Beta Peptide Research Articles

Therapeutic Candidates for Alzheimer’s Disease: Saponins

Drug development for Alzheimer’s disease, the leading cause of dementia, has been a long-standing challenge. Saponins, which are steroid or triterpenoid glycosides with various pharmacological activities, have displayed therapeutic potential in treating Alzheimer’s disease. In a comprehensive review of the literature from May 2007 to May 2023, we identified 63 references involving 40 different types of saponins that have been studied for their effects on Alzheimer’s disease. These studies suggest that saponins have the potential to ameliorate Alzheimer’s disease by reducing amyloid beta peptide deposition, inhibiting tau phosphorylation, modulating oxidative stress, reducing inflammation, and antiapoptosis. Most intriguingly, ginsenoside Rg1 and pseudoginsenoside-F11 possess these important pharmacological properties and show the best promise for the treatment of Alzheimer’s disease. This review provides a summary and classification of common saponins that have been studied for their therapeutic potential in Alzheimer’s disease, showcasing their underlying mechanisms. This highlights the promising potential of saponins for the treatment of Alzheimer’s disease.

State-of-the-Art Technologies for Studying Cellular and Molecular Mechanisms Underlying Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disease and cause of dementia. It is associated with progressive cognitive decline due to the development of cortical and hippocampal atrophy.
 We reviewed key factors in AD pathogenesis, such as synaptic dysfunction, accumulation and aggregation of amyloid beta (A) peptide, tau phosphorylation causing neurofibrillary tangles, mitochondrial dysfunction, and neuroinflammation. We studied the dysbiosis role in AD development and demonstrated how much the bidirectional communication between the gut and brain sheds new light on some pathogenic processes underlying AD. We reviewed state-of-the-art biomedical technologies for studying AD: transgenic models, electrophysiological techniques, optogenetics, multi-omics approaches, neuroimaging, etc. New biomedical technologies significantly expanded our current knowledge of the AD pathogenesis and laid the groundwork for state-of-the-art treatment approaches.

Moderate intrinsic phenotypic alterations in C9orf72 ALS/FTD iPSC-microglia despite the presence of C9orf72 pathological features.

While motor and cortical neurons are affected in C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), it remains largely unknown if and how non-neuronal cells induce or exacerbate neuronal damage. We differentiated C9orf72 ALS/FTD patient-derived induced pluripotent stem cells into microglia (iPSC-MG) and examined their intrinsic phenotypes. Similar to iPSC motor neurons, C9orf72 ALS/FTD iPSC-MG mono-cultures form G4C2 repeat RNA foci, exhibit reduced C9orf72 protein levels, and generate dipeptide repeat proteins. Healthy control and C9orf72 ALS/FTD iPSC-MG equally express microglial specific genes and perform microglial functions, including inflammatory cytokine release and phagocytosis of extracellular cargos, such as synthetic amyloid beta peptides and healthy human brain synaptoneurosomes. RNA sequencing analysis revealed select transcriptional changes of genes associated with neuroinflammation or neurodegeneration in diseased microglia yet no significant differentially expressed microglial-enriched genes. Moderate molecular and functional differences were observed in C9orf72 iPSC-MG mono-cultures despite the presence of C9orf72 pathological features suggesting that a diseased microenvironment may be required to induce phenotypic changes in microglial cells and the associated neuronal dysfunction seen in C9orf72 ALS/FTD neurodegeneration.

Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aβ) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aβ represents a major constituent of the amyloid plaque. The data show that pE3Aβ formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aβ accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aβ3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aβ and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.

New insights into the function and pathophysiology of the ectodomain sheddase A Disintegrin And Metalloproteinase 10 (ADAM10).

The "A Disintegrin And Metalloproteinase 10" (ADAM10) has gained considerable attention due to its discovery as an "α-secretase" involved in the non-amyloidogenic processing of the amyloid precursor protein, thereby possibly preventing the excessive generation of the amyloid beta peptide, which is associated with the pathogenesis of Alzheimer's Disease. ADAM10 was found to exert many additional functions, cleaving about 100 different membrane proteins. ADAM10 is involved in many pathophysiological conditions, ranging from cancer and autoimmune disorders to neurodegeneration and inflammation. ADAM10 cleaves its substrates close to the plasma membrane, a process referred to as ectodomain shedding. This is a central step in the modulation of the functions of cell adhesion proteins and cell surface receptors. ADAM10 activity is controlled by transcriptional and posttranslational events. The interaction of ADAM10 with tetraspanins and the way they functionally and structurally depend on each other is another topic of interest. In this review, we will summarize findings on how ADAM10 is regulated and what is known about the biology of the protease. We will focus on novel aspects of the molecular biology and pathophysiology of ADAM10 that were previously poorly covered, such as the role of ADAM10 on extracellular vesicles, its contribution to virus entry, its involvement in cardiac disease, cancer, inflammation and immune regulation. ADAM10 has emerged as a regulator controlling cell surface proteins during development and in adult life. Its involvement in disease states suggests that ADAM10 may be exploited as a therapeutic target to treat conditions associated with a dysfunctional proteolytic activity.

The microglial challenge to encapsulated cell mediated drug delivery in brain tissue

AbstractBackgroundDementia disorders affect millions of people worldwide, among which Alzheimer’s disease (AD) accounts for 60% cases. Treatment of these disorders are constrained due to inefficient brain availability of peripherally administered drugs. Conversely, direct intracerebroventricular injections are counterintuitive due to unspecific side‐effects. Recently, Aducanumab has shown potential to clear amyloid plaques from AD brain, but its efficacy to revive clinical symptoms are under debate. In this regard, direct delivery of regenerative biological drugs utilizing encapsulated cell biodelivery (ECB) has gained much attention. Conversely, how the pathological microenvironment of the AD brain affects these encapsulated drug‐delivering cells are scarcely understood. The ECB approach nullifies immune reaction from host tissue by protecting drug‐delivering cells from direct physical contact with the host tissue. Thus, in this study we probe the effect of factors released from activated human microglial cells (HMC3 cell line) on ECB devices harboring nerve growth factor (NGF) releasing cells (NGC0211; modified human ARPE‐19 cells).MethodECB devices harboring NGC0211 cells were manufactured and maintained in DMEM/F12 culture medium containing 10% serum until used. HMC3 cells were activated using AD related molecules including 1µM amyloid beta (Aβ40 and Aβ42) peptides and 10ng/mL inflammatory cytokines (interferon‐γ, IFN‐γ; interleukin‐1β, IL‐1β; or both) for 24hrs, and a cell free microglia conditioned medium (MCM) was obtained. ECB devices were then exposed to various MCMs, and NGF release was followed for several weeks assessed by ELISA (DY256, R&D systems). Similarly, non‐encapsulated NGC0211 cells grown in multi‐well slides were exposed to MCMs and cellular proliferation (Ki67 expression) along with senescence parameters (SA‐β‐galactosidase) were evaluated.ResultWe found that factors released from activated microglia hampered NGF release from ECB devices. Simultaneously, MCMs reduced Ki67 expression and elevated SA‐β‐galactosidase activity in NGC0211 cells.ConclusionActivated microglia, a common feature of all neurodegenerative conditions, impair NGF release, cellular proliferation and induce senescence in NGC0211 cells. Similar impact of microglia on other types of drug‐delivering cells (stem cells or genetically modified) needs to be studied. Our study highlights one of the probable underlying causes for impaired long‐term cell mediated drug delivery in neurodegenerative diseases and helps guide improvements in the technologies.

Rapid suppression of macroscopic homomeric Kv1.2 and heteromeric Kv1.1/1.2 currents by amyloid beta (1‐42) peptide: Implications for Alzheimer’s disease

AbstractBackgroundThe roles of Aβ in the pathogenesis of Alzheimer ’s disease (AD) include disruption of 1) Ca2+ homeostasis, 2) synaptic communication and function, and 3) synaptic plasticity mechanisms thought to be critical for learning and memory. Exactly how these abnormalities arise is incompletely understood. Our lab has focused on the potential involvement of voltage‐gated potassium channels (VGKCs) in several of these processes. VGKCs are activated during an action potential and regulate Ca2+ influx, and inhibition of VGKCs can lead to synapto‐ and neuro‐toxicity. In the nervous system, members of the Kv1.x family can be expressed as homomers or select heteromers; e.g., Kv1.1 is often co‐expressed with Kv1.2. Studies in our lab have shown that Aβ peptides suppress VGKC activity, particularly Kv1.1. Here we asked if Aβ suppression is specific to Kv1.1 homomers, or if it affects other members of the Kv1.x family (e.g., Kv1.2) and/or heteromers also?MethodStage V/VI Xenopus laevis oocytes were acquired from Ecocyte Bioscience (Austin, TX) and injected with Kv1.2 cRNA for homomeric expression. Heteromeric expression was evaluated by coinjection of Kv1.1 and 1.2 cRNA. The effects of bath application (1 μM) of Aβ(1‐42) on macroscopic currents from homomeric Kv1.2 and 1.1/1.2 heteromeric channels were assessed using two‐electrode voltage clamp (TEVC). DMSO was used as a control.ResultAs seen for homomeric Kv1.1 channels, Aβ suppressed homomeric Kv1.2 channels by ∼50% in 30 min. About 50% of this suppression was dependent on intracellular Ca2+. Aβ(1‐42) suppressed macroscopic heteromeric Kv1.1/1.2 channels by ∼41% in 30 min. The suppression was independent of changes in activation‐deactivation kinetics or voltage dependency.ConclusionHeterologously expressed homomeric Kv1.2 and heteromeric Kv1.1/1.2 channels were both suppressed by Aβ(1‐42), similar to Kv1.1 homomers. Suppression of homomeric and heteromeric Kv1.x channels could potentially lead to larger and longer action potentials, resulting in a greater influx of Ca2+ into presynaptic terminals of excitatory neurons, and depolarization of dendritic spines. Both could contribute to synapto‐ and neuro‐toxicity via increased glutamate release and depolarization of NMDA receptors.

Chaperone Activity and Protective Effect against Aβ-Induced Cytotoxicity of Artocarpus camansi Blanco and Amaranthus dubius Mart. ex Thell Seed Protein Extracts.

Alzheimer's disease (AD) is the most common type of dementia and is listed as the sixth-leading cause of death in the United States. Recent findings have linked AD to the aggregation of amyloid beta peptides (Aβ), a proteolytic fragment of 39-43 amino acid residues derived from the amyloid precursor protein. AD has no cure; thus, new therapies to stop the progression of this deadly disease are constantly being searched for. In recent years, chaperone-based medications from medicinal plants have gained significant interest as an anti-AD therapy. Chaperones are responsible for maintaining the three-dimensional shape of proteins and play an important role against neurotoxicity induced by the aggregation of misfolded proteins. Therefore, we hypothesized that proteins extracted from the seeds of Artocarpus camansi Blanco (A. camansi) and Amaranthus dubius Mart. ex Thell (A. dubius) could possess chaperone activity and consequently may exhibit a protective effect against Aβ1-40-induced cytotoxicity. To test this hypothesis, the chaperone activity of these protein extracts was measured using the enzymatic reaction of citrate synthase (CS) under stress conditions. Then, their ability to inhibit the aggregation of Aβ1-40 using a thioflavin T (ThT) fluorescence assay and DLS measurements was determined. Finally, the neuroprotective effect against Aβ1-40 in SH-SY5Y neuroblastoma cells was evaluated. Our results demonstrated that A. camansi and A. dubius protein extracts exhibited chaperone activity and inhibited Aβ1-40 fibril formation, with A. dubius showing the highest chaperone activity and inhibition at the concentration assessed. Additionally, both protein extracts showed neuroprotective effects against Aβ1-40-induced toxicity. Overall, our data demonstrated that the plant-based proteins studied in this research work can effectively overcome one of the most important characteristics of AD.

QSAR Classification of Beta-Secretase 1 Inhibitor Activity in Alzheimer's Disease Using Ensemble Machine Learning Algorithms

This study focuses on the development of a machine learning ensemble approach for the classification of Beta-Secretase 1 (BACE1) inhibitors in Quantitative Structure-Activity Relationship (QSAR) analysis. BACE1 is an enzyme linked to the production of amyloid beta peptide, a significant component of Alzheimer's disease plaques. The discovery of effective BACE1 inhibitors is difficult, but QSAR modeling offers a cost-effective alternative by predicting the activity of compounds based on their chemical structures. This study evaluates the performance of four machine learning models (Random Forest, AdaBoost, Gradient Boosting, and Extra Trees) in predicting BACE1 inhibitor activity. Random Forest achieved the highest performance, with a training accuracy of 98.65% and a testing accuracy of 82.53%. In addition, it exhibited superior precision, recall, and F1-score. Random Forest's superior performance was a result of its ability to capture a wide variety of patterns and its randomized ensemble approach. Overall, this study demonstrates the efficacy of ensemble machine learning models, specifically Random Forest, in predicting the activity of BACE1 inhibitors. The findings contribute to ongoing efforts in Alzheimer's disease drug discovery research by providing a cost-effective and efficient strategy for screening and prioritizing potential BACE1 inhibitors.

Design and synthesis of multi-functional small-molecule based inhibitors of amyloid-β aggregation: Molecular modeling and in vitro evaluation.

Amyloid-β1-42 (Aβ42) peptide aggregate formation in the brain plays a crucial role in the onset and progression of Alzheimer's disease. According to published research, the Aβ monomer's amino acid residues KLVFF (16-20) self-associate to create antiparallel β-sheet fibrils. Small compounds can prevent self-assembly and destroy Aβ fibrils by attaching to the Aβ16-20 regions of Aβ42. To enhance biological characteristics and binding affinity to the amyloid beta peptide, β-sheet breaker small molecules can be developed and modified with various scaffolds. In the current study, a novel series of 2,3-disubstitutedbenzofuran derivatives was designed and created by fusing the benzofuran core of a known iron chelator, neuroprotective, and neurorestorative agent, like VK-28, with a motif found in the structure of a known muscarinic inhibitor and amyloid binding agent, like SKF-64346. Measurements of the binding affinity and in vitro aggregation inhibition of the Aβ42 peptide were made using the thioflavin T (ThT) test. Using AutoDock 4.2 software, molecular docking studies of the synthesized compounds were performed on the monomer and fibril structures of amyloid beta peptide. The compounds 8a-8g exhibited strong binding energy and affinity to Aβ fibrils as well as a 50%-67% reduction of the growth of Aβ aggregation. Finally, the positive traits of our recently synthesized compounds make them excellent candidates for additional in vivo testing as a "β-sheet breaking agent."

An approach for in-situ detection of gold colloid aggregates amyloid formations within the hippocampus of the Cohen’s Alzheimer’s disease rat model by surface enhanced raman scattering methods

BackgroundAmyloid beta (Aβ) peptides, such as Aβ1–40 or Aβ1–42 are regarded as hallmark neuropathological biomarkers associated with Alzheimer’s disease (AD). The formation of an aggregates by Aβ1–40 or Aβ1–42-coated gold nano-particles are hypothesized to contain conformation of Aβ oligomers, which could exist only at an initial stage of fibrillogenesis. New MethodThe attempt of in-situ detection of externally initiated gold colloid (ca. 80 nm diameter) aggregates in the middle section of the hippocampus of the Long Evans Cohen’s Alzheimer’s disease rat model was conducted through the Surface Enhanced Raman Scattering (SERS) method. ResultsThe SERS spectral features contained modes associated with β-sheet interactions and a significant number of modes that were previously reported in SERS shifts for Alzheimer diseased rodent and human brain tissues; thereby, strongly implying a containment of amyloid fibrils. The spectral patterns were further examined and compared with those collected from in-vitro gold colloid aggregates which were formed from Aβ1–40 - or Aβ1–42 -coated 80 nm gold colloid under pH ∼4, pH ∼7, and pH ∼10, and the best matched datasets were found with that of the aggregates of Aβ1–42 -coated 80 nm gold colloid at ∼pH 4.0. The morphology and physical size of this specific gold colloid aggregate was clearly different from those found in-vitro. Comparison with Existing Method(s)The amyloid fibril with a β-sheet conformation identified in previously reported in AD mouse/human brain tissues was involved in a formation of the gold colloid aggregates. However, to our surprise, best explanation for the observed SERS spectral features was possible with those in vitro Aβ1–42 -coated 80 nm gold colloid under pH ∼4. ConclusionsA formation of gold colloid aggregates was confirmed in the AD rat hippocampal brain section with unique physical morphology compared to those observed in in-vitro Aβ1–42 or Aβ1–40 mediated gold colloid aggregates. It was concluded that a β-sheet conformation identified in previously reported in AD mouse/human brain tissues was in volved in a formation of the gold colloid aggregates.

Molecular insights into the inhibition of early stages of Aβ peptide aggregation and destabilization of Alzheimer's Aβ protofibril by dipeptide D-Trp-Aib: A molecular modelling approach

Amyloid beta (Aβ) peptide aggregates rapidly into the soluble oligomers, protofibrils and fibrils to form senile plaques, a neurotoxic component and pathological hallmark of Alzheimer's disease (AD). Experimentally, it has been demonstrated the inhibition of an early stages of Aβ aggregation by a dipeptide D-Trp-Aib inhibitor, but its molecular mechanism is still unclear. Hence, in the present study, we used molecular docking and molecular dynamics (MD) simulations to explore the molecular mechanism of inhibition of an early oligomerization and destabilization of preformed Aβ protofibril by D-Trp-Aib. Molecular docking study showed that the D-Trp-Aib binds at the aromatic (Phe19, Phe20) region of Aβ monomer, Aβ fibril and hydrophobic core of Aβ protofibril. MD simulations revealed the binding of D-Trp-Aib at the aggregation prone region (Lys16-Glu22) resulted in the stabilization of Aβ monomer by π-π stacking interactions between Tyr10 and indol ring of D-Trp-Aib, which decreases the β-sheet content and increases the α-helices. The interaction between Lys28 of Aβ monomer to D-Trp-Aib could be responsible to block the initial nucleation and may impede the fibril growth and elongation. The loss of hydrophobic contacts between two β-sheets of Aβ protofibril upon binding of D-Trp-Aib at the hydrophobic cavity resulted in the partial opening of β-sheets. This also disrupts a salt bridge (Asp23-Lys28) leading to the destabilization of Aβ protofibril. Binding energy calculations revealed that van der Waals and electrostatic interactions maximally favours the binding of D-Trp-Aib to Aβ monomer and Aβ protofibril respectively. The residues Tyr10, Phe19, Phe20, Ala21, Glu22, Lys28 of Aβ monomer, whereas Leu17, Val18, Phe19, Val40, Ala42 of protofibril contributing for the interactions with D-Trp-Aib. Thus, the present study provides structural insights into the inhibition of an early oligomerization of Aβ peptides and destabilization of Aβ protofibril, which could be useful to design novel inhibitors for the treatment of AD.

Hypochlorite-induced oxidation promotes aggregation and reduces toxicity of amyloid beta 1-42

Exacerbated hypochlorite (OCl−) production is linked to neurodegenerative processes, but there is growing evidence that lower levels of hypochlorite activity are important to protein homeostasis. In this study we characterise the effects of hypochlorite on the aggregation and toxicity of amyloid beta peptide 1–42 (Aβ1-42), a major component of amyloid plaques that form in the brain in Alzheimer's disease. Our results demonstrate that treatment with hypochlorite promotes the formation of Aβ1-42 assemblies ≥100 kDa that have reduced surface exposed hydrophobicity compared to the untreated peptide. This effect is the result of the oxidation of Aβ1-42 at a single site as determined by mass spectrometry analysis. Although treatment with hypochlorite promotes the aggregation of Aβ1-42, the solubility of the peptide is enhanced and amyloid fibril formation is inhibited as assessed by filter trap assay, thioflavin T assay and transmission electron microscopy. The results of in vitro assays using SH-SY5Y neuroblastoma cells show that pre-treatment of Aβ1-42 with a sub-stoichiometric amount of hypochlorite substantially reduces its toxicity. The results of flow cytometry analysis and internalisation assays indicate that hypochlorite-induced modification of Aβ1-42 reduces its toxicity via at least two-distinct mechanism, reducing the total binding of Aβ1-42 to the surface of cells and facilitating the cell surface clearance of Aβ1-42 to lysosomes. Our data is consistent with a model in which tightly regulated production of hypochlorite in the brain is protective against Aβ-induced toxicity.

Biodegradable Poly(trehalose) Nanoparticle for Preventing Amyloid Beta Aggregation and Related Neurotoxicity.

Trehalose is a disaccharide that is capable of inhibiting protein aggregation and activating cellular autophagy. It has been shown that a polymer or nanoparticle form, terminated with multiple trehalose units, can significantly enhance the anti-amyloidogenic performance and is suitable for the treatment of neurodegenerative diseases. Here, we report a trehalose-conjugated polycarbonate-co-lactide polymer and formulation of its nanoparticles having multiple numbers of trehalose exposed on the surface. The resultant poly(trehalose) nanoparticle inhibits the aggregation of amyloid beta peptides and disintegrates matured amyloid fibrils into smaller fragments. Moreover, the poly(trehalose) nanoparticle lowers extracellular amyloid β oligomer-driven cellular stress and enhances cell viability. The presence of biodegradable polycarbonate components in the poly(trehalose) nanoparticle would enhance their application potential as an anti-amyloidogenic material.

The Roles of the Amyloid Beta Monomers in Physiological and Pathological Conditions.

Amyloid beta peptide is an important biomarker in Alzheimer's disease, with the amyloidogenic hypothesis as one of the central hypotheses trying to explain this type of dementia. Despite numerous studies, the etiology of Alzheimer's disease remains incompletely known, as the pathological accumulation of amyloid beta aggregates cannot fully explain the complex clinical picture of the disease. Or, for the development of effective therapies, it is mandatory to understand the roles of amyloid beta at the brain level, from its initial monomeric stage prior to aggregation in the form of senile plaques. In this sense, this review aims to bring new, clinically relevant data on a subject intensely debated in the literature in the last years. In the first part, the amyloidogenic cascade is reviewed and the possible subtypes of amyloid beta are differentiated. In the second part, the roles played by the amyloid beta monomers in physiological and pathological (neurodegenerative) conditions are illustrated based on the most relevant and recent studies published on this topic. Finally, considering the importance of amyloid beta monomers in the pathophysiology of Alzheimer's disease, new research directions with diagnostic and therapeutic impacts are suggested.

BACE1 regulates expression of Clusterin in astrocytes for enhancing clearance of β-amyloid peptides

BackgroundAbnormal accumulation of amyloid beta peptide (Aβ) in the brain induces a cascade of pathological changes in Alzheimer’s disease (AD), and inhibiting BACE1, which is required for Aβ generation, is therefore being explored for the treatment of AD by reducing Aβ accumulation. As Bace1 knockout mice exhibit increased number of reactive astrocytes and AD brains have reactive astrocytes that surround amyloid plaques, we investigated the role of BACE1 in astrocytes and determined whether BACE1 regulates astrocytic functions.MethodsWe conducted unbiased single cell RNA-seq (scRNA-seq) using purified astrocytes from Bace1 KO mice and wild type control littermates. Similar scRNA-seq was also conducted using AD mice with conditional deletion of Bace1 in the adult stage (5xFAD;Bace1fl/fl;UBC-creER compared to 5xFAD;Bace1fl/fl controls). We compared the transcriptomes of astrocyte and reactive astrocyte clusters and identified several differentially expressed genes, which were further validated using Bace1 KO astrocyte cultures. Mice with astrocyte-specific Bace1 knockout in 5xFAD background were used to compare amyloid deposition. Mechanistic studies using cultured astrocytes were used to identify BACE1 substrates for changes in gene expression and signaling activity. ResultsAmong altered genes, Clusterin (Clu) and Cxcl14 were significantly upregulated and validated by measuring protein levels. Moreover, BACE1 deficiency enhanced both astrocytic Aβ uptake and degradation, and this effect was significantly attenuated by siRNA knockdown of Clu. Mechanistic study suggests that BACE1 deficiency abolishes cleavage of astrocytic insulin receptors (IR), and this may enhance expression of Clu and Cxcl14. Acutely isolated astrocytes from astrocyte-specific knockout of Bace1 mice (Bace1 fl/fl;Gfap-cre) show similar increases in CLU and IR. Furthermore, astrocyte-specific knockout of Bace1 in a 5xFAD background resulted in a significant attenuation in cortical Aβ plaque load through enhanced clearance.ConclusionTogether, our study suggests that BACE1 in astrocytes regulates expression of Clu and Cxcl14, likely via the control of insulin receptor pathway, and inhibition of astrocytic BACE1 is a potential alternative strategy for enhancing Aβ clearance.

Chloramphenicol Derivatization in Its Primary Hydroxyl Group with Basic Amino Acids Leads to New Pharmacophores with High Antimicrobial Activity.

In a previous study published by our group, successful modification of the antibiotic chloramphenicol (CHL) was reported, which was achieved by replacing the dichloroacetyl tail with alpha and beta amino acids, resulting in promising new antibacterial pharmacophores. In this study, CHL was further modified by linking the basic amino acids lysine, ornithine, and histidine to the primary hydroxyl group of CHL via triazole, carbamate, or amide bonding. Our results showed that while linking the basic amino acids retained antibacterial activity, it was somewhat reduced compared to CHL. However, in vitro testing demonstrated that all derivatives were comparable in activity to CHL and competed for the same ribosomal binding site with radioactive chloramphenicol. The amino acid-CHL tethering modes were evaluated either with carbamate (7, 8) derivatives, which exhibited higher activity, or with amide- (4-6) or triazole-bridged compounds (1-3), which were equally potent. Our findings suggest that these new pharmacophores have potential as antimicrobial agents, though further optimization is needed.

The Role of Ryanodine Receptors in Regulating Neuronal Activity and Its Connection to the Development of Alzheimer's Disease.

Research into the early impacts of Alzheimer's disease (AD) on synapse function is one of the most promising approaches to finding a treatment. In this context, we have recently demonstrated that the Abeta42 peptide, which builds up in the brain during the processing of the amyloid precursor protein (APP), targets the ryanodine receptors (RyRs) of mouse hippocampal neurons and potentiates calcium (Ca2+) release from the endoplasmic reticulum (ER). The uncontrolled increase in intracellular calcium concentration ([Ca2+]i), leading to the development of Ca2+ dysregulation events and related excitable and synaptic dysfunctions, is a consolidated hallmark of AD onset and possibly other neurodegenerative diseases. Since RyRs contribute to increasing [Ca2+]i and are thought to be a promising target for AD treatment, the goal of this review is to summarize the current level of knowledge regarding the involvement of RyRs in governing neuronal function both in physiological conditions and during the onset of AD.

Development of a MATLAB-based Toolkit to Quantify Neurodegenerative Histopathology Markers Applied to the Evaluation of a CRISPR-based Therapeutic for Alzheimer’s Disease (P3-6.003)

<h3>Objective:</h3> The objective of this study was to develop a comprehensive, easy-to-use set of MATLAB-based tools to quantify Alzheimer’s disease (AD) associated histopathological markers in immunolabeled brain tissue. We then utilized this MATLAB-based toolkit to assess the efficacy of a novel CRISPR-based, amyloid precursor protein (APP)-editing approach in a mouse model of AD. <h3>Background:</h3> AD is characterized immunohistologically by the presence of extracellular amyloid plaque deposits and associated neuroinflammation throughout the cerebral cortex and hippocampus. Amyloid plaques are aggregates of amyloid beta (Aβ) peptides that are released by successive cleavages of full length APP. Our lab has developed a novel CRISPR-based, APP-editing approach that effectively deletes the YENPTY endocytic domain at the C-terminus of APP, thus attenuating pathologic beta-cleavage and up-regulating neuroprotective alpha-cleavage. In order to assess the efficacy of our approach in vivo, we used CRISPRs to generate germ-line and somatic edits in an APP knock in (APP NL/G/F) mouse model of AD. <h3>Design/Methods:</h3> <h3>Immunohistochemistry (IHC):</h3> Tissues from appropriately aged control and CRISPR-edited APP NL/G/F mice were immunostained for Aβ plaques (6E10 antibodies), astrocytes (anti-GFAP antibodies) and microglia (anti-Iba1 antibodies). <h3>MATLAB analyses:</h3> After images were optimally contrasted, a color-channel filter was applied to isolate cells and plaques from the background. Our MATLAB program was then able to segment individual cells/plaques and measure their area, distance to nearby cells/plaques, and percentage of overlap. Activated microglia were defined as those with an area of 25 μm² or greater. <h3>Results:</h3> MATLAB quantification of IHC-stained sections revealed a robust decrease in Aβ deposition and reactive astrogliosis in CRISPR-treated APP NL/G/F tissues. These results matched supporting qualitative assessments and biochemical analyses. <h3>Conclusions:</h3> Our MATLAB toolkit can reliably quantify common neuropathological IHC markers and may be useful for evaluating the efficacy of neurotherapeutics. <b>Disclosure:</b> Mr. Satyadev has nothing to disclose. Dr. Aulston has nothing to disclose. Miss Branes has nothing to disclose. Miss Checka has nothing to disclose. Dr. Roy has stock in CRISPRAlz. The institution of Dr. Roy has received research support from Beam Therapeutics. Dr. Roy has received intellectual property interests from a discovery or technology relating to health care.

Sideritis scardica Extracts Demonstrate Neuroprotective Activity against Aβ25-35 Toxicity.

Alzheimer's disease (AD) is the most prevalent neurodegenerative condition, primarily affecting seniors. Despite the significant time and money spent over the past few decades, no therapy has been developed yet. In recent years, the research has focused on ameliorating the cytotoxic amyloid beta (Aβ) peptide aggregates and the increased elevated oxidative stress, two interconnected main AD hallmarks. Medicinal plants constitute a large pool for identifying bioactive compounds or mixtures with a therapeutic effect. Sideritis scardica (SS) has been previously characterized as neuroprotective toward AD. We investigated this ability of SS by generating eight distinct solvent fractions, which were chemically characterized and assessed for their antioxidant and neuroprotective potential. The majority of the fractions were rich in phenolics and flavonoids, and all except one showed significant antioxidant activity. Additionally, four SS extracts partly rescued the viability in Aβ25-35-treated SH-SY5Y human neuroblastoma cells, with the initial aqueous extract being the most potent and demonstrating similar activity in retinoic-acid-differentiated cells as well. These extracts were rich in neuroprotective substances, such as apigenin, myricetin-3-galactoside, and ellagic acid. Our findings indicate that specific SS mixtures can benefit the pharmaceutical industry to develop herbal drugs and functional food products that may alleviate AD.