Michael R. Sierks

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.

Trimeric Tau Is Toxic to Human Neuronal Cells at Low Nanomolar Concentrations

In Alzheimers disease (AD), tau aggregates into fibrils and higher order neurofibrillary tangles, a key histopathological feature of AD. However, soluble oligomeric tau species may play a more critical role in AD progression since these tau species correlate better with neuronal loss and cognitive dysfunction. Recent studies show that extracellular oligomeric tau can inhibit memory formation and synaptic function and also transmit pathology to neighboring neurons. However, the specific forms of oligomeric tau involved in toxicity are still unknown. Here, we used two splice variants of recombinant human tau and generated monomeric, dimeric, and trimeric fractions of each isoform. The composition of each fraction was verified chromatographically and also by atomic force microscopy. The toxicity of each fraction toward both human neuroblastoma cells and cholinergic-like neurons was assessed. Trimeric, but not monomeric or dimeric, tau oligomers of both splice variants were neurotoxic at low nanomolar concentrations. Further characterization of tau oligomer species with disease-specific modifications and morphologies is necessary to identify the best targets for the development of biomarker and therapeutic development for AD and related tauopathies.

Characterizing antibody specificity to different protein morphologies by AFM.

Protein misfolding and aggregation can lead to several neurodegenerative diseases including Alzheimers Disease (AD), Parkinsons Disease (PD) and Huntingtons Disease (HD). While the respective proteins involved in each disease differ in their pathological effects and amino acid sequences, the aggregated forms all share a common cross beta-sheet conformation. Substantial controversy exists over the roles of the different aggregate morphologies in disease onset and progression, and analytical tools such as morphology specific antibodies are needed to distinguish between the different protein morphologies in situ. Here we utilize atomic force microscopy (AFM) to characterize the binding of three single chain antibody fragments (scFvs) to different morphologies of alpha-synuclein (alphaS). From the topographic images generated using the AFM, we were able to show that one scFv bound all morphologies of alphaS, a second bound only oligomeric alphaS, and a third bound only fibrillar alphaS by comparing the height distribution of the different alphaS morphologies with and without addition of the different scFvs. These results demonstrate the versatility of the AFM-based technique as an easy tool to characterize specific antigen-antibody binding and the potential applications of scFvs as promising immunodiagnostics for protein misfolding diseases.

Synthesis of the first pseudosugar-C-disaccharide. A potential antigen for eliciting glycoside-bond forming antibodies with catalytic groups

Abstract A number of synthetic routes to the first pseudo-C-disaccharide ever prepared has been studied. The compound, methyl 7-(( 1S , 3R , 4R , 5S , 6S )-1-amino-3-hydroxymethyl-4,5,6trihydroxycyclohexyl) -6,7-dideoxy-α-D- gluco -heptopyranoside ( 1 ), is structurally related to cellobiose, but includes a crucial amino-functionality at the pseudoanomeric centre. It was prepared by 1,2-addition of the anion of methyl 6,7-dideoxy-2,3,4-tri- O -benzyl-α-D- gluco -hept-6ynopyranoside to ( 4R , 5S , 6R )-3-benzyloxymethyl-4,5,6-tribenzyloxy-2-cyclohexenone followed by stereoselective conversion of the tertiary alcohol to azide and finally reduction.

Solvent and Viscosity Effects on the Rate-Limiting Product Release Step of Glucoamylase during Maltose Hydrolysis

Release of product from the active site is the rate‐limiting step in a number of enzymatic reactions, including maltose hydrolysis by glucoamylase (GA) . With GA, an enzymatic conformational change has been associated with the product release step. Solvent characteristics such as viscosity can strongly influence protein conformational changes. Here we show that the rate‐limiting step of GA has a rather complex dependence on solvent characteristics. Seven different cosolvents were added to the GA/maltose reaction solution. Five of the cosolvents, all having an ethylene glycol base, resulted in an increase in activity at low concentration of cosolvent and variable decreases in activity at higher concentrations. The increase in enzyme activity was dependent on polymer length of the cosolvent; the longer the polymer, the lower the concentration needed. The maximum increase in catalytic activity at 45 °C (40–45%) was obtained with the three longest polymers (degree of polymerization from 200 to 8000) . A further increase in activity to 60–65% was obtained at 60 °C. The linear relationship between ln(kcat) and (viscosity) 2 obtained with all the cosolvents provides further evidence that product release is the rate‐limiting step in the GA catalytic mechanism. A substantial increase in the turnover rate of GA by addition of relatively small amounts of a cosolvent has potential applications for the food industry where high‐fructose corn syrup (HFCS) is one of the primary products produced with GA. Since maltodextrin hydrolysis by GA is by far the slowest step in the production of HFCS, increasing the catalytic rate of GA can substantially reduce the process time.

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.

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.

Catalytic mechanism of glucoamylase probed by mutagenesis in conjunction with hydrolysis of alpha-D-glucopyranosyl fluoride and maltooligosaccharides.

The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using alpha-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116-->Ala, Trp120-->Phe, Asp176-->Asn, and Glu400-->Gln. These replacements decreased the activity (kcat/KM) toward maltose by 6-320-fold. Toward GF, however, Tyr116-->Ala and Trp120-->Phe GAs, showed wild-type and twice wild-type level activity, while Asp176-->Asn and Glu400-->Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., & Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400-->Gln and wild-type GA complexed with GF, the pH-activity (kcat) profile shows a pKa of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., & Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu400-->Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis.

Aglycon mimicking: Glycoside bond cleavage transition state mimics based on hydroxypyrrolidine inhibitors

Abstract β-L-xylopyranosides of a number of both known and new hydroxypyrrolidines were prepared.

Protein misfolding and neurodegenerative diseases

This special issue includes fifteen reviews and two original research articles by leading scientists in the fields of neuropathology, biochemistry, and cell biology, dealing with the role of protein aggregation and prion-like propagation of protein misfolding in neurodegenerative diseases. In the review article “Breaking the code of amyloid-β oligomers,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/950783/, S. E. Lesne outlines the “oligomeric” view of the amyloid hypothesis in Alzheimers disease (AD), discussing how structurally different amyloid-β (Aβ) oligomers may contribute to the pathogenesis, and the controversial role of the prion protein (PrP) in Aβ toxicity. He stresses the need to thoroughly characterize the oligomeric Aβ assemblies for dissecting the disease mechanisms and designing specific, effective therapies. Tau oligomers may also play an important neurotoxic role in AD. In the research article “Trimeric tau is toxic to human neuronal cells at low nanomolar concentrations,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/260787/, H. Tian et al. show that two nonphosphorylated human recombinant tau splice variants are neurotoxic at low nanomolar concentrations. They provide evidence that trimeric but not monomeric or dimeric tau is responsible for the toxicity. In the review article “The innate immune system in Alzheimers disease,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/576383/, A. Boutajangout and T. Wisniewski focus on the potential roles of the triggering receptor expressed on myeloid cells 2 protein (TREM2) and Toll-like receptors (TLRs) in AD. They give an overview of TREM2 functions and its involvement in phagocytic and anti-inflammatory pathways. They also review the critical roles of TLR4 and 9 in the innate immune response, the interplay of these pattern recognition receptors, and highlight the importance of microglia-mediated innate immunity in AD pathogenesis. Several articles deal with the cellular processes involved in protein folding and quality control and how their corruption may trigger neurotoxicity. In the review article “Disulfide bonding in neurodegenerative misfolding diseases,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/318319/, M. F. Mossuto discusses the role of disulfide bond formation; in the review article “Role of protein misfolding and proteostasis deficiency in protein misfolding diseases and aging,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/638083/, K. Cuanalo-Contreras et al. review the involvement of the unfolded protein response (UPR), the ubiquitin proteasome system (UPS), autophagy, and aggresome formation in neurodegenerative diseases and aging. In the review article “ER dysfunction and protein folding stress in ALS,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/674751/, S. Matus et al. specifically focus on the role of UPR in amyotrophic lateral sclerosis (ALS), and in the research article “Early delivery of misfolded PrP from ER to lysosomes by autophagy,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/560421/, C. J. Cortes et al. provide experimental evidence of a role of autophagy in the early quality control of misfolded PrP. In the review article “S-Nitrosation and ubiquitin-proteasome system interplay in neuromuscular disorders,” available at the following link: http://www.hindawi.com/journals/ijcb/2014/428764/, S. Rizza et al. give an overview on the mechanisms regulating S-nitrosation and its implication in redox signaling and neurodegeneration. They provide evidence that S-nitrosation is involved in UPS and suggest links with the pathogenesis of neuromuscular disorders and neuropathies. In the review article “Convergence of synapses, endosomes, and prions in the biology of neurodegenerative diseases,” available at the following link: http://www.hindawi.com/journals/ijcb/2013/141083/, G. K. Gouras critically discusses the roles played by synapses and cellular proteolytic systems in generation, accumulation, and prion-like propagation of neurodegenerative disease-specific proteins. He stresses the need to define the physiological functions of the misfolding proteins as well as to understand the cell biology of the synapses and endocytic/exocytic pathways in neurons better. The review article “Prion protein misfolding, strains, and neurotoxicity: an update from studies on mammalian prions” by I. Poggiolini et al. available at the following link: http://www.hindawi.com/journals/ijcb/2013/910314/ is a comprehensive review of the prion diseases, looking at the latest information on current knowledge of the mechanisms of PrP conversion and the molecular basis of prion strains. The review article “From prion diseases to prion-like propagation mechanisms of neurodegenerative diseases” by I. Acquatella-Tran Van Ba et al. available at the following link: http://www.hindawi.com/journals/ijcb/2013/975832/, summarizes the history of prion diseases, from the development of the prion concept to the production of synthetic prions, and discusses recent hypotheses on the mechanisms of de novo prion generation. The review article “Synaptic dysfunction in prion diseases: a trafficking problem?” by A. Senatore et al. http://www.hindawi.com/journals/ijcb/2013/543803/, reviews recent data pointing to intracellular PrP misfolding in synaptic dysfunction and suggests a new model of synaptotoxicity that could explain the phenotypic heterogeneity of prion diseases. The review article “Infectivity versus seeding in neurodegenerative diseases sharing a prion-like mechanism” by N. Fernandez-Borges et al. available at the following link: http://www.hindawi.com/journals/ijcb/2013/583498/, provides a critical appraisal of the current evidence supporting prion-like mechanisms in AD, frontotemporal dementia and other tauopathies, Parkinsons disease, and ALS, discussing crucial differences between these disorders and “real” prion infections. Along this line, the review article “Prions ex vivo: what cell culture models tell us about infectious proteins” by S. Krauss and I. Vorberg available at the following link: http://www.hindawi.com/journals/ijcb/2013/704546/, reviews the data on the cellular propagation of different protein aggregates, demonstrating that not all the typical characteristics of prions are shared by other misfolding proteins. In the review article “Identification of misfolded proteins in body fluids for the diagnosis of prion diseases” available at the following link: http://www.hindawi.com/journals/ijcb/2013/839329/, F. Properzi and M. Pocchiari give an up-to-date and comprehensive overview of the assays for detecting pathological forms of PrP in body fluids, highlighting the technological progress made in recent years. They stress the need to validate these diagnostic tools in blood samples and the importance of understanding prion metabolism in blood for effective diagnosis. The review article “Small-molecule theranostic probes: a promising future in neurodegenerative diseases” by S. Aulic et al. available at the following link: http://www.hindawi.com/journals/ijcb/2013/150952/, reviews the potential diagnostic and therapeutic activity of small molecules that bind and influence the aggregation of several misfolding proteins, and the review article “Gene-based antibody strategies for prion diseases” by A. Cardinale and S. Biocca available at the following link: http://www.hindawi.com/journals/ijcb/2013/710406/, presents an overview of the application of intracellular antibody technology (intrabodies) in prion diseases. They concisely review the concept of intrabodies and provide recent information on in vitro and in vivo studies. They stress the importance of targeting the actual neurotoxic species in prion diseases and improving the in vivo stability and efficacy of vectored antiprion antibody fragments. Despite major progress in our understanding of the pathogenesis of neurodegenerative diseases and the role of protein misfolding, effective treatments are still lacking. We hope that this special issue will help broaden the view of the problem and stimulate further research in the field. Alessio Cardinale Roberto Chiesa Michael Sierks