Extracellular high molecular weight α-synuclein oligomers induce cell death by disrupting the plasma membrane.

α-Synuclein (αS), the causative protein of Parkinson's disease and other α-synucleinopathies, aggregates from a low molecular weight form (LMW-αS) to a high molecular weight αS oligomer (HMW-αSo). Aggregated αS accumulates intracellularly, induces intrinsic apoptosis, is released extracellularly, and appears to propagate disease through prion-like spreading. Whether extracellular αS aggregates are cytotoxic, damage cell wall, or induce cell death is unclear. We investigated cytotoxicity and cell death caused by HMW-αSo or LMW-αS. Extracellular HMW-αSo was more cytotoxic than LMW-αS and was a crucial factor for inducing plasma membrane damage and cell death. HMW-αSo induced reactive oxygen species production and phospholipid peroxidation in the membrane, thereby impairing calcium homeostasis and disrupting plasma membrane integrity. HMW-αSo also induced extrinsic apoptosis and cell death by activating acidic sphingomyelinase. Thus, as extracellular HMW-αSo causes neuronal injury and death via cellular transmission and direct plasma membrane damage, we propose an additional disease progression pathway for α-synucleinopathies.

The effects of enhancing angiotensin converting enzyme in myelomonocytes on ameliorating Alzheimer's-related disease and preserving cognition.

This review examines the role of angiotensin-converting enzyme (ACE) in the context of Alzheimer's disease (AD) and its potential therapeutic value. ACE is known to degrade the neurotoxic 42-residue long alloform of amyloid ß-protein (Aß42), a peptide strongly associated with AD. Previous studies in mice, demonstrated that targeted overexpression of ACE in CD115+ myelomonocytic cells (ACE10 models) improved their immune responses to effectively reduce viral and bacterial infection, tumor growth, and atherosclerotic plaque. We further demonstrated that introducing ACE10 myelomonocytes (microglia and peripheral monocytes) into the double transgenic APPSWE/PS1ΔE9 murine model of AD (AD+ mice), diminished neuropathology and enhanced the cognitive functions. These beneficial effects were dependent on ACE catalytic activity and vanished when ACE was pharmacologically blocked. Moreover, we revealed that the therapeutic effects in AD+ mice can be achieved by enhancing ACE expression in bone marrow (BM)-derived CD115+ monocytes alone, without targeting central nervous system (CNS) resident microglia. Following blood enrichment with CD115+ ACE10-monocytes versus wild-type (WT) monocytes, AD+ mice had reduced cerebral vascular and parenchymal Aß burden, limited microgliosis and astrogliosis, as well as improved synaptic and cognitive preservation. CD115+ ACE10-versus WT-monocyte-derived macrophages (Mo/MΦ) were recruited in higher numbers to the brains of AD+ mice, homing to Aß plaque lesions and exhibiting a highly Aß-phagocytic and anti-inflammatory phenotype (reduced TNFα/iNOS and increased MMP-9/IGF-1). Moreover, BM-derived ACE10-Mo/MΦ cultures had enhanced capability to phagocytose Aß42 fibrils, prion-rod-like, and soluble oligomeric forms that was associated with elongated cell morphology and expression of surface scavenger receptors (i.e., CD36, Scara-1). This review explores the emerging evidence behind the role of ACE in AD, the neuroprotective properties of monocytes overexpressing ACE and the therapeutic potential for exploiting this natural mechanism for ameliorating AD pathogenesis.

Structural Dynamics of Amyloid-ß Protofibrils and Actions of Anti-Amyloid-ß Antibodies as Observed by High-Speed Atomic Force Microscopy.

Amyloid-ß (Aß) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aß42 PF at the single-molecule level and the effect of lecanemab, an anti-Aß PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aß aggregation process.

The Feasibility of Early Alzheimer's Disease Diagnosis Using a Neural Network Hybrid Platform.

Early diagnosis of Alzheimer's Disease (AD) is critical for disease prevention and cure. However, currently, techniques with the required high sensitivity and specificity are lacking. Recently, with the advances and increased accessibility of data analysis tools, such as machine learning, research efforts have increasingly focused on using these computational methods to solve this challenge. Here, we demonstrate a convolutional neural network (CNN)-based AD diagnosis approach using the surface-enhanced Raman spectroscopy (SERS) fingerprints of human cerebrospinal fluid (CSF). SERS and CNN were combined for biomarker detection to analyze disease-associated biochemical changes in the CSF. We achieved very high reproducibility in double-blind experiments for testing the feasibility of our system on human samples. We achieved an overall accuracy of 92% (100% for normal individuals and 88.9% for AD individuals) based on the clinical diagnosis. Further, we observed an excellent correlation coefficient between our test score and the Clinical Dementia Rating (CDR) score. Our findings offer a substantial indication of the feasibility of detecting AD biomarkers using the innovative combination of SERS and machine learning. We are hoping that this will serve as an incentive for future research in the field.

From reaction kinetics to dementia: A simple dimer model of Alzheimer's disease etiology.

Oligomers of the amyloid ß-protein (Aß) have been implicated in the pathogenesis of Alzheimer's disease (AD) through their toxicity towards neurons. Understanding the process of oligomerization may contribute to the development of therapeutic agents, but this has been difficult due to the complexity of oligomerization and the metastability of the oligomers thus formed. To understand the kinetics of oligomer formation, and how that relates to the progression of AD, we developed models of the oligomerization process. Here, we use experimental data from cell viability assays and proxies for rate constants involved in monomer-dimer-trimer kinetics to develop a simple mathematical model linking Aß assembly to oligomer-induced neuronal degeneration. This model recapitulates the rapid growth of disease incidence with age. It does so through incorporation of age-dependent changes in rates of Aß monomer production and elimination. The model also describes clinical progression in genetic forms of AD (e.g., Down's syndrome), changes in hippocampal volume, AD risk after traumatic brain injury, and spatial spreading of the disease due to foci in which Aß production is elevated. Continued incorporation of clinical and basic science data into the current model will make it an increasingly relevant model system for doing theoretical calculations that are not feasible in biological systems. In addition, terms in the model that have particularly large effects are likely to be especially useful therapeutic targets.

Myricetin prevents high molecular weight Aß1-42 oligomer-induced neurotoxicity through antioxidant effects in cell membranes and mitochondria.

Excessive accumulation of amyloid ß-protein (Aß) is one of the primary mechanisms that leads to neuronal death with phosphorylated tau in the pathogenesis of Alzheimer's disease (AD). Protofibrils, one of the high-molecular-weight Aß oligomers (HMW-Aßo), are implicated to be important targets of disease modifying therapy of AD. We previously reported that phenolic compounds such as myricetin inhibit Aß1-40, Aß1-42, and α-synuclein aggregations, including their oligomerizations, which may exert protective effects against AD and Parkinson's disease. The purpose of this study was to clarify the detailed mechanism of the protective effect of myricetin against the neurotoxicity of HMW-Aßo in SH-SY5Y cells. To assess the effect of myricetin on HMW-Aßo-induced oxidative stress, we systematically examined the level of membrane oxidative damage by measuring cell membrane lipid peroxidation, membrane fluidity, and cell membrane potential, and the mitochondrial oxidative damage was evaluated by mitochondrial permeability transition (MPT), mitochondrial reactive oxygen species (ROS), and manganese-superoxide dismutase (Mn-SOD), and adenosine triphosphate (ATP) assay in SH-SY5Y cells. Myricetin has been found to increased cell viability by suppression of HMW-Aßo-induced membrane disruption in SH-SY5Y cells, as shown in reducing membrane phospholipid peroxidation and increasing membrane fluidity and membrane resistance. Myricetin has also been found to suppress HMW-Aßo-induced mitochondria dysfunction, as demonstrated in decreasing MPT, Mn-SOD, and ATP generation, raising mitochondrial membrane potential, and increasing mitochondrial-ROS generation. These results suggest that myricetin preventing HMW-Aßo-induced neurotoxicity through multiple antioxidant functions may be developed as a disease-modifying agent against AD.

Effects of IL-34 on Macrophage Immunological Profile in Response to Alzheimer's-Related Aß42 Assemblies.

Interleukin-34 (IL-34) is a recently discovered cytokine that acts as a second ligand of the colony stimulating factor 1 receptor (CSF1R) in addition to macrophage colony-stimulating factor (M-CSF). Similar to M-CSF, IL-34 also stimulates bone marrow (BM)-derived monocyte survival and differentiation into macrophages. Growing evidence suggests that peripheral BM-derived monocyte/macrophages (BMMO) play a key role in the physiological clearance of cerebral amyloid ß-protein (Aß). Aß42 forms are especially neurotoxic and highly associated with Alzheimer's disease (AD). As a ligand of CSF1R, IL-34 may be relevant to innate immune responses in AD. To investigate how IL-34 affects macrophage phenotype in response to structurally defined and stabilized Aß42 oligomers and preformed fibrils, we characterized murine BMMO cultured in media containing M-CSF, IL-34, or regimens involving both cytokines. We found that the immunological profile and activation phenotype of IL-34-stimulated BMMO differed significantly from those cultured with M-CSF alone. Specifically, macrophage uptake of fibrillar or oligomeric Aß42 was markedly reduced following exposure to IL-34 compared to M-CSF. Surface expression of type B scavenger receptor CD36, known to facilitate Aß recognition and uptake, was modified following treatment with IL-34. Similarly, IL-34 macrophages expressed lower levels of proteins involved in both Aß uptake (triggering receptor expressed on myeloid cells 2, TREM2) as well as Aß-degradation (matrix metallopeptidase 9, MMP-9). Interestingly, intracellular compartmentalization of Aß visualized by staining of early endosome antigen 1 (EEA1) was not affected by IL-34. Macrophage characteristics associated with an anti-inflammatory and pro-wound healing phenotype, including processes length and morphology, were also quantified, and macrophages stimulated with IL-34 alone displayed less process elongation in response to Aß42 compared to those cultured with M-CSF. Further, monocytes treated with IL-34 alone yielded fewer mature macrophages than those treated with M-CSF alone or in combination with IL-34. Our data indicate that IL-34 impairs monocyte differentiation into macrophages and reduces their ability to uptake pathological forms of Aß. Given the critical role of macrophage-mediated Aß clearance in both murine models and patients with AD, future work should investigate the therapeutic potential of modulating IL-34 in vivo to increase macrophage-mediated Aß clearance and prevent disease development.

Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and Structure Observed by High-Speed Atomic Force Microscopy.

Fibril formation is an obligatory process in amyloid diseases and is characterized by nucleation and elongation phases that result in the formation of long filaments with cross-ß sheet structure. The kinetics of this process, as well as that of secondary nucleation, is controlled by a variety of factors, including nucleus (seed) structure, monomer conformation, and biochemical milieu. Some fibrillar amyloid assemblies act as prions, replicating themselves from protein monomers templated by existing prion seeds. Prion strains, which are characterized by distinct physicochemical and pathologic properties, may also form due to perturbation of the templating process within the susceptible organism. Understanding the types and effects of perturbations occurring during the development and progression of Parkinson's disease is an area requiring more study. Here, we used high-speed atomic force microscopy to determine the kinetics and structural dynamics of α-synuclein fibril elongation initiated by self-seeding or cross-seeding of wild-type (WT) or mutant α-synuclein with WT or mutant α-synuclein seeds. We found that cross-seeding modulated not only elongation rates but also the structures of the growing fibrils. Some fibrils produced in this manner had structures distinct from their "parent" seeds. In other cases, cross-seeding was not observed at all. These findings suggest that α-synuclein sequence variants can produce different types of strains by self- or cross-seeding. Perpetuation of specific strains then would depend on the relative rates of fibril growth and the relative stabilities of the fibrils formed by each strain.

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aß42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid ß-protein (Aß42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aß42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aß-phagocytic macrophages. We found that cortical neurons were more susceptible to Aß42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aß42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aß42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aß42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD.

Ultrasensitive amyloid ß-protein quantification with high dynamic range using a hybrid graphene-gold surface-enhanced Raman spectroscopy platform.

Surface enhanced Raman spectroscopy (SERS) holds great promise in biosensing because of its single-molecule, label-free sensitivity. We describe here the use of a graphene-gold hybrid plasmonic platform that enables quantitative SERS measurement. Quantification is enabled by normalizing analyte peak intensities to that of the graphene G peak. We show that two complementary quantification modes are intrinsic features of the platform, and that through their combined use, the platform enables accurate determination of analyte concentration over a concentration range spanning seven orders of magnitude. We demonstrate, using a biologically relevant test analyte, the amyloid ß-protein (Aß), a seminal pathologic agent of Alzheimer's disease (AD), that linear relationships exist between (a) peak intensity and concentration at a single plasmonic hot spot smaller than 100 nm, and (b) frequency of hot spots with observable protein signals, i.e. the co-location of an Aß protein and a hot spot. We demonstrate the detection of Aß at a concentration as low as 10-18 M after a single 20 µl aliquot of the analyte onto the hybrid platform. This detection sensitivity can be improved further through multiple applications of analyte to the platform and by rastering the laser beam with smaller step sizes.

Peripherally derived angiotensin converting enzyme-enhanced macrophages alleviate Alzheimer-related disease.

Targeted overexpression of angiotensin-converting enzyme (ACE), an amyloid-ß protein degrading enzyme, to brain resident microglia and peripheral myelomonocytes (ACE10 model) substantially diminished Alzheimer's-like disease in double-transgenic APPSWE/PS1ΔE9 (AD+) mice. In this study, we explored the impact of selective and transient angiotensin-converting enzyme overexpression on macrophage behaviour and the relative contribution of bone marrow-derived ACE10 macrophages, but not microglia, in attenuating disease progression. To this end, two in vivo approaches were applied in AD+ mice: (i) ACE10/GFP+ bone marrow transplantation with head shielding; and (ii) adoptive transfer of CD115+-ACE10/GFP+ monocytes to the peripheral blood. Extensive in vitro studies were further undertaken to establish the unique ACE10-macrophage phenotype(s) in response to amyloid-ß1-42 fibrils and oligomers. The combined in vivo approaches showed that increased cerebral infiltration of ACE10 as compared to wild-type monocytes (∼3-fold increase; P < 0.05) led to reductions in cerebral soluble amyloid-ß1-42, vascular and parenchymal amyloid-ß deposits, and astrocytosis (31%, 47-80%, and 33%, respectively; P < 0.05-0.0001). ACE10 macrophages surrounded brain and retinal amyloid-ß plaques and expressed 3.2-fold higher insulin-like growth factor-1 (P < 0.01) and ∼60% lower tumour necrosis factor-α (P < 0.05). Importantly, blood enrichment with CD115+-ACE10 monocytes in symptomatic AD+ mice resulted in pronounced synaptic and cognitive preservation (P < 0.05-0.001). In vitro analysis of macrophage response to well-defined amyloid-ß1-42 conformers (fibrils, prion rod-like structures, and stabilized soluble oligomers) revealed extensive resistance to amyloid-ß1-42 species by ACE10 macrophages. They exhibited 2-5-fold increased surface binding to amyloid-ß conformers as well as substantially more effective amyloid-ß1-42 uptake, at least 8-fold higher than those of wild-type macrophages (P < 0.0001), which were associated with enhanced expression of surface scavenger receptors (i.e. CD36, scavenger receptor class A member 1, triggering receptor expressed on myeloid cells 2, CD163; P < 0.05-0.0001), endosomal processing (P < 0.05-0.0001), and ∼80% increased extracellular degradation of amyloid-ß1-42 (P < 0.001). Beneficial ACE10 phenotype was reversed by the angiotensin-converting enzyme inhibitor (lisinopril) and thus was dependent on angiotensin-converting enzyme catalytic activity. Further, ACE10 macrophages presented distinct anti-inflammatory (low inducible nitric oxide synthase and lower tumour necrosis factor-α), pro-healing immune profiles (high insulin-like growth factor-1, elongated cell morphology), even following exposure to Alzheimer's-related amyloid-ß1-42 oligomers. Overall, we provide the first evidence for therapeutic roles of angiotensin-converting enzyme-overexpressing macrophages in preserving synapses and cognition, attenuating neuropathology and neuroinflammation, and enhancing resistance to defined pathognomonic amyloid-ß forms.

High molecular weight amyloid ß1-42 oligomers induce neurotoxicity via plasma membrane damage.

Amyloid ß-protein (Aß) molecules tend to aggregate and subsequently form low MW (LMW) oligomers, high MW (HMW) aggregates such as protofibrils, and ultimately fibrils. These Aß species can generally form amyloid plaques implicated in the neurodegeneration of Alzheimer disease (AD), but therapies designed to reduce plaque load have not demonstrated clinical efficacy. Recent evidence implicates amyloid oligomers in AD neuropathology, but the precise mechanisms are uncertain. We examined the mechanisms of neuronal dysfunction from HMW-Aß1-42 exposure by measuring membrane integrity, reactive oxygen species (ROS) generation, membrane lipid peroxidation, membrane fluidity, intracellular calcium regulation, passive membrane electrophysiological properties, and long-term potentiation (LTP). HMW-Aß1-42 disturbed membrane integrity by inducing ROS generation and lipid peroxidation, resulting in decreased membrane fluidity, intracellular calcium dysregulation, depolarization, and impaired LTP. The damaging effects of HMW-Aß1-42 were significantly greater than those of LMW-Aß1-42. Therapeutic reduction of HMW-Aß1-42 may prevent AD progression by ameliorating direct neuronal membrane damage.-Yasumoto, T., Takamura, Y., Tsuji, M., Watanabe-Nakayama, T., Imamura, K., Inoue, H., Nakamura, S., Inoue, T., Kimura, A., Yano, S., Nishijo, H., Kiuchi, Y., Teplow, D. B., Ono, K. High molecular weight amyloid ß1-42 oligomers induce neurotoxicity via plasma membrane damage.

Preparation of Pure Populations of Amyloid ß-Protein Oligomers of Defined Size.

Protein and peptide oligomers are thought to play important roles in the pathogenesis of a number of neurodegenerative diseases. For this reason, considerable effort has been devoted to understanding the oligomerization process and to determining structure-activity relationships among the many types of oligomers that have been described. We discuss here a method for producing pure populations of amyloid ß-protein (Aß) of specific sizes using the most pathologic form of the peptide, Aß42. This work was necessitated because Aß oligomerization produces oligomers of many different sizes that are non-covalently associated, which means that dissociation or further assembly may occur. These characteristics preclude rigorous structure-activity determinations. In studies of Aß40, we have used the method of photo-induced cross-linking of unmodified proteins (PICUP) to produce zero-length carbon-carbon bonds among the monomers comprising each oligomer, thus stabilizing the oligomers. We then isolated pure populations of oligomers by fractionating the oligomers by size using SDS-PAGE and then extracting each population from the stained gel bands. Although this procedure worked well with the shorter Aß40 peptide, we found that a significant percentage of Aß42 oligomers had not been stabilized. Here, we discuss a new method capable of yielding stable Aß42 oligomers of sizes from dimer through dodecamer.

Surface enhanced Raman spectroscopy distinguishes amyloid Β-protein isoforms and conformational states.

Amyloid ß-protein (Aß) self-association is one process linked to the development of Alzheimer's disease (AD). Aß peptides, including its most abundant forms, Aß40 and Aß42, are associated with the two predominant neuropathologic findings in AD, vascular and parenchymal amyloidosis, respectively. Efforts to develop therapies for AD often have focused on understanding and controlling the assembly of these two peptides. An obligate step in these efforts is the monitoring of assembly state. We show here that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) readily distinguishes Aß40 and Aß42. We show further, through comparison of assembly dependent changes in secondary structure and morphology, that the SERS/PCA approach unambiguously differentiates closely related assembly stages not readily differentiable by circular dichroism spectroscopy, electron microscopy, or other techniques. The high discriminating power of SERS/PCA is based on the rich structural information present in its spectra, which comprises not only on interatomic resonances between covalently associated atoms and hydrogen bond interactions important in controlling secondary structure, but effects of protein orientation relative to the substrate surface. Coupled with the label-free, single molecule sensitivity of SERS, the approach should prove useful for determining structure activity relationships, suggesting target sites for drug development, and for testing the effects of such drugs on the assembly process. The approach also could be of value in other systems in which assembly dependent changes in protein structure correlate with the formation of toxic peptide assemblies.