Brian O'Nuallain

Conformational Abs recognizing a generic amyloid fibril epitope

Disease-related amyloid fibrils appear to share a common, but poorly understood, structure. We describe here the generation and preliminary characterization of two conformation-specific mAbs, WO1 and WO2, that bind to the amyloid fibril state of the Alzheimers peptide Aβ(1–40) but not to its soluble, monomeric state. Surprisingly, these Abs also bind to other disease-related amyloid fibrils and amyloid-like aggregates derived from other proteins of unrelated sequence, such as transthyretin, islet amyloid polypeptide, β2-microglobulin, and polyglutamine. At the same time, WO1 and WO2 do not bind to the native protein precursors of these amyloids, nor do they bind to other kinds of protein aggregates. This new class of Abs associated with a fundamental amyloid-folding motif appear to recognize a common conformational epitope with little apparent dependence on amino acid side chain information. These Abs should contribute to the understanding of amyloid structure, assembly, and toxicity and also may benefit the development of diagnostic and therapeutic agents for amyloid diseases.

Amyloid β-Protein Dimers Rapidly Form Stable Synaptotoxic Protofibrils

Nonfibrillar, water-soluble low-molecular weight assemblies of the amyloid β-protein (Aβ) are believed to play an important role in Alzheimers disease (AD). Aqueous extracts of human brain contain Aβ assemblies that migrate on SDS-polyacrylamide gels and elute from size exclusion as dimers (∼8 kDa) and can block long-term potentiation and impair memory consolidation in the rat. Such species are detected specifically and sensitively in extracts of Alzheimer brain suggesting that SDS-stable dimers may be the basic building blocks of AD-associated synaptotoxic assemblies. Consequently, understanding the structure and properties of Aβ dimers is of great interest. In the absence of sufficient brain-derived dimer to facilitate biophysical analysis, we generated synthetic dimers designed to mimic the natural species. For this, Aβ(1-40) containing cysteine in place of serine 26 was used to produce disulphide cross-linked dimer, (AβS26C)2. Such dimers had no detectable secondary structure, produced an analytical ultracentrifugation profile consistent for an ∼8.6 kDa protein, and had no effect on hippocampal long-term potentiation (LTP). However, (AβS26C)2 aggregated more rapidly than either AβS26C or wild-type monomers and formed parastable β-sheet rich, thioflavin T-positive, protofibril-like assemblies. Whereas wild-type Aβ aggregated to form typical amyloid fibrils, the protofibril-like structures formed by (AβS26C)2 persisted for prolonged periods and potently inhibited LTP in mouse hippocampus. These data support the idea that Aβ dimers may stabilize the formation of fibril intermediates by a process distinct from that available to Aβ monomer and that higher molecular weight prefibrillar assemblies are the proximate mediators of Aβ toxicity.

Kinetics and thermodynamics of amyloid assembly using a high-performance liquid chromatography-based sedimentation assay

Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered structures that appear to assemble according to ordered mechanisms. Some of these fibrils, as illustrated here with the Alzheimers plaque peptide amyloid beta, assemble to an endpoint that is a dynamic equilibrium between monomers and fibrils exhibiting a characteristic equilibrium constant with an associated free energy of formation. Some fibrils, as illustrated here with the polyglutamine repeat sequences associated with Huntingtons disease, assemble via highly regular mechanisms exhibiting nucleated growth polymerization kinetics. Here, we describe a series of linked methods for quantitative analysis of such aggregation kinetics and thermodynamics, focusing on a robust high-performance liquid chromatography (HPLC)-based sedimentation assay. An integrated group of protocols is provided for peptide disaggregation, setting up the HPLC sedimentation assay, the preparation of fibril seed stocks and determination of the average functional molecular weight of the fibrils, elongation and nucleation kinetics analysis, and the determination of the critical concentration describing the thermodynamic endpoint of fibril elongation.

Diagnostic and therapeutic potential of amyloid-reactive IgG antibodies contained in human sera

Passive immunotherapy using fibril-reactive mAbs has been shown experimentally to reduce amyloid formation and also accelerate amyloidolysis. We now report that human sera, as well as various sources of pooled human IgG, including pharmacologic formulations of immune globulin i.v. (IGIV), contain Abs that specifically recognize fibrils formed from light chains and other amyloidogenic precursor proteins, including serum amyloid A, transthyretin, islet amyloid polypeptide, and amyloid β 1–40 peptide, but notably, do not react with these molecules in their native nonfibrillar forms. After isolation of the Abs from IGIV via fibril-conjugated affinity column chromatography, the EC50-binding value for light chains and amyloid β 1–40 peptide fibrils was ∼15 nM–a magnitude ∼200 and 70 times less than that of the unbound fraction and unfractionated product, respectively. Comparable reactivity was found in the case of those formed from serum amyloid A, transthyretin, and islet amyloid polypeptide. The purified Abs immunostained human amyloid tissue deposits and could inhibit fibrillogenesis, as shown in fibril formation and extension assays. Most importantly, in vivo reactivity was evidenced in a murine model when the enriched Abs were used to image amyloid, as well as expedite its removal. These promising experimental results suggest that fibril affinity-purified IGIV has potential as a diagnostic and therapeutic agent for patients with amyloid-associated disease.

Quantitative high-resolution microradiographic imaging of amyloid deposits in a novel murine model of AA amyloidosis

The mouse model of experimentally induced systemic AA amyloidosis is long established, well validated, and closely analogous to the human form of this disease. However, the induction of amyloid by experimental inflammation is unpredictable, inconsistent, and difficult to modulate. We have previously shown that murine AA amyloid deposits can be imaged using iodine-123 labeled SAP scintigraphy and report here substantial refinements in both the imaging technology and the mouse model itself. In this regard, we have generated a novel prototype of AA amyloid in which mice expressing the human interleukin 6 gene, when given amyloid enhancing factor, develop extensive and progressive systemic AA deposition without an inflammatory stimulus, i.e., a transgenic rapidly inducible amyloid disease (TRIAD) mouse. Additionally, we have constructed high-resolution micro single photon emission computed tomography (SPECT)/computed tomography (CT) instrumentation that provides images revealing the precise anatomic location of amyloid deposits labeled by radioiodinated serum amyloid P component (SAP). Based on reconstructed microSPECT/CT images, as well as autoradiographic, isotope biodistribution, and quantitative histochemical analyses, the 125I-labeled SAP tracer bound specifically to hepatic and splenic amyloid in the TRIAD animals. The ability to discern radiographically the extent of amyloid burden in the TRIAD model provides a unique opportunity to evaluate the therapeutic efficacy of pharmacologic compounds designed to inhibit fibril formation or effect amyloid resolution.

Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid

Background: Some antibodies express serine protease activity. Transthyretin misfolding causes accumulation of pathogenic amyloid. Results: Constitutively produced IgM but not IgG class antibodies selectively hydrolyzed and dissolved misfolded transthyretin without hydrolyzing physiologically folded transthyretin. Some IgMs were oligoreactive with amyloids and superantigens. Conclusion: Catalytic IgMs may clear misfolded TTR and delay amyloidosis. Significance: The innate antibody repertoire is a source of selective catabodies to toxic proteins. Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function.

Tau immunization: a cautionary tale?

The amyloid β (Aβ)-protein and microtubule-associated protein, tau, are the major components of the amyloid plaques and neurofibrillary tangles that typify Alzheimers disease (AD) pathology. As such both Aβ and tau have long been proposed as therapeutic targets. Immunotherapy, particularly targeting Aβ, is currently the most advanced clinical strategy for treating AD. However, several Aβ-directed clinical trials have failed, and there is concern that targeting this protein may not be useful. In contrast, there is a growing optimism that tau immunotherapy may prove more efficacious. Here, for the first time, we studied the effects of chronic administration of an anti-tau monoclonal antibody (5E2) in amyloid precursor protein transgenic mice. For our animal model, we chose the J20 mouse line because prior studies had shown that the cognitive deficits in these mice require expression of tau. Despite the fact that 5E2 was present and active in the brains of immunized mice and that this antibody appeared to engage with extracellular tau, 5E2-treatment did not recover age-dependent spatial reference memory deficits. These results indicate that the memory impairment evident in J20 mice is unlikely to be mediated by a form of extracellular tau recognized by 5E2. In addition to the lack of positive effect of anti-tau immunotherapy, we also documented a significant increase in mortality among J20 mice that received 5E2. Because both the J20 mice used here and tau transgenic mice used in prior tau immunotherapy trials are imperfect models of AD our results recommend extensive preclinical testing of anti-tau antibody-based therapies using multiple mouse models and a variety of different anti-tau antibodies.

A human monoclonal IgG that binds aβ assemblies and diverse amyloids exhibits anti-amyloid activities in vitro and in vivo.

Alzheimers disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potential in vitro and in vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formation in vitro and abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβ in vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformers binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAbs activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformers binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAbs activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

ANTI-N-TERMINAL ABETA MAB 3A1 PREFERENTIALLY RECOGNIZES ABETA AGGREGATES AND DOES NOT CROSS-REACT WITH APP

from 4 different groups of mice depending on the TBI paradigm and time point after TBI at which the brains were collected: 1) sacrificed at 24 hours after a 50 psi single blast treatment, 2) sacrificed at 3 weeks after a 50 psi single blast treatment, 3) sacrificed at 3 weeks after repeated blast treatment (6 blasts over two weeks), 4) sacrificed at 3 weeks following anesthesia (sham group). The isolated tau oligomers were analyzed biochemically via PK digestion, western blotting and seeding. Moreover, their toxicity, internalization and spreading were tested in cell culture. Results:Results show that the TBI brain-derived Tau samples resemble tau oligomeric strains in terms of PK digestion pattern and reactivity with tau oligomer monoclonal antibodies. In addition, some of the samples were found to be toxic, and express different internalization levels into SY5Y cells in culture. Conclusions: Results suggest that different TBI brain-derived Tau oligomeric-like strains contribute to the neuronal death observed after TBI. Future experiments will investigate the samples’ differential ability to induce cell toxicity and neurodegeneration in vitro and in vivo. Results hold substantial translational implications towards applying Tau-immunotherapy techniques to reduce the risk for late-life dementia.