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.

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 (Aβ) 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).

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 (Aβ) 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).

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).

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.

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

Conformationally distinct aggregates of the amyloid β (Aβ) 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 Aβ. Here, we utilize these scFvs to localize the presence of these Aβ variants in human AD brain and to demonstrate their potential as therapeutic agents for treating AD.

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.

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.

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

The amyloid β (Aβ) peptide, correlated with development of Alzheimer's disease (AD), is produced by sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. Alternative proteolytic cleavage of APP by α-secretase prevents formation of Aβ 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 β-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.

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, β-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.

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

The amyloid cascade hypothesis of Alzheimer's disease (AD) proposes amyloid- β (Aβ) is a chief pathological element of dementia. AD therapies have targeted monomeric and oligomeric Aβ 1-40 and 1-42 peptides. However, alternative APP proteolytic processing produces a complex roster of Aβ species.

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 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 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.

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.

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.

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 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.

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.

A novel nicotinic mechanism underlies β-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 Aβ up-regulated neuronal α7-nAChRs and increased neuronal excitability in cultured hippocampal neurons. However, the impact of the up-regulated α7-nAChRs on Aβ-induced neurotoxicity remains unclear.

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.

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.

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.

Probing Antibody-Antigen Interactions.

Antibodies are biological molecules generated by the host immune system in response to the invasion of foreign bodies or antigens. Therefore, antibodies must possess high specificity toward target antigens in order for the antigen to be recognized and subsequently destroyed. Because of this specificity, antibodies or antibody fragments that maintain binding specificity are heavily used in diagnostic assays and are becoming increasingly important in many therapeutic applications.

Human α4β2 nicotinic acetylcholine receptor as a novel target of oligomeric α-synuclein.

Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined.

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 Alzheimer's 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.

Bispecific tandem single chain antibody simultaneously inhibits β-secretase and promotes α-secretase processing of AβPP.

Misfolding and aggregation of amyloid-β (Aβ) is an important early event in the pathogenesis of Alzheimer's disease. Aβ is produced by sequential proteolysis of the amyloid-β protein precursor (AβPP) by β- and γ-secretases. A third protease, α-secretase, cleaves AβPP in the middle of the Aβ sequence precluding formation of Aβ.

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 Alzheimer's 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.