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Amyloids, with their ß-sheet-rich structure, contribute to diabetes, neurodegenerative diseases, and amyloidosis by aggregating within diverse anatomical compartments. Insulin amyloid (IA), sharing structural resemblances with amyloids linked to neurological disorders, acts as a prototype, while compounds capable of degrading these fibrils hold promise as therapeutic agents for amyloidosis intervention. In this research, liposomal nanoformulated iota carrageenan (nCG) was formulated to disrupt insulin amyloids, demonstrating about a 17-20 % higher degradation efficacy compared to conventional carrageenan through thioflavin T fluorescence, dynamic light scattering analysis, and turbidity quantification. The biocompatibility of the nCG and nCG-treated insulin amyloids was evaluated through MTT assay, live-dead cell assay on V79 cells, and hemolysis testing on human blood samples to establish their safety for use in vitro. Zebrafish embryos were utilized to assess in vivo biocompatibility, while adult zebrafish were employed to monitor the degradation capacity of IA post subcutaneous injection, with fluorescence emitted by the fish captured via IVIS. This demonstrated that the formulated nCG exhibited superior anti-amyloid efficacy compared to carrageenan alone, while both materials demonstrated biocompatibility. Furthermore, through docking simulations, an exploration was conducted into the molecular mechanisms governing the inhibition of the target protein pancreatic insulin by carrageenan.

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Background: Reduction of the production of amyloid-ß (Aß) species has been intensively investigated as potential therapeutic approaches for Alzheimer's disease (AD). However, the degradation of Aß species, another potential beneficial approach, has been far less explored. Objective: To investigate the potential of multi-copper oxidases (MCOs) in degrading Aß peptides and their potential benefits for AD treatment. Methods: We investigated the degradation efficiency of MCOs by using electrophoresis and validated the ceruloplasmin (CP)-Aß interaction using total internal reflection fluorescence microscopy, fluorescence photometer, and fluorescence polarization measurement. We also investigated the therapeutic effect of ascorbate oxidase (AO) by using induced pluripotent stem (iPS) neuron cells and electrophysiological analysis with brain slices. Results: We discovered that CP, an important MCO in human blood, could degrade Aß peptides. We also found that other MCOs could induce Aß degradation as well. Remarkably, we revealed that AO had the strongest degrading effect among the tested MCOs. Using iPS neuron cells, we observed that AO could rescue neuron toxicity which induced by Aß oligomers. In addition, our electrophysiological analysis with brain slices suggested that AO could prevent an Aß-induced deficit in synaptic transmission in the hippocampus. Conclusions: To the best of our knowledge, our report is the first to demonstrate that MCOs have a degrading function for peptides/proteins. Further investigations are warranted to explore the possible benefits of MCOs for future AD treatment.

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BACKGROUND: Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. Impaired autophagy in plaque-associated microglia (PAM) has been reported to accelerate amyloid plaque deposition and cognitive impairment in AD pathogenesis. Recent evidence suggests that the transcription factor EB (TFEB)-mediated activation of the autophagy-lysosomal pathway is a promising treatment approach for AD. Moreover, the complementary therapy of intermittent hypoxia therapy (IHT) has been shown to upregulate autophagy and impart beneficial effects in patients with AD. However, the effect of IHT on PAM remains unknown. METHODS: 8-Month-old APP/PS1 mice were treated with IHT for 28 days. Spatial learning memory capacity and anxiety in mice were investigated. AD pathology was determined by the quantity of nerve fibers and synapses density, numbers of microglia and neurons, Aß plaque deposition, pro-inflammatory factors, and the content of Aß in the brain. TFEB-mediated autophagy was determined by western blot and qRT-PCR. Primary microglia were treated with oligomeric Aß 1-42 (oAß) combined with IHT for mechanism exploration. Differential genes were screened by RNA-seq. Autophagic degradation process of intracellular oAß was traced by immunofluorescence. RESULTS: In this study, we found that IHT ameliorated cognitive function by attenuating neuronal loss and axonal injury in an AD animal model (APP/PS1 mice) with beta-amyloid (Aß) pathology. In addition, IHT-mediated neuronal protection was associated with reduced Aß accumulation and plaque formation. Using an in vitro PAM model, we further confirmed that IHT upregulated autophagy-related proteins, thereby promoting the Aß autophagic degradation by PAM. Mechanistically, IHT facilitated the nuclear localization of TFEB in PAM, with TFEB activity showing a positive correlation with Aß degradation by PAM in vivo and in vitro. In addition, IHT-induced TFEB activation was associated with the inhibition of the AKT-MAPK-mTOR pathway. CONCLUSIONS: These results suggest that IHT alleviates neuronal damage and neuroinflammation via the upregulation of TFEB-dependent Aß clearance by PAM, leading to improved learning and memory in AD mice. Therefore, IHT may be a promising non-pharmacologic therapy in complementary medicine against AD.

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Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

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BACKGROUND: Alzheimer's disease (AD) is diagnosed with the deposition of insoluble ß-amyloid (Aß) peptides in the neuropil of the brain leading to dementia. The extracellular deposition of the fibrillar Aß peptide on the neurons is known as senile plaques. Therefore, Aß degradation and clearance from the human body is a promising therapeutic approach in the medication of AD. METHODS: In the current study, the enzyme lumbrokinase (LK) was extracted and purified from earthworm and its activity was utilized toward Aß 1-42 amyloids degradation in vitro alongside with an additional enzyme serratiopeptidase (SP) considering nattokinase (NK) as a standard. RESULTS: The output of this study revealed that preformed Aß 1-42 amyloids was disintegrated by both LK and SP, as demonstrated from fluorescence assay using Thioflavin T dye. In addition, dynamic light scattering study revealed the lower size of the preformed fibrils Aß 1-42 at various time intervals after incubation with the enzymes LK and SP. Furthermore, in silico approach showed high affinity thermodynamically favorable interaction of LK as well as SP toward Aß 1-42 amyloid. Finally, the toxicity of degraded preformed Aß 1-42 amyloid was assessed by MTT assay which showed reduced toxicity of enzyme treated Aß 1-42 amyloid compared to only Aß 1-42 amyloid. CONCLUSION: The findings of the present study indicated that LK and SP, not only had Aß 1-42 amyloid degrading potential, but also could reduce the toxicity which can make them a suitable drug candidate for AD. Furthermore, the in vivo studies are needed to be executed in future.

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It is known that insulin-degrading-enzyme (IDE) plays a crucial role in the clearance of Alzheimer's amyloid-ß (Aß). The cysteine-free IDE mutant (cf-E111Q-IDE) is catalytically inactive against insulin, but its effect on Aß degradation is unknown that would help in the allosteric modulation of the enzyme activity. Herein, the degradation of Aß(1-40) by cf-E111Q-IDE via a non-chaperone mechanism is demonstrated by NMR and LC-MS, and the aggregation of fragmented peptides is characterized using fluorescence and electron microscopy. cf-E111Q-IDE presented a reduced effect on the aggregation kinetics of Aß(1-40) when compared with the wild-type IDE. Whereas LC-MS and diffusion ordered NMR spectroscopy revealed the generation of Aß fragments by both wild-type and cf-E111Q-IDE. The aggregation propensities and the difference in the morphological phenotype of the full-length Aß(1-40) and its fragments are explained using multi-microseconds molecular dynamics simulations. Notably, our results reveal that zinc binding to Aß(1-40) inactivates cf-E111Q-IDE's catalytic function, whereas zinc removal restores its function as evidenced from high-speed AFM, electron microscopy, chromatography, and NMR results. These findings emphasize the catalytic role of cf-E111Q-IDE on Aß degradation and urge the development of zinc chelators as an alternative therapeutic strategy that switches on/off IDE's function.

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The protein misfolded structure called amyloids is related with extensive range of pathologies like local amyloidosis and neurodegenerative diseases. Several studies have reported the potential of insulin to generate local amyloidosis under certain state. Reports also showed that fibrils of insulin generated local amyloid mass due to continuous subcutaneous injection in mouse as well as rat. The present study was designed to examine the consequence of insulin fibril injections in rats, as well as the ability of enzymes, Lumbrokinase (LK) and Serratiopeptidase (SP) in diminishing this amyloid mass progression. The results showed that insulin fibrils generated amyloid masses in rats after subcutaneous injection for two weeks which was significantly condensed in size for the groups injected with insulin fibrils combined with LK or SP. At higher doses of LK and SP, the absence of amyloid structure was observed in histopathological studies. Light microscopy, polarized microscopy as well as Lumia live in vivo imaging system was used to analyze the results. In conclusion, the overall outcome of this study showed the anti-amyloid potential of enzyme LK and SP in the attenuation of local amyloidosis.

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BACKGROUND: PrPC is a host-encoded prion protein, which gets post translationally modified into a transmissible, ß-sheet rich disease associated protein called PrPSc, responsible for the Prion disease including mad cow disease in cattle and CJD in humans. The PrP 106-126 region in PrPSc peptide initiates the conformational change in that protein leading to fibrillation. Any agent that can destabilize or disintegrate such proteins can be served as a potential drug candidate for Prion diseases. METHODS: In the present study, an enzyme Lumbrokinase (LK) was isolated from earthworm and its activity was exploited towards PrP 106-126 amyloids in vitro along with another enzyme Serratiopeptidase (SP) taking Nattokinase (NK) as a standard. RESULTS: The results showed that PrP 106-126 amyloid formation was inhibited by both LK and SP, as evidenced from Thioflavin T fluorescence assay. Further, the size of fibrils as estimated by dynamic light scattering, was also found to be lower at different time intervals after incubation of the prion amyloids with LK and SP. Additionally, the molecular dynamics simulation revealed the thermodynamically favorable interaction of PrP 106-126 with LK as well as with SP with high affinity. CONCLUSION: Finally, the toxicity of the disintegrated amyloids was assessed using PC12 cell lines which showed higher cell viability in case of LK and SP treated amyloids compared to only PrP 106- 126 amyloid treatment. Altogether, the study concluded that the serine proteases like LK and SP have the potential to disintegrate PrP 106-126 amyloids with improved cell viability. The in vivo studies are needed to be executed in future.

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An electrokinetic potential (ζ-potential) based approach was introduced to address the amyloid degradation on ZnO-nanoflower platform. The hallmark of neurodegenerative disorders like Alzheimer's disease, Parkinson's disease (PD), Creutzfeldt-Jakob Disease (CJD), Prion- associated diseases, type-II diabetes, etc. is the deposition of misfolded protein aggregates predominantly ß-sheeted in structure and fibrillar morphology, known as amyloids, in the brain and different parts of the body. Agents that can degrade these amyloids can be potential candidate for the therapy of amyloidosis. Ultrasmall nanoparticles are gaining interest due to their ability to cross blood brain barrier (BBB) which is favorable for the treatment of neurodegenerative disorders. Considering the influence of Zn2+ in the formation of Aß aggregates instead of fibrillation, the present study was designed based on the ZnO nanoparticles (ZnO-NP) and ZnO nanoflowers (ZnO-NF) to compare the anti amyloid ability using a model huminsulin amyloid. Fluorescence study, atomic force microscopy (AFM), IR spectroscopy (FTIR) and reduction of fibril size using dynamic light scattering showed that ZnO-NF can degrade amyloids with a higher capacity than their nanoparticle counterpart. Significant reduction in magnitude of ζ-potential in ZnO-NF treated huminsulin amyloid supported the notion to come to the consensus and became the new indicator for anti-amyloidosis. The cell viability assay of ZnO-NP and ZnO-NF at a higher dose than that used for amyloid degradation using PC12 and HaCaT cell lines showed their biocompatibility in a safe manner. Thus, it can be suggested that ZnO-NF would be a better candidate for amyloid degradation compared to ZnO-NPs due to higher surface to volume ratio of the petals.

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Alzheimer's disease (AD) is characterized by neuritic plaques and neurofibrillary tangles. It is reported that enzymatic degradation of amyloid-ß (Aß) plays a pivotal role in Aß accumulation and type-2 cannabinoid receptor (CB2R) participates in Aß processing in the brain; however, the underlying mechanisms remain unclear. We determined that Aß degradation-related proteins are significantly different between CB2R-/- mice and wild-type (WT) mice via proteomic analysis. Moreover, the data demonstrated that the angiotensin converting enzyme (ACE) and insulin-degrading enzyme (IDE) levels are substantially attenuated, and the Aß level is significantly enhanced in CB2R-/--Aß1 - 42 mice compared with that of WT-Aß1 - 42 mice. Furthermore, Aß-mediated synaptic dysfunction, the loss of memory associated proteins, and the suppression of glutamatergic transmission are more severe in CB2R-/--Aß1 - 42 mice than that in WT-Aß1 - 42 mice. CB2R activation could decrease Aß1 - 40 and Aß1 - 42 levels and enhance ACE and IDE levels with its selective agonist JWH133; however, AM630 (CB2R antagonist) abrogates all changes induced by JWH133 in N2a cells with AßPP overexpression. Taken together, our study demonstrated that the deletion of CB2R reduces exogenous Aß degradation and aggravates the toxicity of Aß via the reduction of ACE and IDE, which suggests that CB2R is involved in the onset of AD and a potential therapeutic target for AD.

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