Philip Schulz

Detecting Morphologically Distinct Oligomeric Forms of α-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.

CSF levels of oligomeric alpha-synuclein and beta-amyloid as biomarkers for neurodegenerative disease

Protein misfolding and aggregation is a critically important feature in many devastating neurodegenerative diseases, therefore characterization of the CSF concentration profiles of selected key forms and morphologies of proteins involved in these diseases, including β-amyloid (Aβ) and α-synuclein (a-syn), can be an effective diagnostic assay for these diseases. CSF levels of tau and Aβ have been shown to have great promise as biomarkers for Alzheimers disease. However since the onset and progression of many neurodegenerative diseases have been strongly correlated with the presence of soluble oligomeric aggregates of proteins including various Aβ and a-syn aggregate species, specific detection and quantification of levels of each of these different toxic protein species in CSF may provide a simple and accurate means to presymptomatically diagnose and distinguish between these diseases. Here we show that the presence of different protein morphologies in human CSF samples can be readily detected using highly selective morphology specific reagents in conjunction with a sensitive electronic biosensor. We further show that these morphology specific reagents can readily distinguish between post-mortem CSF samples from AD, PD and cognitively normal sources. These studies suggest that detection of specific oligomeric aggregate species holds great promise as sensitive biomarkers for neurodegenerative disease.

Nanobody specific for oligomeric beta-amyloid stabilizes nontoxic form

While accumulation and deposition of beta amyloid (Aβ) is a primary pathological feature of Alzheimers disease (AD), increasing evidence has implicated small, soluble oligomeric aggregates of Aβ as the neurotoxic species in AD. Reagents that specifically recognize oligomeric morphologies of Aβ have potential diagnostic and therapeutic value. Using a novel biopanning technique that combines phage display technology and atomic force microscopy, we isolated the nanobody E1 against oligomeric Aβ. Here we show that E1 specifically recognizes a small oligomeric Aβ aggregate species distinct from the species recognized by the A4 nanobody previously reported by our group. While E1, like A4, blocks assembly of Aβ into larger oligomeric and fibrillar forms and prevents any Aβ induced toxicity toward neuronal cells, it does so by binding a small Aβ oligomeric species, directing its assembly toward a stable nontoxic conformation. The E1 nanobody selectively recognizes naturally occurring Aβ aggregates produced in human AD brain tissue indicating that a variety of morphologically distinct Aβ aggregate forms occur naturally and that a stable low-n nontoxic Aβ form exists that does not readily aggregate into larger forms. Because E1 catalyses the formation of a stable nontoxic low-n Aβ species it has potential value as a therapeutic reagent for AD which can be used in combination with other therapeutic approaches.

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.

Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples

We developed atomic force microscope (AFM)‐based protocols that enable isolation and characterization of antibody‐based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single‐chain antibody fragments (scFvs) that specifically recognize an oligomeric beta‐amyloid (Aβ) species correlated with Alzheimers disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We used several subtractive panning steps to remove all phage binding nondesired antigens and then used a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain‐derived oligomeric Aβ. The protocols described are readily adapted to isolating antibody‐based reagents against other antigenic targets with limited availability.

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

Oligomeric forms of α‐synuclein and β‐amyloid are toxic protein variants that are thought to contribute to the onset and progression of Parkinsons disease (PD) and Alzheimers 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 β‐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 β‐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 β‐amyloid pathology, and, very intriguingly, these PD cases also had a history of dementia. Detection of different oligomeric α‐synuclein and β‐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.

Improved affinity selection using phage display technology and off-rate based selection

Flow systems such as a BIAcore biosensor can be very efficient tools to isolate high affinity antibody fragments from affinity matured phage display libraries. Here we show that using flow based selection, we can readily isolate a variant with a 35-fold higher affinity, especially with a 7 fold better off-rate, compared to the parent clone after only a single round of selection from a second generation affinity matured phage display library. The flow system represents a fast method to isolate affinity improved antibody fragments and can be particularly useful for isolating antibodies to antigens that have poor solubility, are toxic to the host cell, or prone to aggregation.

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.

Isolation and characterization of antibody fragments selective for toxic oligomeric tau

Oligomeric tau species are important in the onset and progression of Alzheimers 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.

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

Oligomeric forms of amyloid-β (Aβ), tau, and TDP-43 play important roles in Alzheimers 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 Aβ 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 Aβ variant levels, and AD samples by high Aβ 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 Aβ 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.