Toxic Oligomers Research Articles

Ultrastructural evidence for self-replication of Alzheimer-associated Aβ42 amyloid along the sides of fibrils

The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aβ42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aβ42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).

Starting at the beginning: endoplasmic reticulum proteostasis and systemic amyloid disease.

Systemic amyloid diseases are characterized by the deposition of an amyloidogenic protein as toxic oligomers and amyloid fibrils on tissues distal from the site of protein synthesis. Traditionally, these diseases have been viewed as disorders of peripheral target tissues where aggregates are deposited, and toxicity is observed. However, recent evidence highlights an important role for endoplasmic reticulum (ER) proteostasis pathways within tissues synthesizing and secreting amyloidogenic proteins, such as the liver, in the pathogenesis of these disorders. Here, we describe the pathologic implications of ER proteostasis and its regulation on the toxic extracellular aggregation of amyloidogenic proteins implicated in systemic amyloid disease pathogenesis. Furthermore, we discuss the therapeutic potential for targeting ER proteostasis to reduce the secretion and toxic aggregation of amyloidogenic proteins to mitigate peripheral amyloid-associated toxicity involved in the onset and progression of systemic amyloid diseases.

Structural Characterization of Individual α-Synuclein Oligomers Formed at Different Stages of Protein Aggregation by Atomic Force Microscopy-Infrared Spectroscopy.

Aberrant α-synuclein aggregation is strongly associated with the onset and development of Parkinson's disease (PD). Therefore, characterizing the structure of toxic intermediate oligomers plays an essential role in better understanding their neurotoxicity. Using atomic force microscopy-infrared spectroscopy (AFM-IR), we were able to reveal the structure of α-synuclein oligomers present at different stages of protein aggregation and establish a relationship between morphology and structure on the single oligomer level. We were also able to probe the secondary structure evolution of individual oligomers. Moreover, the IR spectra of individual oligomers suggest structural rearrangement that is necessary for oligomers with an antiparallel β-sheet to propagate into fibrils that have a parallel-β-sheet secondary structure. Detailed investigation of structural organization of α-synuclein oligomers reported in this study is critically important to understand the toxicity of these protein species. We also anticipate that this work will help developing approaches for oligomer detection and consequently presymptomatic diagnostic of PD.

Active Immunotherapy with 12-mer Aβ1-42-like Assembly Vaccine Shows Efficacy in Aged 3×Tg-AD Mice.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder characterized by senile plaques and neurofibrillary tangles (NFTs). The amyloid-oligomer hypothesis indicates that the buildup of toxic oligomers in vivo is likely to impair memory and synaptic function. In our study, a kind of novel recombinant chimeric 12×(Aβ1-15-Th) antigen was developed as 12-mer Aβ1-42-like assembly vaccine. We designed this 12×(Aβ1-15- Th) antigen to mimic the assembly states of Aβ1-42 using twelvefold Aβ1-15 (B cell epitopes of human Aβ1-42) and foreign human T helper (Th) epitopes (as the T cell epitopes of Aβ1-42) constructs. Its immunogenicity as a subunit vaccine was tested on C57/BL6 mice, and the efficacy was shown by applying it to AD mice. This 12×(Aβ1-15-Th) vaccine induced robust Aβ-specific antibodies in 3×Tg- AD and C57/BL6 mice. As early immunotherapeutic agent of AD, the 12×(Aβ1-15-Th) vaccine significantly improved the behavior performance of aged 3 × Tg-AD mice, and reduced the levels of soluble Aβ oligomers and soluble Aβ in the brain. In aged 3 × Tg- AD mice, immunotherapy with the 12×(Aβ1-15-Th) vaccine could prevent Aβ-induced decrease of synaptic proteins, which suggested that it had neuroprotective effects on the brain. The novel recombinant 12×(Aβ1-15-Th) chimeric vaccine targeting of pathological conformations of Aβ oligomers has shown obvious neuroprotective benefits in the preclinical AD model mouse, which indicates that it is a good candidate vaccine for the prophylaxis of AD.

Neuroligin-1 is altered in the hippocampus of Alzheimer\u2019s disease patients and mouse models, and modulates the toxicity of amyloid-beta oligomers

Synapse loss occurs early and correlates with cognitive decline in Alzheimer’s disease (AD). Synaptotoxicity is driven, at least in part, by amyloid-beta oligomers (Aβo), but the exact synaptic components targeted by Aβo remain to be identified. We here tested the hypotheses that the post-synaptic protein Neuroligin-1 (NLGN1) is affected early in the process of neurodegeneration in the hippocampus, and specifically by Aβo, and that it can modulate Aβo toxicity. We found that hippocampal NLGN1 was decreased in patients with AD in comparison to patients with mild cognitive impairment and control subjects. Female 3xTg-AD mice also showed a decreased NLGN1 level in the hippocampus at an early age (i.e., 4 months). We observed that chronic hippocampal Aβo injections initially increased the expression of one specific Nlgn1 transcript, which was followed by a clear decrease. Lastly, the absence of NLGN1 decreased neuronal counts in the dentate gyrus, which was not the case in wild-type animals, and worsens impairment in spatial learning following chronic hippocampal Aβo injections. Our findings support that NLGN1 is impacted early during neurodegenerative processes, and that Aβo contributes to this effect. Moreover, our results suggest that the presence of NLGN1 favors the cognitive prognosis during Aβo-driven neurodegeneration.

Nonlinear and mixed inhibitory effect of matrine on the cytotoxicity of oligomeric amyloid-β protein

The formation of amyloid β-protein (1–42) (Aβ42) oligomers and Aβ42 oligomer cytotoxicity are two defining characteristics of the etiology of Alzheimer's disease (AD). In this study, we found that matrine (Mat) could maintain or even enhance the cytotrophic effect of Aβ42 monomers by inhibiting their aggregation and by working in a manner similar to synergy with Aβ42 monomers. Moreover, Mat could also exert a cytoprotective effect by actively promoting the disaggregation of immature Aβ42 oligomers in a concentration-dependent manner. Although Mat at intermediate concentrations (1–50 μM) exhibited both cytotrophic and cytoprotective effects on SH-SY5Y cells, Mat at higher concentrations (100 μM) only exhibited a cytoprotective effect. Molecular docking studies reveal that these differences are a result of the different interactions between Mat and Aβ42 oligomers that occur at different molecular ratios. Our results support the hypothesis that there may be a Mat-like metabolite in the human brain that acts as a molecular chaperone for Aβ42 monomers. A deficiency in this chaperone would result in the gradual aggregation of Aβ42 monomers, and eventually, formation of toxic Aβ42 oligomers. In addition, reduction or clearance of Aβ42 aggregates or deposits and inhibition or elimination of the toxicity of oligomeric Aβ42, were not always directly correlated. Finally, the site(s) responsible for cytotoxicity in Aβ42 oligomers may be located in the integrated region of the N-terminal fragments of Aβ42 chains. This study provides valuable insights into the mechanisms involved in the development of natural drugs for the treatment of Alzheimer's disease.

Susceptibility to cellular stress in PS1 mutant N2a cells is associated with mitochondrial defects and altered calcium homeostasis

Presenilin 1 (PS1) mutations are the most common cause of familial Alzheimer’s disease (FAD). PS1 also plays a role in cellular processes such as calcium homeostasis and autophagy. We hypothesized that mutant presenilins increase cellular vulnerability to stress. We stably expressed human PS1, mutant PS1E280A and mutant PS1Δ9 in mouse neuroblastoma N2a cells. We examined early signs of stress in different conditions: endoplasmic reticulum (ER) stress, calcium overload, oxidative stress, and Aβ 1–42 oligomers toxicity. Additionally, we induced autophagy via serum starvation. PS1 mutations did not have an effect in ER stress but PS1E280A mutation affected autophagy. PS1 overexpression influenced calcium homeostasis and generated mitochondrial calcium overload modifying mitochondrial function. However, the opening of the mitochondrial permeability transition pore (MPTP) was affected in PS1 mutants, being accelerated in PS1E280A and inhibited in PS1Δ9 cells. Altered autophagy in PS1E280A cells was neither modified by inhibition of γ-secretase, nor by ER calcium retention. MPTP opening was directly regulated by γ-secretase inhibitors independent on organelle calcium modulation, suggesting a novel direct role for PS1 and γ-secretase in mitochondrial stress. We identified intrinsic cellular vulnerability to stress in PS1 mutants associated simultaneously with both, autophagic and mitochondrial function, independent of Aβ pathology.

Reduction of the RNA Binding Protein TIA1 Exacerbates Neuroinflammation in Tauopathy.

Neuroinflammatory processes play an integral role in the exacerbation and progression of pathology in tauopathies, a class of neurodegenerative disease characterized by aggregation of hyperphosphorylated tau protein. The RNA binding protein (RBP) T-cell Intracellular Antigen 1 (TIA1) is an important regulator of the innate immune response in the periphery, dampening cytotoxic inflammation and apoptosis during cellular stress, however, its role in neuroinflammation is unknown. We have recently shown that TIA1 regulates tau pathophysiology and toxicity in part through the binding of phospho-tau oligomers into pathological stress granules, and that haploinsufficiency of TIA1 in the P301S mouse model of tauopathy results in reduced accumulation of toxic tau oligomers, pathologic stress granules, and the development of downstream pathological features of tauopathy. The putative role of TIA1 as a regulator of the peripheral immune response led us to investigate the effects of TIA1 on neuroinflammation in the context of tauopathy, a chronic stressor in the neural environment. Here, we evaluated indicators of neuroinflammation including; reactive microgliosis and phagocytosis, pro-inflammatory cytokine release, and oxidative stress in hippocampal neurons and glia of wildtype and P301S transgenic mice expressing TIA1+/+, TIA1+/–, and TIA1–/– in both early (5 month) and advanced (9 month) disease states through biochemical, ultrastructural, and histological analyses. Our data show that both TIA1 haploinsufficiency and TIA1 knockout exacerbate neuroinflammatory processes in advanced stages of tauopathy, suggesting that TIA1 dampens the immune response in the central nervous system during chronic stress.

Oligomerization and Conformational Change Turn Monomeric β-Amyloid and Tau Proteins Toxic: Their Role in Alzheimer's Pathogenesis.

The structural polymorphism and the physiological and pathophysiological roles of two important proteins, β-amyloid (Aβ) and tau, that play a key role in Alzheimer’s disease (AD) are reviewed. Recent results demonstrate that monomeric Aβ has important physiological functions. Toxic oligomeric Aβ assemblies (AβOs) may play a decisive role in AD pathogenesis. The polymorph fibrillar Aβ (fAβ) form has a very ordered cross-β structure and is assumed to be non-toxic. Tau monomers also have several important physiological actions; however, their oligomerization leads to toxic oligomers (TauOs). Further polymerization results in probably non-toxic fibrillar structures, among others neurofibrillary tangles (NFTs). Their structure was determined by cryo-electron microscopy at atomic level. Both AβOs and TauOs may initiate neurodegenerative processes, and their interactions and crosstalk determine the pathophysiological changes in AD. TauOs (perhaps also AβO) have prionoid character, and they may be responsible for cell-to-cell spreading of the disease. Both extra- and intracellular AβOs and TauOs (and not the previously hypothesized amyloid plaques and NFTs) may represent the novel targets of AD drug research.

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions.

Expanded CAG nucleotide repeats are the underlying genetic cause of at least 14 incurable diseases, including Huntington’s disease (HD). The toxicity associated with many CAG repeat expansions is thought to be due to the translation of the CAG repeat to create a polyQ protein, which forms toxic oligomers and aggregates. However, recent studies show that HD CAG repeats undergo a non-canonical form of translation called Repeat-associated non-AUG dependent (RAN) translation. RAN translation of the CAG sense and CUG anti-sense RNAs produces six distinct repeat peptides: polyalanine (polyAla, from both CAG and CUG repeats), polyserine (polySer), polyleucine (polyLeu), polycysteine (polyCys), and polyglutamine (polyGln). The toxic potential of individual CAG-derived RAN polypeptides is not well understood. We developed pure C. elegans protein models for each CAG RAN polypeptide using codon-varied expression constructs that preserve RAN protein sequence but eliminate repetitive CAG/CUG RNA. While all RAN polypeptides formed aggregates, only polyLeu was consistently toxic across multiple cell types. In GABAergic neurons, which exhibit significant neurodegeneration in HD patients, codon-varied (Leu)38, but not (Gln)38, caused substantial neurodegeneration and motility defects. Our studies provide the first in vivo evaluation of CAG-derived RAN polypeptides in a multicellular model organism and suggest that polyQ-independent mechanisms, such as RAN-translated polyLeu peptides, may have a significant pathological role in CAG repeat expansion disorders.

Effects of Boron Nitride Nanotube on the Secondary Structure of Aβ(1-42) Trimer: Possible Inhibitory Effect on Amyloid Formation.

Aggregation of amyloid-β (Aβ) peptides into prefibrillar toxic oligomers is responsible for Alzheimer's disease, which is one of the most commonly known forms of neurodegenerative diseases. It has been reported that the low-molecular weight Aβ(1-42) trimer and its higher order oligomers are responsible for the cytotoxicity in the neuronal cells, leading to their death. Various experimental studies have shown that boron nitride nanotube (BNNTs) are noncytotoxic for health and environment and biocompatible in living cells and can be used as delivery vehicles for brain anticancer drugs. Here, we investigate the effects of BNNT on the secondary structure of Aβ(1-42) in the amyloid oligomerization process. We have performed long atomistic molecular dynamics simulations of 4.0 μs in total to study the structural stability of Aβ(1-42) trimer both in presence and absence of BNNT in explicit solvent. It is found that in the absence of BNNT, Aβ(1-42) trimer aggregates, leading to α-helix to β-sheet transition, whereas BNNT provides structural stability to the peptides by keeping them separated and preserving the initial monomeric helical conformation of the Aβ(1-42) trimer. It is found through a free-energy decomposition analysis that the key residues (Lys28, Ile31, Ile32, Leu34, Val40, and Ile41) from the C-terminal that are more responsible for β-sheet fibril formation bind with the BNNT surface and block their structural conversion. Due to the presence of polar B-N bonds, BNNT is less hydrophobic as compared with the carbonaceous nanomaterials where large hydrophobicity of these carbonaceous nanomaterials mostly destabilizes the secondary structure of peptides. The current study reveals that the less hydrophobicity of BNNT provides stability of the initial secondary structure of the Aβ(1-42) trimer and hinders their aggregation. Hence, the secondary structure stabilization in presence of BNNT provides new possibilities for the design of different nanoparticles as therapeutic agents for different types of amyloidogenesis by tuning their hydrophobicity.

Evaluation of Toxic Amyloid 42 Oligomers in Rat Primary Cerebral Cortex Cells and Human iPS-derived Neurons Treated with 10-Me-Aplog-1, a New PKC Activator.

Amyloid β42 (Aβ42), a causative agent of Alzheimer’s disease (AD), is derived extracellularly from Aβ precursor protein (APP) following the latter’s cleavage by β-secretase, but not α-secretase. Protein kinase Cα (PKCα) activation is known to increase α-secretase activity, thereby suppressing Aβ production. Since Aβ42 oligomer formation causes potent neurotoxicity, APP modulation by PKC ligands is a promising strategy for AD treatment. Although bryostatin-1 (bryo-1) is a leading compound for this strategy, its limited natural availability and the difficulty of its total synthesis impedes further research. To address this limitation, Irie and colleagues have developed a new PKC activator with few side effects, 10-Me-Aplog-1, (1), which decreased Aβ42 in the conditioned medium of rat primary cerebral cortex cells. These results are associated with increased α-secretase but not PKCε-dependent Aβ-degrading enzyme. The amount of neuronal embryonic lethal abnormal vision (nELAV), a known β-secretase stabilizer, was reduced by treatment with 1. Notably, 1 prevented the formation of intracellular toxic oligomers. Furthermore, 1 suppressed toxic oligomerization within human iPS-derived neurons such as bryo-1. Given that 1 was not neurotoxic toward either cell line, these findings suggest that 1 is a potential drug lead for AD therapy.

Targeting Hsc70-based autophagy to eliminate amyloid β oligomers

Amyloid β (Aβ) oligomers may be a real culprit in the pathogenesis of Alzheimer’s disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aβ become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aβ oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aβ oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aβ oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aβ oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aβ oligomers and have superior neuroprotective activity.

Transthyretin Inhibits Primary and Secondary Nucleations of Amyloid-β Peptide Aggregation and Reduces the Toxicity of Its Oligomers.

Alzheimer’s disease is associated with the deposition of the amyloid-β peptide (Aβ) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aβ scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aβ40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aβ40, of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aβ40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aβ40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aβ40 oligomers, as well as with oligomers of Aβ42, while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aβ oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aβ oligomers and the ability of the fibrils to proliferate.

Effect of Melatonin on Lipid Membrane Structure and Membrane Interactions with Amyloid. An NMR and LSPR Study

Alzheimer's Disease (AD) is a neurodegenerative disease and leading cause of dementia resulting in memory loss and cognition decline. The molecular mechanism of AD is not well understood, which has delayed the development of effective treatments. Amyloid-ß (Aß) is the suspected cause of AD, as nonspecific interactions of toxic amyloid oligomers with the plasma membrane cause neuronal cell death. Melatonin is a hormone produced by pineal gland to regulate sleep cycle and has been shown to be protective against Aß in cellular and animal studies, but the molecular mechanism is not well understood. Melatonin can partition into lipid membranes and has remarkable effects on their biophysical properties; this may be important in explaining its protective effects. In this study, we aimed to determine the effect of melatonin on the phase behaviour of model lipid membranes and its ability to inhibit amyloid interaction with lipid bilayers, using nuclear magnetic resonance (NMR) and localized surface plasmon resonance (LSPR), respectively. We used a tertiary mixture of DPPC, POPC, and cholesterol bilayers as a lipid model. Fully hydrated multilamellar lipid stacks were used in solid-state ²H-NMR experiments, showing that melatonin competes with cholesterol to associate with certain lipids and promotes the coexistence of liquid ordered and liquid disordered phases in a temperature-dependent manner. We studied membrane interactions with Aß (1-42) using LSPR and determined that the incorporation of melatonin into the lipid bilayer leads to a decrease in the nonspecific binding of Aß to the supported lipid bilayer. Combining the results from both techniques, we conclude that melatonin's protective effect against amyloid toxicity in lipid membranes is due to its ability to change the structural properties of the membrane and to reduce amyloid affinity to such membranes.

Use of Giant Plasma Membrane Vesicles (GPMV) to Examine the Lo/Ld Phase Preference of the C99 Domain of the Amyloid Precursor Protein

Alzheimer's disease (AD) is the most common form of dementia at the moment cannot be treated, prevented, or even delayed. Despite decades of intensive research efforts, there are still many mysteries regarding the etiology of AD. The predominant hypothesis for AD is that disease pathology begins decades before clinically-detectable symptoms due to the formation of toxic oligomers and aggregates composed mainly of amyloid-β (Aβ) peptides. Amyloid-β is generated when the amyloid precursor protein (APP) transmembrane C-terminal fragment, C99, is cleaved in the membrane by γ-secretase. Cholesterol has long been implicated in AD and shown to increase the generation of Aβ peptides, but it is not known how cholesterol or cholesterol-rich environments impact C99 membrane localization. We are examining the membrane phase preference of C99 localization in membranes using giant plasma membrane-derived vesicles (GPMVs). These cell-derived giant vesicles exhibit phase separation into cholesterol-rich Lo and Ld-like domains that can be directly observed using confocal microscopy. We compare and contrast various methods for preparing GPMVs and report the phase partitioning behavior of both WT C99 and cholesterol binding site-disrupted mutant forms.

Biophysical Characterization of Covalently Modified Protein Tau: Oligomers, Aggregation, and Tubulin Interactions

The molecular interactions that drive the onset and spread of Alzheimer's disease (AD) remain poorly understood. In AD, tau neurofibrillary tangles correlate with cognitive decline and have historically been associated with tau's toxicity. Recently, tau oligomers have been implicated as stable toxic subunits that contribute to the formation of paired helical filaments and eventual neurofibrillary tangles. Covalently modified tau is a major component of neurofibrillary tangles and plays a significant role in tau's ability to dissociate from microtubules, aggregate, and become toxic. Elucidating the effects of covalent modifications on oligomer formation could aide our understanding of the molecular mechanisms behind tau's toxicity. The complexity of tau, partially due to its intrinsically disordered nature, allows for phosphorylation, acetylation, and succinylation to occur at multiple sites and influence the physiological and pathological roles of tau. Specific residue modifications can play a major role in modulating tau function and aggregation. This has led to the use of mimics in cell, mouse, and in-vitro models of tau pathology. Site-specific mimics of phosphorylation, acetylation, and succinylation can aide our ability to biophysically characterize the effect of covalently modified tau on oligomer formation, aggregation propensity, and tubulin interactions; Ultimately aiding our understanding of tau function and structure. We apply nuclear magnetic resonance, fluorescence, and light scattering to show that covalently modified tau mimics of phosphorylation, acetylation, and succinylation perturb tau-T2R interactions (a model of tau-tubulin interactions), induce filament formation, and form stable toxic oligomers with different levels of efficiency. Our results reinforce phosphorylation as a contributor to filament formation, acetylation as a driver of oligomer formation, and introduces tau succinylation as a novel modifier of tau structure and function.

Melatonin Changes Domain Structure and Protects Model Neuronal Membranes Against Damage Caused by Amyloid-Beta

The amyloid-β(1-42) peptide is a major player in Alzheimer's disease—a debilitating neurodegenerative disorder that causes memory loss and neuronal damage in elderly patients. Amyloid-β(1-42) forms toxic oligomers that bind to neuronal membranes through interactions with cholesterol-rich lipid rafts. These interactions result in damage to the lipid membrane through the insertion of the amyloid-β(1-42) peptide and subsequent formation of an ion channel or pore. Melatonin, a hormone naturally produced in the brain during sleep, has been identified as a potentially protective native molecule in cellular and animal studies however the molecular mechanisms of its protection are not well understood. Due to its lipophilicity, melatonin partitions into neural model membranes and associates with the lipid headgroups. In this study we used atomic force microscopy (AFM), and Kelvin probe force microscopy (KPFM) techniques to elucidate the effect of melatonin on the structure and domain organization in neuronal model membranes composed of DOPC, POPC, gangliosides, sphingomyelin, and cholesterol. In addition we measured membrane permeability using black lipid membrane (BLM) techniques on the same neuronal models to elucidate the protective effect of melatonin against the damage induced by amyloid-β peptide ion channels. We demonstrated that melatonin changes the structure of neuronal model membranes (both topographical and electrostatic changes in the nanoscale domains) and reduces the ion current (a measure of damage) across model membranes induced by the binding of the amyloid-β(1-42) peptide.

Sulforaphane Attenuates Aβ Oligomers Mediated Decrease in Phagocytic Activity of Microglial Cells

Microglia are the brain mononuclear phagocytes which plays a key role in neurodegenerative diseases, like Alzheimer’s. Till date, microglia have been explored mostly for their neuro-inflammatory functions. Recent studies have shifted their focus towards less explored functions which involve non-autonomous clearance of protein aggregates. However, these functions are significantly affected by aging and neurodegeneration. In Alzheimer’s disease (AD), microglia have been reported to clear amyloid beta (Aβ) deposits via phagocytosis or release various pro-inflammatory cytokines. Whether microglia could be beneficial or detrimental to the brain, it all depends upon the type and strength of stimulus. So, if their beneficial properties could be selectively harnessed without activating pro-inflammatory response, a potential therapeutic strategy could be developed to check the formation of protein aggregates like Aβ. In the present study, we have checked the effect of toxic amyloid beta oligomers (Aβo) on the microglial phagocytic activity. Our findings revealed that at lower concentrations, Aβo are not toxic to the cells and they can survive even with longer exposures but with decreased phagocytic activity. However, at higher concentrations Aβo become toxic and resulted in modulation of various genes which regulates microglial phagocytic activity. Sulforaphane (SFN) treatment has shown to induce the phagocytic activity of Aβo treated microglial cells. In addition, low dose Aβo and SFN treatment have not shown modulation in the levels of pro-inflammatory mediators of microglia. Taken together, these findings suggest that SFN treatment may ameliorate the Aβo mediated decrease in microglial phagocytic activity.

Correction of Systematic Bias in Single Molecule Photobleaching Measurements

Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such “prebleaching” can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.