Shanta Boddapati

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

BackgroundOverexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinsons disease (PD) and other synucleinopathies. αS 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 αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models.ResultsHere we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra- or extracellulary.ConclusionsSince 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.

Cyclodextrins promote protein aggregation posing risks for therapeutic applications

The presence of neurofibrillary tangles (NFTs) is a hallmark feature of various neurodegenerative disorders including Alzheimers (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.

Inhibiting β-secretase activity in Alzheimer's disease cell models with single-chain antibodies specifically targeting APP.

The Amyloid-β (Aβ) peptide is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage of APP first by β-secretase and then by γ-secretase. β-Site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in β-secretase processing of APP and is a primary therapeutic target for treatment of Alzheimers disease. While inhibiting BACE-1 activity has obvious therapeutic advantages, BACE-1 also cleaves numerous other substrates with important physiological activity. Thus, blanket inhibition of BACE-1 function may have adverse side effects. We isolated a single chain variable fragment (scFv) from a human-based scFv yeast display library that selectively inhibits BACE-1 activity toward APP by binding the APP substrate at the proteolytic site. We selected the iBSEC1 scFv, since it recognizes the BACE-1 cleavage site on APP but does not bind the adjacent highly antigenic N-terminal of Aβ, and thus it will target APP but not soluble Aβ. When added to 7PA2 cells, a mammalian cell line that overexpresses APP, the iBSEC1 scFv binds APP on the cell surface, reduces toxicity induced by APP overexpression, and reduces both intracellular and extracellular Aβ levels by around 50%. Since the iBSEC1 scFv does not contain the antibody F(c) region, this construct does not pose the risk of exacerbating inflammation in the brain as faced with full-length monoclonal antibodies for potential therapeutic applications.

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.

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.

Engineered Proteolytic Nanobodies Reduce Aβ Burden and Ameliorate Aβ-Induced Cytotoxicity

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimers 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.

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 Alzheimers 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. The levels of Aβ generated from AβPP can therefore be controlled by tailoring activity of these proteases toward AβPP. We previously showed that β-secretase proteolysis of AβPP could be selectively inhibited using the single chain antibody fragment (scFv) iBSEC1, which blocks the cleavage site on AβPP, and α-secretase proteolysis of AβPP could be selectively enhanced using a proteolytic scFv (Asec1A) engineered to have α-secretase-like activity. Here we show that DIA10D, a novel tandem bispecific scFv combining iBSEC1 with the ASec1A can control amyloidogenic processing of AβPP by simultaneously inhibiting β-secretase and increasing α-secretase processing of AβPP. When expressed in H4 (neuroglioma) cells overexpressing AβPP, DIA10D potently reduces levels of extracellular Aβ by around 50% while also increasing levels of neuroprotective sAβPPα. DIA10D activity has been designed to selectively target AβPP, so this modulation of AβPP processing should not affect endogenous activity of α-and β-secretases towards other substrates.

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta-amyloid.

Although Alzheimers disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation.