staining with TDP in FTD brain tissue slice. FTD-TDP1, FTD-TDP2, FTD-TDP4 and FTD-TDP5 all protected neuronal cells against FTD TDP induced toxicity suggesting potential therapeutic value. CONCLUSIONS: These results show existence of different disease specific TDP variants in FTD individuals. We have identified a panel of scFvs capable of recognizing these disease specific TDP variants in postmortem FTD tissue and sera samples over age matched controls and can thus serve as a biomarker tool.

Isolation and characterization of antibody fragment selective for human Alzheimer's disease brain-derived tau variants.

Reagents that can selectively recognize specific toxic tau variants associated with onset and progression of Alzheimer's disease (AD) and other tauopathies can be effective diagnostic and therapeutic tools. We utilized a novel atomic force microscopy-based biopanning protocol to isolate antibody fragments (single chain variable fragments, scFvs) that selectively bind tau variants present in human AD but not cognitively normal age-matched brain tissue. We identified 6 scFvs [Alzheimer's disease tau (ADT)-1 through 6] that readily distinguished between AD and control tissue and sera samples. We utilized 3 of the scFvs (ADT-2, ADT-4, and ADT-6) to analyze longitudinal plasma samples from 50 human patients, 25 patients which converted to AD during the study and 25 that remained cognitively normal. All 3 scFvs could distinguish the AD from control samples with higher tau levels in apolipoprotein E3/3 AD cases compared to apolipoprotein E3/4. Immunohistochemical analyses of human AD brain slices indicated several but not all tau variants overlapping with phosphorylated tau staining. Several reagents also showed therapeutic potential, protecting neuronal cells against AD tau-induced toxicity.

Bispecific Antibody Fragment Targeting APP and Inducing α-Site Cleavage Restores Neuronal Health in an Alzheimer's Mouse Model.

The amyloid ß (Aß) peptide, correlated with development of Alzheimer's disease (AD), is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. Alternative proteolytic cleavage of APP by α-secretase prevents formation of Aß peptide and produces a neuroprotective protein, a soluble fragment of APPα (sAPPα). We previously generated a single-chain variable domain antibody fragment (scFv) that binds APP at the ß-secretase cleavage site and blocks cleavage of APP (iBsec1), and a second scFv which has been engineered to have α-secretase-like activity that increases α-secretase cleavage of APP (Asec1a) and showed that a bispecific antibody (Diab) combining both iBsec1 and Asec1a constructs protects mammalian cells from oxidative stress. Here, we show that the diabody is an effective therapeutic agent in a mouse model of AD. An apolipoprotein B (ApoB) binding domain peptide was genetically added to the diabody to facilitate transfer across the blood-brain barrier, and a recombinant human adeno-associated virus 2/8 (rAAV2/8) was used as a vector to express the gene constructs in a APP/PS1 mouse model of AD. The diabody increased levels of sAPPα, decreased Aß deposits and levels of oligomeric Aß, increased neuronal health as indicated by MAP2 and synaptophysin staining, increased hippocampal neurogenesis, and most importantly dramatically increased survival rates compared with untreated mice or mice treated only with the ß-secretase inhibitor. These results indicate that altering APP processing to inhibit ß-site activity while simultaneously promoting α-secretase processing provides substantially increased neuronal benefits compared with inhibition of ß-secretase processing alone and represents a promising new therapeutic approach for treating AD.

CNS disease-related protein variants as blood-based biomarkers in traumatic brain injury.

OBJECTIVE: To utilize a panel of 11 single chain variable fragments (scFvs) that selectively bind disease-related variants of TAR DNA-binding protein (TDP)-43, ß-amyloid, tau, and α-synuclein to assess damage following traumatic brain injury (TBI), and determine if the presence of protein variants could account for the increased risk of various neurodegenerative diseases following TBI. METHODS: We utilized the panel of 11 scFvs in a sensitive ELISA format to analyze sera from 43 older veterans, 25 who had experienced at least 1 TBI incident during their lifetime (∼29.4 years after TBI), and 18 controls who did not incur TBI, in a cross-sectional study. RESULTS: Each of the 11 scFvs individually could significantly distinguish between TBI and control samples, though they did not detect each TBI sample. Comparing the levels of all 11 variants, all 25 TBI cases displayed higher reactivity compared to the controls and receiver operating characteristic analysis revealed 100% sensitivity and specificity. Higher total protein variants levels correlated with TBI severity and with loss of consciousness. Oligomeric tau levels distinguished between single and multiple TBI incidents. While all TBI cases were readily selected with the panel, the binding pattern varied from patient to patient, suggesting subgroups that are at increased risk for different neurodegenerative diseases. CONCLUSION: The panel of protein variants-specific scFvs can be used to identify blood-based biomarkers indicative of TBI even 20 years or more after the initial TBI. Being able to identify subgroups of biomarker profiles allows for the possibility of individually targeted treatments.

APP/Aß structural diversity and Alzheimer's disease pathogenesis.

The amyloid cascade hypothesis of Alzheimer's disease (AD) proposes amyloid- ß (Aß) is a chief pathological element of dementia. AD therapies have targeted monomeric and oligomeric Aß 1-40 and 1-42 peptides. However, alternative APP proteolytic processing produces a complex roster of Aß species. In addition, Aß peptides are subject to extensive posttranslational modification (PTM). We propose that amplified production of some APP/Aß species, perhaps exacerbated by differential gene expression and reduced peptide degradation, creates a diverse spectrum of modified species which disrupt brain homeostasis and accelerate AD neurodegeneration. We surveyed the literature to catalog Aß PTM including species with isoAsp at positions 7 and 23 which may phenocopy the Tottori and Iowa Aß mutations that result in early onset AD. We speculate that accumulation of these alterations induce changes in secondary and tertiary structure of Aß that favor increased toxicity, and seeding and propagation in sporadic AD. Additionally, amyloid-ß peptides with a pyroglutamate modification at position 3 and oxidation of Met35 make up a substantial portion of sporadic AD amyloid deposits. The intrinsic physical properties of these species, including resistance to degradation, an enhanced aggregation rate, increased neurotoxicity, and association with behavioral deficits, suggest their emergence is linked to dementia. The generation of specific 3D-molecular conformations of Aß impart unique biophysical properties and a capacity to seed the prion-like global transmission of amyloid through the brain. The accumulation of rogue Aß ultimately contributes to the destruction of vascular walls, neurons and glial cells culminating in dementia. A systematic examination of Aß PTM and the analysis of the toxicity that they induced may help create essential biomarkers to more precisely stage AD pathology, design countermeasures and gauge the impacts of interventions.

Blood-Based Oligomeric and Other Protein Variant Biomarkers to Facilitate Pre-Symptomatic Diagnosis and Staging of Alzheimer's Disease.

Oligomeric forms of amyloid-ß (Aß), tau, and TDP-43 play important roles in Alzheimer's disease (AD), and therefore are promising biomarkers. We previously generated single chain antibody fragments (scFvs) that selectively bind disease-related variants of these proteins including A4, C6T, and E1, which bind different oligomeric Aß variants; D11C, which binds oligomeric tau; and AD-TDP1 and AD-TDP2, which bind disease related TDP-43 variants. To determine the utility of these disease-related variants as early biomarkers, we first analyzed 11 human sera samples obtained ∼2 years prior to an initial mild cognitive impairment (MCI) diagnosis. While the subsequent diagnoses for the cases covered several different conditions, all samples had elevated protein variant levels relative to the plasma controls although with different individual biomarker profiles. We then analyzed a set of longitudinal human plasma samples from four AD (encompassing time points prior to MCI diagnosis and continuing until after conversion to AD) and two control cases. Pre-MCI samples were characterized by high TDP-43 variant levels, MCI samples by high Aß variant levels, and AD samples by high Aß and tau variant levels. Sample time points ranged from ∼7 years pre-MCI to ∼9 years after AD conversion. Bivariate correlations showed a negative correlation with TDP-43 levels and positive correlations with cumulative Aß and oligomeric tau levels indicating an increase in neurodegenerative processes with time in AD. Detection of disease related protein variants not only readily selects AD cases from controls, but also stages progression of AD and holds promise for a pre-symptomatic blood-based biomarker profile for AD.

TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis.

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.

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

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.

Oligomeric α-synuclein and ß-amyloid variants as potential biomarkers for Parkinson's and Alzheimer's diseases.

Oligomeric forms of α-synuclein and ß-amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. The detection of toxic variants in human cerebrospinal fluid (CSF) and blood has great promise for facilitating early and accurate diagnoses of these devastating diseases. Two hurdles that have impeded the use of these protein variants as biomarkers are the availability of reagents that can bind the different variants and a sensitive assay to detect their very low concentrations. We previously isolated antibody-based reagents that selectively bind two different oligomeric variants of α-synuclein and two of ß-amyloid, and developed a phage-based capture enzyme-linked immunosorbent assay (ELISA) with subfemtomolar sensitivity to quantify their presence. Here, we used these reagents to show that these oligomeric α-synuclein variants are preferentially present in PD brain tissue, CSF and serum, and that the oligomeric ß-amyloid variants are preferentially present in AD brain tissue, CSF, and serum. Some AD samples also had α-synuclein pathology and some PD samples also had ß-amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α-synuclein and ß-amyloid species is an effective method for identifying tissue, CSF and sera from PD and AD samples, respectively, and samples that also contained early stages of other protein pathologies, indicating their potential value as blood-based biomarkers for neurodegenerative diseases.

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

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.

A novel nicotinic mechanism underlies ß-amyloid-induced neurotoxicity.

Loss of basal forebrain cholinergic neurons (BFCN) correlates with cognitive deficits in Alzheimer disease (AD). Our recent evidence suggests that chronic exposure to Aß up-regulated neuronal α7-nAChRs and increased neuronal excitability in cultured hippocampal neurons. However, the impact of the up-regulated α7-nAChRs on Aß-induced neurotoxicity remains unclear. In this study, we investigated the role of α7-nAChRs in the mediation of Aß-induced neurotoxicity. The effects of Aß exposure on α7-nAChRs and cytotoxicity were examined using whole-cell patch clamp recordings, atomic force microscope (AFM) imaging, immunoprecipitation, and lactate dehydrogenase (LDH) release assay in primary cultured hippocampal neurons as well as differentiated human neuroblastoma (SH-SY5Y) cells with cholinergic characteristics. We found that α7-nAChRs are necessary for Aß-induced neurotoxicity in hippocampal neurons because chronic Aß significantly increased LDH level in hippocampal cultures, which was prevented by either α7-nAChR antagonist methyllycaconitine (MLA) or by α7 subunit gene deletion (cultures prepared from nAChR α7 subunit KO mice), whereas ß2-containing nAChR antagonist (dihydro-ß-erythroidine, DhßE) or the genetic deletion of nAChR ß2 subunit (cultures prepared from ß2 KO mice) failed to prevent Aß-induced toxicity. In SH-SY5Y cells, larger aggregates of Aß preferentially up-regulated α7-nAChR expression and function accompanied by a significant decrease in cell viability. Co-treatment MLA, but not mecamylamine (MEC), prevented Aß exposure-induced neurotoxicity. Our results suggest a detrimental role of upregulated α7-nAChRs in the mediation of Aß-induced neurotoxicity.

Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis.

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Isolation and characterization of antibody fragments selective for toxic oligomeric tau.

Oligomeric tau species are important in the onset and progression of Alzheimer's disease (AD), as they are neurotoxic and can propagate tau-tangle pathology. Therefore, reagents that selectively recognize different key morphologies of tau are needed to help define the role of tau in AD and related diseases. We utilized a biopanning protocol that combines the binding diversity of phage-displayed antibody libraries with the powerful imaging capability of atomic force microscopy to isolate single-chain antibody fragments (scFvs) that selectively bind toxic oligomeric tau. We isolated 3 different antibody fragments that bind oligomeric but not monomeric or fibrillar tau. The scFvs differentiate brain tissue homogenates of both 3×TG and tau-AD mice from wild-type mice, detecting oligomeric tau at much earlier ages than when neurofibrillary tangles are typically detected. The scFvs also distinguish human postmortem AD brain tissue from cognitively normal postmortem human brain tissue, demonstrating the potential of this approach for developing biomarkers for early detection and progression of AD.

A sensitive phage-based capture ELISA for sub-femtomolar detection of protein variants directly from biological samples.

To determine the role of proteins, and in particular protein variants, in human health, it may often be necessary to quantitatively determine the concentration of a specific protein variant present in complex biological samples such as blood, cerebral spinal fluid (CSF), or tissue. Many protein variants are present only at trace levels and therefore a simple assay with very high sensitivity and reliability would greatly facilitate correlation of the presence of particular protein variants with the progression of specific diseases. We have developed a simple phage based capture ELISA system that enables femtomolar or better detection of individual protein variants directly from complex biological samples. The protocol utilizes a capture reagent that selectively recognizes a unique epitope of the protein variant and a phage based detection reagent that binds to a second epitope present in all forms of the target protein. The phage based detection reagent is essentially a self-assembling nanoparticle consisting of several thousand coat proteins that can each be labeled to amplify the detection signal by several orders of magnitude. Here we demonstrate that we can achieve subfemtomolar detection of individual protein variants that have been implicated in neurodegenerative disease directly from complex tissue homogenates and sera. The ELISA system should facilitate identification of disease specific protein variants or other compounds even when present at trace amounts in samples including blood, CSF, saliva and urine.

ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.