Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the ß-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

Associations of CSF BACE1 with amyloid pathology, neurodegeneration, and cognition in Alzheimer's disease.

Β-site amyloid precursor protein (APP) cleaving enzyme (BACE1) is a crucial protease in the production of amyloid-ß (Aß) in Alzheimer's disease (AD) patients. However, the side effects observed in clinical trials of BACE1 inhibitors, including reduction in brain volume and cognitive worsening, suggest that the exact role of BACE1 in AD pathology is not fully understood. To further investigate this, we examined cerebrospinal fluid (CSF) levels of BACE1 and its cleaved product sAPPß that reflects BACE1 activity in the China Aging and Neurodegenerative Disorder Initiative cohort. We found significant correlations between CSF BACE1 or sAPPß levels and CSF Aß40, Aß42, and Aß42/Aß40 ratio, but not with amyloid deposition detected by 18F-Florbetapir PET. Additionally, CSF BACE1 and sAPPß levels were positively associated with cortical thickness in multiple brain regions, and higher levels of sAPPß were linked to increased cortical glucose metabolism in frontal and supramarginal areas. Interestingly, individuals with higher baseline levels of CSF BACE1 exhibited slower rates of brain volume reduction and cognitive worsening over time. This suggests that increased levels and activity of BACE1 may not be the determining factor for amyloid deposition, but instead, may be associated with increased neuronal activity and potentially providing protection against neurodegeneration in AD.

Navigating the Maze of Alzheimer's disease by exploring BACE1: Discovery, current scenario, and future prospects.

Alzheimer's disease (AD) is a chronic neurological condition that has become a leading cause of cognitive decline in elder individuals. Hardly any effective medication has been developed to halt the progression of AD due to the disease's complexity. Several theories have been put forward to clarify the mechanisms underlying AD etiology. The identification of amyloid plaques as a hallmark of AD has sparked the development of numerous drugs targeting the players involved in the amyloidogenic pathway, such as the ß-site of amyloid precursor protein cleavage enzyme 1 (BACE1) blockers. Over the last ten years, preclinical and early experimental research has led several pharmaceutical companies to prioritize producing BACE1 inhibitors. Despite all these efforts, earlier discovered inhibitors were discontinued in consideration of another second-generation small molecules and recent BACE1 antagonists failed in the final stages of clinical trials because of the complications associated either with toxicity or effectiveness. In addition to discussing the difficulties associated with development of BACE1 inhibitors, this review aims to provide an overview of BACE1 and offer perspectives on the causes behind the failure of five recent BACE1 inhibitors, that would be beneficial for choosing effective treatment approaches in the future.

Box-Behnken Design-Based Optimization of Phytochemical Extraction from Diplazium esculentum (Retz.) Sw. Associated with Its Antioxidant and Anti-Alzheimer's Properties.

A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, ß-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

ER-to-lysosome-associated degradation acts as failsafe mechanism upon ERAD dysfunction.

The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.

A Strategy for Allowing Earlier Diagnosis and Rigorous Evaluation of BACE1 Inhibitors in Preclinical Alzheimer's Disease.

Given continued failure of BACE1 inhibitor programs at symptomatic and prodromal stages of Alzheimer's disease (AD), clinical trials need to target the earlier preclinical stage. However, trial design is complex in this population with negative diagnosis of classical hippocampal amnesia on standard memory tests. Besides recent advances in brain imaging, electroencephalogram, and fluid-based biomarkers, new cognitive markers should be established for earlier diagnosis that can optimize recruitment to BACE1 inhibitor trials in presymptomatic AD. Notably, accelerated long-term forgetting (ALF) is emerging as a sensitive cognitive measure that can discriminate between asymptomatic individuals with high risks for developing AD and healthy controls. ALF is a form of declarative memory impairment characterized by increased forgetting rates over longer delays (days to months) despite normal storage within the standard delays of testing (20-60 min). Therefore, ALF may represent a harbinger of preclinical dementia and the impairment of systems memory consolidation, during which memory traces temporarily stored in the hippocampus become gradually integrated into cortical networks. This review provides an overview of the utility of ALF in a rational design of next-generation BACE1 inhibitor trials in preclinical AD. I explore potential mechanisms underlying ALF and relevant early-stage biomarkers useful for BACE1 inhibitor evaluation, including synaptic protein alterations, astrocytic dysregulation and neuron hyperactivity in the hippocampal-cortical network. Furthermore, given the physiological role of the isoform BACE2 as an AD-suppressor gene, I also discuss the possible association between the poor selectivity of BACE1 inhibitors and their side effects (e.g., cognitive worsening) in prior clinical trials.

Melatonin Inhibits Hypoxia-Induced Alzheimer's Disease Pathogenesis by Regulating the Amyloidogenic Pathway in Human Neuroblastoma Cells.

Stroke and Alzheimer's disease (AD) are prevalent age-related diseases; however, the relationship between these two diseases remains unclear. In this study, we aimed to investigate the ability of melatonin, a hormone produced by the pineal gland, to alleviate the effects of ischemic stroke leading to AD by observing the pathogenesis of AD hallmarks. We utilized SH-SY5Y cells under the conditions of oxygen-glucose deprivation (OGD) and oxygen-glucose deprivation and reoxygenation (OGD/R) to establish ischemic stroke conditions. We detected that hypoxia-inducible factor-1α (HIF-1α), an indicator of ischemic stroke, was highly upregulated at both the protein and mRNA levels under OGD conditions. Melatonin significantly downregulated both HIF-1α mRNA and protein expression under OGD/R conditions. We detected the upregulation of ß-site APP-cleaving enzyme 1 (BACE1) mRNA and protein expression under both OGD and OGD/R conditions, while 10 µM of melatonin attenuated these effects and inhibited beta amyloid (Aß) production. Furthermore, we demonstrated that OGD/R conditions were able to activate the BACE1 promoter, while melatonin inhibited this effect. The present results indicate that melatonin has a significant impact on preventing the aberrant development of ischemic stroke, which can lead to the development of AD, providing new insight into the prevention of AD and potential stroke treatments.

Synthesis of new N-(5,6-methylenedioxybenzothiazole-2-yl)-2-[(substituted)thio/piperazine]acetamide/propanamide derivatives and evaluation of their AChE, BChE, and BACE-1 inhibitory activities.

In this study, the synthesis of N-(5,6-methylenedioxybenzothiazole-2-yl)-2-[(substituted)thio/piperazine]acetamide/propanamide derivatives (3a-3k) and to investigate their acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and ß-secretase 1 (BACE-1) inhibition activity were aimed. Mass, 1H NMR, and 13C NMR spectra were utilized to determine the structure of the synthesized compounds. Compounds 3b, 3c, 3f, and 3j showed AChE inhibitory activity which compound 3c (IC50 = 0.030 ± 0.001 µM) showed AChE inhibitory activity as high as the reference drug donepezil (IC50 = 0.0201 ± 0.0010 µM). Conversely, none of the compounds showed BChE activity. Compounds 3c and 3j showed the highest BACE-1 inhibitory activity and IC50 value was found as 0.119 ± 0.004 µM for compound 3j whereas IC50 value was 0.110 ± 0.005 µM for donepezil, which is one of the reference substance. Molecular docking studies have been carried out using the data retrieved from the server of the Protein Data Bank (PDBID: 4EY7 and 2ZJM). Using in silico approach behavior active compounds (3c and 3j) and their binding modes clarified.

STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells.

Staufen-1 (STAU1) is a double-stranded RNA-binding protein (RBP) involved in a variety of pathological conditions. In this study, we investigated the potential role of STAU1 in Alzheimer's disease (AD), in which two hallmarks are well-established as cerebral ß-amyloid protein (Aß) deposition and Tau-centered neurofibrillary tangles. We found that STAU1 protein level was significantly increased in cells that stably express full-length APP and the brain of APP/PS1 mice, an animal model of AD. STAU1 knockdown, as opposed to overexpression, significantly decreased the protein levels of ß-amyloid converting enzyme 1 (BACE1) and Aß. We further found that STAU1 extended the half-life of the BACE1 mRNA through binding to the 3' untranslated region (3'UTR). Transcriptome analysis revealed that STAU1 enhanced the expression of growth arrest and DNA damage 45 ß (GADD45B) upstream of P38 MAPK signaling, which contributed to STAU1-induced regulation of Tau phosphorylation at Ser396 and Thr181. Together, STAU1 promoted amyloidogenesis by inhibiting BACE1 mRNA decay, and augmented Tau phosphorylation through activating GADD45B in relation to P38 MAPK. Targeting STAU1 that acts on both amyloidogenesis and tauopathy may serve as an optimistic approach for AD treatment.

Short-term regulation of TSFM level does not alter amyloidogenesis and mitochondrial function in type-specific cells.

BACKGROUND: Mitochondrial Ts translation elongation factor (TSFM) is an enzyme that catalyzes exchange of guanine nucleotides. By forming a complex with mitochondrial Tu translation elongation factor (TUFM), TSFM participates in mitochondrial protein translation. We have previously reported that TUFM regulates translation of beta-site APP cleaving enzyme 1 (BACE1) via ROS (reactive oxygen species)-dependent mechanism, suggesting a potential role in amyloid precursor protein (APP) processing associated with Alzheimer's disease (AD), which led to the speculation that TSFM may regulate APP processing in a similar way to TUFM. METHODS AND RESULTS: Here, we report that in cultured cells, knockdown or overexpression TSFM did not change protein levels in BACE1 and APP. Besides, the levels of cytoplasmic ROS and mitochondrial superoxide, in addition to ATP level, cell viability and mitochondrial membrane potential were not significantly altered by TSFM knockdown in the short term. Further transcriptome analysis revealed that expression of majority of mitochondrial genes were not remarkably changed by TSFM silencing. The possibility of TSFM involved in cardiomyopathy and cancer development was uncovered using bioinformatics analysis. CONCLUSIONS: Collectively, short-term regulation of TSFM level in cultured cells does not cause a significant change in proteins involved in APP processing, levels in ROS and ATP associated with mitochondrial function. Whereas our study could contribute to comprehend certain clinical features of TSFM mutations, the roles of TSFM in cardiomyopathy and cancer development might deserve further investigation.

Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy.

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), ß secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aß aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 µM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 µM) along with good anti-Aß aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 µM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aß-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand-protein confirmation throughout the simulation run.

Biomimetic Nanovesicles as a Dual Gene Delivery System for the Synergistic Gene Therapy of Alzheimer's Disease.

The association between dysfunctional microglia and amyloid-ß (Aß) is a fundamental pathological event and increases the speed of Alzheimer's disease (AD). Additionally, the pathogenesis of AD is intricate and a single drug may not be enough to achieve a satisfactory therapeutic outcome. Herein, we reported a facile and effective gene therapy strategy for the modulation of microglia function and intervention of Aß anabolism by ROS-responsive biomimetic exosome-liposome hybrid nanovesicles (designated as TSEL). The biomimetic nanovesicles codelivery ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) siRNA (siBACE1) and TREM2 plasmid (pTREM2) gene drug efficiently penetrate the blood-brain barrier and enhance the drug accumulation at AD lesions with the help of exosomes homing ability and angiopep-2 peptides. Specifically, an upregulation of TREM2 expression can reprogram microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype while also restoring its capacity to phagocytose Aß and its nerve repair function. In addition, siRNA reduces the production of Aß plaques at the source by knocking out the BACE1 gene, which is expected to further enhance the therapeutic effect of AD. The in vivo study suggests that TSEL through the synergistic effect of two gene drugs can ameliorate APP/PS1 mice cognitive impairment by regulating the activated microglial phenotype, reducing the accumulation of Aß, and preventing the retriggering of neuroinflammation. This strategy employs biomimetic nanovesicles for the delivery of dual nucleic acids, achieving synergistic gene therapy for AD, thus offering more options for the treatment of AD.

Assessment of fragment docking and scoring with the endothiapepsin model system.

Fragment-based screening has become indispensable in drug discovery. Yet, the weak binding affinities of these small molecules still represent a challenge for the reliable detection of fragment hits. The extent of this issue was illustrated in the literature for the aspartic protease endothiapepsin: When seven biochemical and biophysical in vitro screening methods were applied to screen a library of 361 fragments, very poor overlap was observed between the hit fragments identified by the individual approaches, resulting in high levels of false positive and/or false negative results depending on the mutually compared methods. Here, the reported in vitro findings are juxtaposed with the results from in silico docking and scoring approaches. The docking programs GOLD and Glide were considered with the scoring functions ASP, ChemScore, ChemPLP, GoldScore, DSXCSD, and GlideScore. First, the ranking power and scoring power were assessed for the named scoring functions. Second, the capability of reproducing the crystallized fragment binding modes was tested in a structure-based redocking approach. The redocking success notably depended on the ligand efficiency of the considered fragments. Third, a blinded virtual screening approach was employed to evaluate whether in silico screening can compete with in vitro methods in the enrichment of fragment databases.

Reducing Nogo-B Improves Hepatic Fibrosis by Inhibiting BACe1-Mediated Autophagy.

BACKGROUND: Hepatic fibrosis (HF) is a histopathological change in the process of long-term liver injury caused by cytokine secretion and internal environment disturbance, resulting in excessive liver repair and fiber scar. Nogo-B protein is widely distributed in peripheral tissues and organs and can regulate the migration of endothelial cells by activating TGF-ß1 in vascular remodeling after injury. Nogo-B has been shown to promote organ fibrosis. This study was to determine the role of Nogo-B in HF. METHODS: An HF model was built by intraperitoneal injections with 20% carbon tetrachloride. Localization of Nogo-B was detected by FISH. The interaction between Nogo-B and BACE1 was confirmed by Co-IP. Autophagy flux was analyzed using tandem mRFP-GFP-LC3 fluorescence microscopy, electron microscopy, and western blotting. Detection of serum AST and ALT and H&E staining were utilized to detect the degree of liver injury. The HF was evaluated by Masson trichromatic staining. RT-qPCR, western blotting, and immunofluorescence were employed to detect relevant indicators. RESULTS: Reducing Nogo-B suppressed AST and ALT levels, the accumulation of collagen I and α-SMA, and expressions of pro-fibrotic genes in mouse liver. BACE1 was a potential downstream target of Nogo-B. Nogo-B was upregulated in TGF-ß1-activated hepatic stellate cells (HSCs). Knocking down Nogo-B caused the downregulation of pro-fibrotic genes and inhibited viability of HSCs. Nogo-B knockdown prevented CCL4-induced fibrosis, accompanied by downregulation of extracellular matrix. Nogo-B inhibited HSC autophagy and increased lipid accumulation. BACE1 knockdown inhibited HSC autophagy and activation in LX-2 cells. CONCLUSION: Nogo-B knockdown prevents HF by directly inhibiting BACe1-mediated autophagy.

[Expression relationship and significance of NEAT1 and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease].

OBJECTIVE: To explore the expression relationship and significance of long chain non-coding RNA nuclear-enriched abundant transcript 1 (LncRNA NEAT1) and miR-27a-3p in serum and cerebrospinal fluid of patients with Alzheimer disease (AD). METHODS: Sixty-six AD patients received by the department of neurology of our hospital from October 2019 to September 2021 were gathered, according to the clinical dementia rating scale score, they were grouped into mild group (≤1 point, n=41) and moderate-to-severe group (>1 point, n=25). Another 66 cases of serum and cerebrospinal fluid samples from outpatient physical examination personnel were regarded as the control group. The general information on all subjects was recorded and cognition was assessed; real-time quantitative PCR was performed to measure the expression levels of miR-27a-3p and NEAT1 in serum and cerebrospinal fluid; enzyme-linked immunosorbent assay was performed to measure the protein levels of ß-amyloid precursor protein cleaving enzyme 1 (BACE1), ß-amyloid (Aß) 40 and Aß42 in cerebrospinal fluid; Spearman' s method was performed to analyze the correlation of serum miR-27a-3p and NEAT1 levels with mini-mental state examination (MMSE) and montreal cognitive assessment (MoCA) scores; Pearson method was performed to analyze the correlation between serum miR-27a-3p and NEAT1 levels and Aß deposition standard uptake value ratio (SUVR) and cerebrospinal fluid miR-27a-3p, NEAT1, BACE1, Aß42 and Aß40 levels. RESULTS: The MMSE score [21 (17, 25), 9(7, 11) vs. 27 (21, 34)], MoCA score [17 (12, 21), 10 (7, 13) vs. 27 (21, 31)], serum miR-27a-3p level (0.55±0.13, 0.46±0.06 vs. 0.97±0.22), cerebrospinal fluid miR-27a-3p (0.48±0.10, 0.35±0.10 vs. 1.03±0.31), Aß42 levels [(303.55±36.77) ng/L, (231.45±34.14) ng/L vs. (499.99±53.63) ng/L] and Aß42/Aß40 ratio (0.030±0.008, 0.022±0.007 vs. 0.048±0.010) of AD patients in mild group and moderate-to-severe group were all lower than those in the control group, and the moderate-to-severe group were lower than the mild group (all P < 0.05); the serum NEAT1 level (2.31±0.64, 3.13±0.76 vs. 1.05±0.20), SUVR (1.50±0.29, 1.76±0.52 vs. 0.74±0.15), and cerebrospinal fluid NEAT1 (3.51±1.24, 4.30±1.65 vs. 1.01±0.23) and BACE1 levels [(55.78±5.98) µg/L, (72.32±16.08) µg/L vs. (21.39±3.73) µg/L] were higher than those in the control group, and the moderate-to-severe group were higher than the mild group (all P < 0.05). Serum NEAT1 level in AD patients was positively correlated with SUVR, cerebrospinal fluid NEAT1 and BACE1 (r=0.350, 0.606, 0.341, P < 0.05), and negatively correlated with MMSE score and MoCA score (r=-0.473, -0.482, all P < 0.05); serum miR-27a-3p level was positively correlated with cerebrospinal fluid miR-27a-3p level, MMSE score and MoCA score (r=0.695, 0.424, 0.412, all P < 0.05), and negatively correlated with SUVR and cerebrospinal fluid BACE1 level (r=-0.521, -0.447, all P < 0.05). CONCLUSION: The expression trends of NEAT1 and miR-27a-3p in the serum and cerebrospinal fluid of AD patients are consistent, the level of NEAT1 is increased, and the level of miR-27a-3p is decreased. The levels of the two are negatively correlated, which is related to the degree of Aß deposition in the brain of AD patients and is involved in the progression of AD.

Genome-Wide Identification of the SPP/SPPL Gene Family and BnaSPPL4 Regulating Male Fertility in Rapeseed (Brassica napus L.).

Signal peptide peptidase (SPP) and its homologs, signal peptide peptidase-like (SPPL) proteases, are members of the GxGD-type aspartyl protease family, which is widespread in plants and animals and is a class of transmembrane proteins with significant biological functions. SPP/SPPLs have been identified; however, the functions of SPP/SPPL in rapeseed (Brassica napus L.) have not been reported. In this study, 26 SPP/SPPLs were identified in rapeseed and categorized into three groups: SPP, SPPL2, and SPPL3. These members mainly contained the Peptidase_A22 and PA domains, which were distributed on 17 out of 19 chromosomes. Evolutionary analyses indicated that BnaSPP/SPPLs evolved with a large number of whole-genome duplication (WGD) events and strong purifying selection. Members are widely expressed and play a key role in the growth and development of rapeseed. The regulation of rapeseed pollen fertility by the BnaSPPL4 gene was further validated through experiments based on bioinformatics analysis, concluding that BnaSPPL4 silencing causes male sterility. Cytological observation showed that male infertility caused by loss of BnaSPPL4 gene function occurs late in the mononucleate stage due to microspore dysplasia.

In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context.

Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707.

BACE1 Inhibition Utilizing Organic Compounds Holds Promise as a Potential Treatment for Alzheimer's and Parkinson's Diseases.

Background: Neurological disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) manifest through gradually deteriorating cognitive functions. An encouraging strategy for addressing these disorders involves the inhibition of precursor-cleaving enzyme 1 (BACE1). Objectives: In the current research, a virtual screening technique was employed to identify potential BACE1 inhibitors among selected herbal isolates. Methods: This study evaluated 79 flavonoids, anthraquinones (AQs), and cinnamic acid derivatives for their potential blood-brain barrier (BBB) permeability. Using the AutoDock 4.0 tool, molecular docking analysis was conducted to determine the binding affinity of BBB permeable compounds to the BACE1 active site. Molecular dynamics (MD) simulations were performed to assess the stability of the docked poses of the most potent inhibitors. The interactions between the most effective plant-based inhibitors and the residues within the BACE1 catalytic site were examined before and after MD simulations. Results: Ponciretin, danthron, chrysophanol, and N-p-coumaroyltyramine were among the highest-ranking BACE1 inhibitors, with inhibition constant values calculated in the nanomolar range. Furthermore, during 10 ns simulations, the docked poses of these ligands were observed to be stable. Conclusion: The findings propose that ponciretin, danthron, chrysophanol, and N-p-coumaroyltyramine might serve as potential choices for the treatment of AD and PD, laying the groundwork for the creation of innovative BACE1 inhibitors.