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.

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.

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.

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.

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.

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.

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.

Acute sleep deprivation in mice generates protein pathology consistent with neurodegenerative diseases.

Introduction: Insufficient or disturbed sleep is strongly associated with adverse health conditions, including various neurodegenerative disorders. While the relationship between sleep and neurodegenerative disease is likely bidirectional, sleep disturbances often predate the onset of other hallmark clinical symptoms. Neuronal waste clearance is significantly more efficient during sleep; thus, disturbed sleep may lead to the accumulation of neuronal proteins that underlie neurodegenerative diseases. Key pathological features of neurodegenerative diseases include an accumulation of misfolded or misprocessed variants of amyloid beta (Aß), tau, alpha synuclein (α-syn), and TarDNA binding protein 43 (TDP-43). While the presence of fibrillar protein aggregates of these neuronal proteins are characteristic of neurodegenerative diseases, the presence of small soluble toxic oligomeric variants of these different proteins likely precedes the formation of the hallmark aggregates. Methods: We hypothesized that sleep deprivation would lead to accumulation of toxic oligomeric variants of Aß, tau, α-syn, and TDP-43 in brain tissue of wild-type mice. Adult mice were subjected to 6 h of sleep deprivation (zeitgeber 0-6) for 5 consecutive days or were left undisturbed as controls. Following sleep deprivation, brains were collected, and protein pathology was assessed in multiple brain regions using an immunostain panel of reagents selectively targeting neurodegenerative disease-related variants of Aß, tau, α-syn, and TDP-43. Results: Overall, sleep deprivation elevated levels of all protein variants in at least one of the brain regions of interest. The reagent PDTDP, targeting a TDP-43 variant present in Parkinson's disease, was elevated throughout the brain. The cortex, caudoputamen, and corpus callosum brain regions showed the highest accumulation of pathology following sleep deprivation. Discussion: These data provide a direct mechanistic link between sleep deprivation, and the hallmark protein pathologies of neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases.

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.

A novel nicotinic acetylcholine receptor subtype in basal forebrain cholinergic neurons with high sensitivity to amyloid peptides.

Nicotinic acetylcholine receptors (nAChRs) containing alpha7 subunits are thought to assemble as homomers. alpha7-nAChR function has been implicated in learning and memory, and alterations of alpha7-nAChR have been found in patients with Alzheimer's disease (AD). Here we report findings consistent with a novel, naturally occurring nAChR subtype in rodent, basal forebrain cholinergic neurons. In these cells, alpha7 subunits are coexpressed, colocalize, and coassemble with beta2 subunit(s). Compared with homomeric alpha7-nAChRs from ventral tegmental area neurons, functional, presumably heteromeric alpha7beta2-nAChRs on cholinergic neurons freshly dissociated from medial septum/diagonal band (MS/DB) exhibit relatively slow kinetics of whole-cell current responses to nicotinic agonists and are more sensitive to the beta2 subunit-containing nAChR-selective antagonist, dihydro-beta-erythroidine (DHbetaE). Interestingly, presumed, heteromeric alpha7beta2-nAChRs are highly sensitive to functional inhibition by pathologically relevant concentrations of oligomeric, but not monomeric or fibrillar, forms of amyloid beta(1-42) (Abeta(1-42)). Slow whole-cell current kinetics, sensitivity to DHbetaE, and specific antagonism by oligomeric Abeta(1-42) also are characteristics of heteromeric alpha7beta2-nAChRs, but not of homomeric alpha7-nAChRs, heterologously expressed in Xenopus oocytes. Moreover, choline-induced currents have faster kinetics and less sensitivity to Abeta when elicited from MS/DB neurons derived from nAChR beta2 subunit knock-out mice rather than from wild-type mice. The presence of novel, functional, heteromeric alpha7beta2-nAChRs on basal forebrain cholinergic neurons and their high sensitivity to blockade by low concentrations of oligomeric Abeta(1-42) suggests possible mechanisms for deficits in cholinergic signaling that could occur early in the etiopathogenesis of AD and might be targeted by disease therapies.

Engineered proteolytic nanobodies reduce Abeta burden and ameliorate Abeta-induced cytotoxicity.

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD), and reduction of Abeta levels in the brain could be a viable therapeutic approach. A potentially noninflammatory route to facilitate clearance and reduce toxicity of Abeta is to degrade the peptide using proteolytic nanobodies. Here we show that a proteolytic nanobody engineered to cleave Abeta at its alpha-secretase site has potential therapeutic value. The Asec-1A proteolytic nanobody, derived from a parent catalytic light chain antibody, prevents aggregation of monomeric Abeta, inhibits further aggregation of preformed Abeta aggregates, and reduces Abeta-induced cytotoxicity toward a human neuroblastoma cell line. The nanobody also reduces toxicity induced by overexpression of the human amyloid precursor protein (APP) in a Chinese hamster ovary (CHO) cell line by cleaving APP at the alpha-secretase site which precludes formation of Abeta. Targeted proteolysis of APP and Abeta with catalytic nanobodies represents a novel therapeutic approach for treating AD where potentially harmful side effects can be minimized.

Curcumin reduces alpha-synuclein induced cytotoxicity in Parkinson's disease cell model.

BACKGROUND: Overexpression and abnormal accumulation of aggregated alpha-synuclein (alphaS) have been linked to Parkinson's disease (PD) and other synucleinopathies. alphaS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase alphaS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. RESULTS: Here we show that curcumin can alleviate alphaS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of alphaS and extracellular addition of oligomeric alphaS increase ROS which induces apoptosis, suggesting that aggregated alphaS may induce similar toxic effects whether it is generated intra- or extracellulary. CONCLUSIONS: Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.

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.

Cyclodextrins promote protein aggregation posing risks for therapeutic applications.

The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimer's (AD) and Niemann-Pick type C (NPC) diseases. NFTs have been correlated with elevated cholesterol levels and a cholesterol-scavenging compound, cyclodextrin, effectively modulates and traffics cholesterol from cell bodies in NPC disease models. Cyclodextrins are also used as drug carriers to the blood-brain barrier (BBB) and other tissues. While cyclodextrins have potential value in treating brain diseases, it is important to determine how cyclodextrins affect natively unfolded proteins such as beta-amyloid (Abeta) whose aggregation has been correlated with AD. We show that cyclodextrins drastically alter Abeta aggregation kinetics and induce morphological changes to Abeta that can enhance toxicity towards SH-SY5Y human neuroblastoma cells. These results suggest that care must be taken when using cyclodextrins for BBB delivery or for treatment of brain disease because cyclodextrins can promote toxic aggregation of Abeta.

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.

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates.

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity.

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.

Characterization of an antibody scFv that recognizes fibrillar insulin and beta-amyloid using atomic force microscopy.

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.

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.