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Scholarly Works (50 results)

  • Thesis
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UC Irvine Electronic Theses and Dissertations (2018)

ABSTRACT OF THE DISSERTATION

Characterization of a pathogenic conformation of amyloid immunoreactivity in Alzheimer’s Disease and Cerebral Amyloid Angiopathy

By

Ricardo Albay III

Doctor of Philosophy in Biological Sciences

University of California, Irvine, 2018

Professor Charles G. Glabe, Chair

The deposition of the amyloid β protein (Aβ) in the brain as plaques and the formation of neurofibrillary tangles (NFT) filled with hyper-phosphorylated tau are the canonical histopathological hallmarks of Alzheimer’s disease (AD). The genetics of early onset, familial Alzheimer’s disease (fAD) supports a causal role of Aβ in AD pathogenesis. However, plaques do not correlate with dementia and therapeutic strategies inhibiting the production of soluble Aβ or leveraging monoclonal antibodies to target the removal of monomeric Aβ have produced disappointing results. Despite the therapeutic pitfalls that sequence specific antibodies have faced, conformation dependent monoclonal antibodies provide an invaluable tool to investigate specific conformations of protein aggregates whose relative abundance may prove significant to the pathogenesis of disease. By investigating the pathological significance of a unique subpopulation of vascular amyloid we addressed a knowledge gap that exists in understanding the role conformational diversity of amyloid structure plays in Alzheimer’s pathology. The data presented here demonstrate that monoclonal antibodies recognize deposits in human and transgenic mouse brain that are spatially, temporally, and morphologically diverse (Chapter 1). We report a unique immunoreactivity in vascular smooth muscle cells (VSMC) that correlates with plaque pathology and a loss of α-actin immunoreactivity in AD brains and transgenic mouse models of AD (Chapter 2). We investigated whether mOC31 immunoreactivity was associated with markers of inflammation, including the activation of microglia, presence of pro-inflammatory signals interleukin-1β (IL1β) and high-mobility group box 1 (HMGB1); we found that HMGB1 is colocalized with mOC31 immunoreactivity in the cytosol of VSMC (Chapter 2). To address the causative link between Aβ and mOC31 immunoreactivity with the observed loss of α-actin immunoreactivity we developed an in vitro model utilizing primary human brain vascular smooth muscle cells (HBVSMC). In this model, we found that exogenous treatment with Aβ40-WT and Aβ40-A21G was sufficient to induce the loss of α-actin immunoreactivity observed in human and transgenic mouse brain (Chapter 3). Furthermore, we report a loss of membrane integrity and a direct interaction between Aβ and α-actin. These results suggest that Aβ may have both indirect and direct effects on VSMC phenotype. Our findings demonstrate that amyloid heterogeneity is a significant factor to consider in the development of disease modifying therapeutics.

    Cover page: Characterization of a pathogenic conformation of amyloid immunoreactivity in Alzheimer’s Disease and Cerebral Amyloid Angiopathy
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2014)
    Amyloidogenic proteins generally form intermolecularly hydrogen-bonded β-sheet aggregates, including parallel, in-register β-sheets (recognized by antiserum OC) or antiparallel β-sheets, β-solenoids, β-barrels, and β-cylindrins (recognized by antiserum A11). Although these groups share many common properties, some amyloid sequences have been reported to form polymorphic structural variants or strains. We investigated the humoral immune response to Aβ42 fibrils and produced 23 OC-type monoclonal antibodies recognizing distinct epitopes differentially associated with polymorphic structural variants. These mOC antibodies define at least 18 different immunological profiles represented in aggregates of amyloid-β (Aβ). All of the antibodies strongly prefer amyloid aggregates over monomer, indicating that they recognize conformational epitopes. Most of the antibodies react with N-terminal linear segments of Aβ, although many recognize a discontinuous epitope consisting of an N-terminal domain and a central domain. Several of the antibodies that recognize linear Aβ segments also react with fibrils formed from unrelated amyloid sequences, indicating that reactivity with linear segments of Aβ does not mean the antibody is sequence-specific. The antibodies display strikingly different patterns of immunoreactivity in Alzheimer disease and transgenic mouse brain and identify spatially and temporally unique amyloid deposits. Our results indicate that the immune response to Aβ42 fibrils is diverse and reflects the structural polymorphisms in fibrillar amyloid structures. These polymorphisms may contribute to differences in toxicity and consequent effects on pathological processes. Thus, a single therapeutic monoclonal antibody may not be able to target all of the pathological aggregates necessary to make an impact on the overall disease process.
      Cover page: Monoclonal Antibodies against Aβ42 Fibrils Distinguish Multiple Aggregation State Polymorphisms in Vitro and in Alzheimer Disease Brain*
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2017)
      Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.
        Cover page: Familial Alzheimer’s Disease Mutations within the Amyloid Precursor Protein Alter the Aggregation and Conformation of the Amyloid-β Peptide* * This work was supported by National Institutes of Health Grants AG033069 and AG00538 and a grant from the Cure Alzheimer’s fund. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2022)

        Background

        Many identified mechanisms could be upstream of the prominent amyloid-β (Aβ) plaques in Alzheimer's disease (AD).

        Objective

        To profile the progression of pathology in AD.

        Methods

        We monitored metabolic signaling, redox stress, intraneuronal amyloid-β (iAβ) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan.

        Results

        Intracellular accumulation of aggregated Aβ in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAβ. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAβ and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAβ accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine.

        Conclusion

        The order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by a pAkt/tAkt metabolic shift in CA1, iAβ accumulation in CA1, and extracellular Aβ deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes.
          Cover page: Age-Related Oxidative Redox and Metabolic Changes Precede Intraneuronal Amyloid-β Accumulation and Plaque Deposition in a Transgenic Alzheimer’s Disease Mouse Model
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2014)
          Type 2 diabetes (T2D) is characterized by a deficiency in β cell mass, increased β cell apoptosis, and extracellular accumulation of islet amyloid derived from islet amyloid polypeptide (IAPP), which β cells coexpress with insulin. IAPP expression is increased in the context of insulin resistance, the major risk factor for developing T2D. Human IAPP is potentially toxic, especially as membrane-permeant oligomers, which have been observed to accumulate within β cells of patients with T2D and rodents expressing human IAPP. Here, we determined that β cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation. Induction of high levels of human IAPP in mouse β cells resulted in accumulation of this amyloidogenic protein as relatively inert fibrils within cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers. Mice hemizygous for transgenic expression of human IAPP did not develop diabetes; however, loss of β cell-specific autophagy in these animals induced diabetes, which was attributable to accumulation of toxic human IAPP oligomers and loss of β cell mass. In human IAPP-expressing mice that lack β cell autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased β cell apoptosis. These findings indicate that autophagy/lysosomal degradation defends β cells against proteotoxicity induced by oligomerization-prone human IAPP.
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2022)
            Strong evidence indicates that amyloid beta (Aβ) inflicts its toxicity in Alzheimers disease (AD) by promoting uncontrolled elevation of cytosolic Ca2+ in neurons. We have previously shown that synthetic Aβ42 oligomers stimulate abnormal intracellular Ca2+ release from the endoplasmic reticulum stores, suggesting that a similar mechanism of Ca2+ toxicity may be common to the endogenous Aβs oligomers. Here, we use human postmortem brain extracts from AD-affected patients and test their ability to trigger Ca2+ fluxes when injected intracellularly into Xenopus oocytes. Immunological characterization of the samples revealed the elevated content of soluble Aβ oligomers only in samples from AD patients. Intracellular injection of brain extracts from control patients failed to trigger detectable changes in intracellular Ca2+. Conversely, brain extracts from AD patients triggered Ca2+ events consisting of local and global Ca2+ fluorescent transients. Pre-incubation with either the conformation-specific OC antiserum or caffeine completely suppressed the brain extracts ability to trigger cytosolic Ca2+ events. Computational modeling suggests that these Ca2+ fluxes may impair cells bioenergetic by affecting ATP and ROS production. These results support the hypothesis that Aβ oligomers contained in neurons of AD-affected brains may represent the toxic agents responsible for neuronal malfunctioning and death associated with the disruption of Ca2+ homeostasis.
              Cover page: Intracellular Injection of Brain Extracts from Alzheimers Disease Patients Triggers Unregulated Ca2+ Release from Intracellular Stores That Hinders Cellular Bioenergetics.
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2020)
              This work provides new insight into the age-related basis of Alzheimer's disease (AD), the composition of intraneuronal amyloid (iAβ), and the mechanism of an age-related increase in iAβ in adult AD-model mouse neurons. A new end-specific antibody for Aβ45 and another for aggregated forms of Aβ provide new insight into the composition of iAβ and the mechanism of accumulation in old adult neurons from the 3xTg-AD model mouse. iAβ levels containing aggregates of Aβ45 increased 30-50-fold in neurons from young to old age and were further stimulated upon glutamate treatment. iAβ was 8 times more abundant in 3xTg-AD than non-transgenic neurons with imaged particle sizes following the same log-log distribution, suggesting a similar snow-ball mechanism of intracellular biogenesis. Pathologically misfolded and mislocalized Alz50 tau colocalized with iAβ and rapidly increased following a brief metabolic stress with glutamate. AβPP-CTF, Aβ45, and aggregated Aβ colocalized most strongly with mitochondria and endosomes and less with lysosomes and autophagosomes. Differences in iAβ by sex were minor. These results suggest that incomplete carboxyl-terminal trimming of long Aβs by gamma-secretase produced large intracellular deposits which limited completion of autophagy in aged neurons. Understanding the mechanism of age-related changes in iAβ processing may lead to application of countermeasures to prolong dementia-free health span.
                Cover page: Age-Related Intraneuronal Aggregation of Amyloid-β in Endosomes, Mitochondria, Autophagosomes, and Lysosomes
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2018)
                The monoclonal antibodies 6E10 and 4G8 are among the first anti-amyloid monoclonal antibodies against Aβ and the most widely used antibodies in Alzheimers disease research. Although the epitopes for 6E10 and 4G8 have been reported to correspond to residues 1-16 and 17-24, a more recent high-resolution mapping approach indicates that 6E10 maps to residues 4-10 while 4G8 maps to residues 18-23. To characterize the binding specificity of both antibodies in greater detail, we used immunoselection of random sequences from phage display library followed by deep sequencing and analysis of resulting patterns from thousands of immunoselected sequences. We found that the minimum sequence required for 6E10 binding is R-x-D with over half (53%) of the immunoselected sequences conforming to this pattern. The vast majority of these sequences contain an H at position x (R-H-D), corresponding to residues 5-7 of the Aβ target sequences, but Y is also permitted at this position in a minority of sequences. For 4G8 we found that the most frequent pattern is F-x-A contained in approximately 30% of the sequences, followed by F-A, L-x(3)-A, L-x-F, and F-F each accounting for approximately 18% of the sequences. The F-x-A motif also occurs in islet amyloid poly peptide which may explain why 4G8 also recognizes amyloid fibrils of this peptide. Immunoselection of random sequences and deep sequencing may also be a facile and efficient means of determining residues critical for antibody binding and validating the specificity of monoclonal antibodies and polyclonal antisera.
                  Cover page: Epitomic Characterization of the Specificity of the Anti-Amyloid Aβ Monoclonal Antibodies 6E10 and 4G8.
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2012)

                  Background/aims

                  Oligomerization of amyloid beta (Aβ) is a hypothesized step in the formation of plaques in Alzheimer's disease (AD) but has been difficult to demonstrate in vivo in humans. As persons destined to develop familial AD (FAD) due to fully penetrant autosomal dominant mutations are essentially certain to develop the disease, they provide the opportunity to identify oligomers during the presymptomatic stage of the disease.

                  Methods

                  We measured levels of Aβ(42) using a conventional immunoassay and prefibrillar, fibrillar, and annular protofibrillar oligomers using polyclonal conformation-dependent antibodies in the cerebrospinal fluid (CSF) of 7 persons at risk for inheriting FAD mutations. Levels of oligomers were compared between FAD mutation carriers and noncarriers.

                  Results

                  Compared to 2 noncarriers, annular protofibrillar oligomers were elevated, prefibrillar and fibrillar oligomers trended towards elevation and Aβ(42) monomer trended towards being decreased in 5 FAD mutation carriers.

                  Conclusion

                  Our data provide evidence for an identifiable elevation of CSF oligomers during the presymptomatic phase of FAD.
                    Cover page: Conformation-Dependent Oligomers in Cerebrospinal Fluid of Presymptomatic Familial Alzheimer’s Disease Mutation Carriers
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2006)
                    The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area compressibility of the lipid has a very large effect. Membranes with a large area compressibility modulus are almost insensitive to amyloid oligomers, but membranes formed from soft, highly compressible lipids are highly susceptible to amyloid oligomer-induced conductance changes. Furthermore, membranes formed using the solvent decane (instead of squalane) are completely insensitive to the presence of oligomers. One simple explanation for these effects on bilayer conductance is that amyloid oligomers increase the area per molecule of the membrane-forming lipids, thus thinning the membrane, lowering the dielectric barrier, and increasing the conductance of any mechanism sensitive to the dielectric barrier.
                      Cover page: Soluble Amyloid Oligomers Increase Bilayer Conductance by Altering Dielectric Structure
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