Structural analysis of the pyroglutamate‐modified isoform of the Alzheimer's disease‐related amyloid‐β using NMR spectroscopy

The aggregation of the Aβ plays a fundamental role in the pathology of AD. Recently, N‐terminally modified Aβ species, pE‐Aβ, have been described as major constituents of Aβ deposits in the brains of AD patients. pE‐Aβ has an increased aggregation propensity and shows increased toxicity compared with Aβ1‐40 and Aβ1‐42. In the present work, high‐resolution NMR spectroscopy was performed to study pE‐Aβ3‐40 in aqueous TFE‐containing solution. Two‐dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE‐Aβ3‐40 was shown to contain two helical regions formed by residues 14–22 and 30–36. This is similar as previously described for Aβ1‐40. However, the secondary chemical shift data indicate decreased helical propensity in pE‐Aβ3‐40 when compared with Aβ1‐40 under exactly the same conditions. This is in agreement with the observation that pE‐Aβ3‐40 shows a drastically increased tendency to form β‐sheet‐rich structures under more physiologic conditions. Structural studies of pE‐Aβ are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE‐Aβ for the pathogenesis of AD. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Annexin A1 restores Aβ1‐42‐induced blood–brain barrier disruption through the inhibition of RhoA‐ROCK signaling pathway

The blood–brain barrier (BBB) is composed of brain capillary endothelial cells and has an important role in maintaining homeostasis of the brain separating the blood from the parenchyma of the central nervous system (CNS). It is widely known that disruption of the BBB occurs in various neurodegenerative diseases, including Alzheimer's disease (AD). Annexin A1 (ANXA1), an anti‐inflammatory messenger, is expressed in brain endothelial cells and regulates the BBB integrity. However, its role and mechanism for protecting BBB in AD have not been identified. We found that β‐Amyloid 1‐42 (Aβ42)‐induced BBB disruption was rescued by human recombinant ANXA1 (hrANXA1) in the murine brain endothelial cell line bEnd.3. Also, ANXA1 was decreased in the bEnd.3 cells, the capillaries of 5XFAD mice, and the human serum of patients with AD. To find out the mechanism by which ANXA1 recovers the BBB integrity in AD, the RhoA‐ROCK signaling pathway was examined in both Aβ42‐treated bEnd.3 cells and the capillaries of 5XFAD mice as RhoA was activated in both cases. RhoA inhibitors alleviated Aβ42‐induced BBB disruption and constitutively overexpressed RhoA‐GTP (active form of RhoA) attenuated the protective effect of ANXA1. When pericytes were cocultured with bEnd.3 cells, Aβ42‐induced RhoA activation of bEnd.3 cells was inhibited by the secretion of ANXA1 from pericytes. Taken together, our results suggest that ANXA1 restores Aβ42‐induced BBB disruption through inhibition of RhoA‐ROCK signaling pathway and we propose ANXA1 as a therapeutic reagent, protecting against the breakdown of the BBB in AD.     

Penetration and differentiation of cephalic neural crest‐derived cells in the developing mouse telencephalon

Neural crest (NC) cells originate from the neural folds and migrate into the various embryonic regions where they differentiate into multiple cell types. A population of cephalic neural crest‐derived cells (NCDCs) penetrates back into the developing forebrain to differentiate into microvascular pericytes, but little is known about when and how cephalic NCDCs invade the telencephalon and differentiate into pericytes. Using a transgenic mouse line in which NCDCs are genetically labeled with enhanced green fluorescent protein (EGFP), we observed that NCDCs started to invade the telencephalon together with endothelial cells from embryonic day (E) 9.5. A majority of NCDCs located in the telencephalon expressed pericyte markers, that is, PDGFRβ and NG2, and differentiated into pericytes around E11.5. Surprisingly, many of the NC‐derived pericytes express p75, an undifferentiated NCDC marker at E11.5, as well as NCDCs in the mesenchyme. At the same time, a minor population of NCDCs that located separately from blood vessels in the telencephalon were NG2‐negative and some of these NCDCs also expressed p75. Proliferation and differentiation of pericytes appeared to occur in a specific mesenchymal region where blood vessels penetrated into the telencephalon. These results indicate that (i) NCDCs penetrate back into the telencephalon in parallel with angiogenesis, (ii) many NC‐derived pericytes may be still in pre‐mature states even though after differentiation into pericytes in the early developing stages, (iii) a small minority of NCDCs may retain undifferentiated states in the developing telencephalon, and (iv) a majority of NCDCs proliferate and differentiate into pericytes in the mesenchyme around the telencephalon.     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Sirtuin 3 is essential for hypertension‐induced cardiac fibrosis via mediating pericyte transition

Hypertension is the key factor for the development of cardiac fibrosis and diastolic dysfunction. Our previous study showed that knockout of sirtuin 3 (SIRT3) resulted in diastolic dysfunction in mice. In the present study, we explored the role of SIRT3 in angiotensin II (Ang‐II)–induced cardiac fibrosis and pericyte‐myofibroblast transition. NG2 tracing reporter NG2‐DsRed mouse was crossed with wild‐type (WT) mice and SIRT3KO mice. Cardiac function, cardiac fibrosis and reactive oxygen species (ROS) were measured. Mice infused with Ang‐II for 28 days showed a significant reduction of SIRT3 expression in the mouse hearts. Knockout of SIRT3 sensitized Ang‐II‐induced elevation of isovolumic relaxation time (IVRT) and reduction of ejection fraction (EF) and fractional shortening (FS). Ang‐II‐induced cardiac fibrosis, capillary rarefaction and hypertrophy were further enhanced by knockout of SIRT3. NG2 pericyte tracing reporter mice infused with Ang‐II had a significantly increased number of NG2‐DsRed pericyte in the heart. Knockout of SIRT3 further enhanced Ang‐II‐induced increase of pericytes. To examine pericyte‐myofibroblast/fibroblast transition, DsRed pericytes were co‐stained with FSP‐1 and α‐SMA. Ang‐II infusion led to a significant increase in numbers of DsRed⁺/FSP‐1⁺ and DsRed⁺/α‐SMA⁺ cells, while SIRT3KO further developed pericyte‐myofibroblast/fibroblast transition. In addition, knockout of SIRT3 promoted Ang‐II‐induced NADPH oxidase‐derived ROS formation together with increased expression of transforming growth factor beta 1 (TGF‐β1). We concluded that Ang‐II induced cardiac fibrosis partly by the mechanisms involving SIRT3‐mediated pericyte‐myofibroblast/fibroblast transition and ROS‐TGF‐β1 pathway.     

Calpain cleavage and inactivation of the sodium calcium exchanger‐3 occur downstream of Aβ in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β‐amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase‐3, activity is a prominent feature of AD brain. In addition, we observe increased calpain‐mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain‐dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.     

Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse

Tightly regulated crosstalk between endothelial cells and pericytes is required for formation and maintenance of functional blood vessels. When the NG2 proteoglycan is absent from pericyte surfaces, vascularization of syngeneic tumors growing in the C57Bl/6 mouse brain is aberrant in several respects, resulting in retardation of tumor progression. In the NG2 null mouse brain, pericyte investment of the tumor vascular endothelium is reduced, causing deficiencies in both pericyte and endothelial cell maturation, as well as reduced basal lamina assembly. While part of this deficit may be due to the previously-identified role of NG2 in β1 integrin-dependent periyte/endothelial cell crosstalk, the ablation of NG2 also appears responsible for loss of collagen VI anchorage, in turn leading to reduced collagen IV deposition. Poor functionality of tumor vessels in NG2 null brain is reflected by reduced vessel patency and increased vessel leakiness, resulting in large increases in tumor hypoxia. These findings demonstrate the importance of NG2-dependent pericyte/endothelial cell interaction in the development and maturation of tumor blood vessels, identifying NG2 as a potential target for anti-angiogenic cancer therapy.     

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß‐Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti‐Aβ‐antibodies, termed Aβ‐nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ‐specific nanobodies was identified by proteolytic epitope extraction‐ and excision‐mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC‐protease, and LysC‐protease). Matrix‐assisted laser desorption ionization – mass spectrometric analysis of the affinity – elution fraction provided the epitope, Aβ(17–28), in the mid‐ to carboxy‐terminal domain of Aβ, which has been shown to exert an Aß‐fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17–28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1–40) or Aβ‐peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ‐nanobodies and Aβ(1–40) and the Aβ(17–28) epitope provided K_D values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ‐aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.     

Biotin,coenzyme Q10, and their combination ameliorate aluminium chloride‐induced Alzheimer's disease via attenuating neuroinflammation and improving brain insulin signaling

Insulin is important for brain function and neuronal survival. Insulin signaling is initiated by the phosphorylation of insulin receptor substrate‐1 (IRS‐1) at tyrosine (pTyr) residue. However, IRS‐1 is inhibited by phosphorylation at serine (pSer). In Alzheimer's disease (AD), oxidative stress and accumulation of amyloid beta (Aβ) induce neuroinflammation, which augments pSer‐IRS‐1 and reduces pTyr‐IRS‐1 disturbing insulin signaling pathway. Coenzyme Q10 (CoQ10) and biotin possess antioxidant and anti‐inflammatory properties, and, in this study, their impact on insulin signaling is investigated in an aluminium chloride (AlCl₃) model of AD. AD was induced by oral administration of AlCl₃ (75 mg/kg) for 60 days. Biotin (2 mg/kg), CoQ10 (10 mg/kg), and their combination were supplemented concomitantly with AlCl₃ for 60 days. Memory test and histological examination were performed. Brain levels of lipid peroxides, antioxidants (reduced glutathione and superoxide dismutase), inflammatory markers (tumor necrosis factor‐α, interleukin‐6 [IL‐6], IL‐1, and nuclear factor κB), and phosphorylated Akt (survival kinase) as well as protein levels of Aβ, IRS‐1 (pTyr and pSer), and caspase‐3 (apoptotic marker) were determined. AlCl₃ resulted in impaired memory, significant increase in Aβ, lipid peroxides, inflammatory markers, caspase‐3, and pSer‐IRS‐1, with significant reduction of the antioxidants, pTyr‐IRS‐1, and p‐Akt reflecting Aβ‐induced inflammation and defective insulin signaling. Histological examination revealed focal aggregations of inflammatory cells and neuronal degeneration. The biochemical deviations and histological changes were attenuated by the concomitant treatment with biotin and, to greater extent, with CoQ10 and the combination. In conclusion, biotin and CoQ10 could protect against AD via attenuating inflammatory response and enhancing insulin signaling.     

Peripheral nerve pericytes modify the blood–nerve barrier function and tight junctional molecules through the secretion of various soluble factors

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen (tsA58) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.     

Retracted: S14G‐humanin inhibits Aβ1–42 fibril formation,disaggregates preformed fibrils,and protects against Aβ‐induced cytotoxicity in vitro

The aggregation of soluble amyloid‐beta (Aβ) peptide into oligomers/fibrils is one of the key pathological features in Alzheimer's disease (AD). The Aβ aggregates are considered to play a pivotal role in the pathogenesis of AD. Therefore, inhibiting Aβ aggregation and destabilizing preformed Aβ fibrils would be an attractive therapeutic target for prevention and treatment of AD. S14G‐humanin (HNG), a synthetic derivative of Humanin (HN), has been shown to be a strong neuroprotective agent against various AD‐related insults. Recent studies have shown that HNG can significantly improve cognitive deficits and reduce insoluble Aβ levels as well as amyloid plaque burden without affecting amyloid precursor protein processing and Aβ production in transgenic AD models. However, the potential mechanisms by which HNG reduces Aβ‐related pathology in vivo remain obscure. In the present study, we found that HNG could significantly inhibit monomeric Aβ1–42 aggregation into fibrils and destabilize preformed Aβ1–42 fibrils in a concentration‐dependent manner by Thioflavin T fluorescence assay. In transmission electron microscope study, we observed that HNG was effective in inhibiting Aβ1–42 fibril formation and disrupting preformed Aβ1–42 fibrils, exhibiting various types of amorphous aggregates without identifiable Aβ fibrils. Furthermore, HNG‐treated monomeric or fibrillar Aβ1–42 was found to significantly reduce Aβ1–42‐mediated cytotoxic effects on PC12 cells in a dose‐dependent manner by MTT assay. Collectively, our results demonstrate for the first time that HNG not only inhibits Aβ1–42 fibril formation but also disaggregates preformed Aβ1–42 fibrils, which provides the novel evidence that HNG may have anti‐Aβ aggregation and fibrillogenesis, and fibril‐destabilizing properties. Together with previous studies, we concluded that HNG may have promising therapeutic potential as a multitarget agent for the prevention and/or treatment of AD. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Conformation‐specific antibodies against multiple amyloid protofibril species from a single amyloid immunogen

We engineered and employed a chaperone‐like amyloid‐binding protein Nucleobindin 1 (NUCB1) to stabilize human islet amyloid polypeptide (hIAPP) protofibrils for use as immunogen in mice. We obtained multiple monoclonal antibody (mAb) clones that were reactive against hIAPP protofibrils. A secondary screen was carried out to identify clones that cross‐reacted with amyloid beta‐peptide (Aβ42) protofibrils, but not with Aβ40 monomers. These mAbs were further characterized in several in vitro assays, in immunohistological studies of a mouse model of Alzheimer's disease (AD) and in AD patient brain tissue. We show that mAbs obtained by immunizing mice with the NUCB1‐hIAPP complex cross‐react with Aβ42, specifically targeting protofibrils and inhibiting their further aggregation. In line with conformation‐specific binding, the mAbs appear to react with an intracellular antigen in diseased tissue, but not with amyloid plaques. We hypothesize that the mAbs we describe here recognize a secondary or quaternary structural epitope that is common to multiple amyloid protofibrils. In summary, we report a method to create mAbs that are conformation‐sensitive and sequence‐independent and can target more than one type of protofibril species.     

Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism

Background

Clearance at the blood-brain barrier (BBB) plays an important role in removal of Alzheimer’s amyloid-β (Aβ) toxin from brain both in humans and animal models. Apolipoprotein E (apoE), the major genetic risk factor for AD, disrupts Aβ clearance at the BBB. The cellular and molecular mechanisms, however, still remain unclear, particularly whether the BBB-associated brain capillary pericytes can contribute to removal of aggregated Aβ from brain capillaries, and whether removal of Aβ aggregates by pericytes requires apoE, and if so, is Aβ clearance on pericytes apoE isoform-specific.

Methods

We performed immunostaining for Aβ and pericyte biomarkers on brain capillaries (<?6 μm in diameter) on tissue sections derived from AD patients and age-matched controls, and APP^Swe/0 mice and littermate controls. Human Cy3-Aβ42 uptake by pericytes was studied on freshly isolated brain slices from control mice, pericyte LRP1-deficient mice (Lrp^lox/lox;Cspg4-Cre) and littermate controls. Clearance of aggregated Aβ42 by mouse pericytes was studied on multi-spot glass slides under different experimental conditions including pharmacologic and/or genetic inhibition of the low density lipoprotein receptor related protein 1 (LRP1), an apoE receptor, and/or silencing mouse endogenous Apoe in the presence and absence of human astrocyte-derived lipidated apoE3 or apoE4. Student’s t-test and one-way ANOVA followed by Bonferroni's post-hoc test were used for statistical analysis.

Results

First, we found that 35% and 60% of brain capillary pericytes accumulate Aβ in AD patients and 8.5-month-old APP^Sw/0 mice, respectively, compared to negligible uptake in controls. Cy3-Aβ42 species were abundantly taken up by pericytes on cultured mouse brain slices via LRP1, as shown by both pharmacologic and genetic inhibition of LRP1 in pericytes. Mouse pericytes vigorously cleared aggregated Cy3-Aβ42 from multi-spot glass slides via LRP1, which was inhibited by pharmacologic and/or genetic knockdown of mouse endogenous apoE. Human astrocyte-derived lipidated apoE3, but not apoE4, normalized Aβ42 clearance by mouse pericytes with silenced mouse apoE.

Conclusions

Our data suggest that BBB-associated pericytes clear Aβ aggregates via an LRP1/apoE isoform-specific mechanism. These data support the role of LRP1/apoE interactions on pericytes as a potential therapeutic target for controlling Aβ clearance in AD.

     

New mechanism of nerve injury in Alzheimer’s disease: β‐amyloid‐induced neuronal pyroptosis

The present study was designed to investigate the role of β‐amyloid (Aβ_1‐42) in inducing neuronal pyroptosis and its mechanism. Mice cortical neurons (MCNs) were used in this study, LPS + Nigericin was used to induce pyroptosis in MCNs (positive control group), and Aβ_1‐42 was used to interfere with MCNs. In addition, propidium iodide (PI) staining was used to examine cell permeability, lactate dehydrogenase (LDH) release assay was employed to detect cytotoxicity, immunofluorescence (IF) staining was used to investigate the expression level of the key protein GSDMD, Western blot was performed to detect the expression levels of key proteins, and enzyme‐linked immunosorbent assay (ELISA) was utilized to determine the expression levels of inflammatory factors in culture medium, including IL‐1β, IL‐18 and TNF‐α. Small interfering RNA (siRNA) was used to silence the mRNA expression of caspase‐1 and GSDMD, and Aβ_1‐42 was used to induce pyroptosis, followed by investigation of the role of caspase‐1‐mediated GSDMD cleavage in pyroptosis. In addition, necrosulfonamide (NSA), an inhibitor of GSDMD oligomerization, was used for pre‐treatment, and Aβ_1‐42 was subsequently used to observe the pyroptosis in MCNs. Finally, AAV9‐siRNA‐caspase‐1 was injected into the tail vein of APP/PS1 double transgenic mice (Alzheimer's disease mice) for caspase‐1 mRNA inhibition, followed by observation of behavioural changes in mice and measurement of the expression of inflammatory factors and pyroptosis‐related protein. As results, Aβ_1‐42 could induce pyroptosis in MCNs, increase cell permeability and enhance LDH release, which were similar to the LPS + Nigericin‐induced pyroptosis. Meanwhile, the expression levels of cellular GSDMD and p30‐GSDMD were up‐regulated, the levels of NLRP3 inflammasome and GSDMD‐cleaved protein caspase‐1 were up‐regulated, and the levels of inflammatory factors in the medium were also up‐regulated. siRNA intervention in caspase‐1 or GSDMD inhibited Aβ_1‐42‐induced pyroptosis, and NSA pre‐treatment also caused the similar inhibitory effects. The behavioural ability of Alzheimer's disease (AD) mice was relieved after the injection of AAV9‐siRNA‐caspase‐1, and the expression of pyroptosis‐related protein in the cortex and hippocampus was down‐regulated. In conclusion, Aβ_1‐42 could induce pyroptosis by GSDMD protein, and NLRP3‐caspase‐1 signalling was an important signal to mediate GSDMD cleavage, which plays an important role in Aβ_1‐42‐induced pyroptosis in neurons. Therefore, GSDMD is expected to be a novel therapeutic target for AD.     

Reduced amyloid‐β degradation in early Alzheimer's disease but not in the APPswePS1dE9 and 3xTg‐AD mouse models

Alzheimer's disease (AD) is hallmarked by amyloid‐β (Aβ) peptides accumulation and aggregation in extracellular plaques, preceded by intracellular accumulation. We examined whether intracellular Aβ can be cleared by cytosolic peptidases and whether this capacity is affected during progression of sporadic AD (sAD) in humans and in the commonly used APPswePS1dE9 and 3xTg‐AD mouse models. A quenched Aβ peptide that becomes fluorescent upon degradation was used to screen for Aβ‐degrading cytoplasmic peptidases cleaving the aggregation‐prone KLVFF region of the peptide. In addition, this quenched peptide was used to analyze Aβ‐degrading capacity in the hippocampus of sAD patients with different Braak stages as well as APPswePS1dE9 and 3xTg‐AD mice. Insulin‐degrading enzyme (IDE) was found to be the main peptidase that degrades cytoplasmic, monomeric Aβ. Oligomerization of Aβ prevents its clearance by IDE. Intriguingly, the Aβ‐degrading capacity decreases already during the earliest Braak stages of sAD, and this decline correlates with IDE protein levels, but not with mRNA levels. This suggests that decreased IDE levels could contribute to early sAD. In contrast to the human data, the commonly used APPswePS1dE9 and 3xTg‐AD mouse models do not show altered Aβ degradation and IDE levels with AD progression, raising doubts whether mouse models that overproduce Aβ peptides are representative for human sAD.     

Cyclooxygenase‐1 inhibition reduces amyloid pathology and improves memory deficits in a mouse model of Alzheimer's disease

Several epidemiological and preclinical studies suggest that non‐steroidal anti‐inflammatory drugs (NSAIDs), which inhibit cyclooxygenase (COX), reduce the risk of Alzheimer's disease (AD) and can lower β‐amyloid (Aβ) production and inhibit neuroinflammation. However, follow‐up clinical trials, mostly using selective cyclooxygenase (COX)‐2 inhibitors, failed to show any beneficial effect in AD patients with mild to severe cognitive deficits. Recent data indicated that COX‐1, classically viewed as the homeostatic isoform, is localized in microglia and is actively involved in brain injury induced by pro‐inflammatory stimuli including Aβ, lipopolysaccharide, and interleukins. We hypothesized that neuroinflammation is critical for disease progression and selective COX‐1 inhibition, rather than COX‐2 inhibition, can reduce neuroinflammation and AD pathology. Here, we show that treatment of 20‐month‐old triple transgenic AD (3 × Tg‐AD) mice with the COX‐1 selective inhibitor SC‐560 improved spatial learning and memory, and reduced amyloid deposits and tau hyperphosphorylation. SC‐560 also reduced glial activation and brain expression of inflammatory markers in 3 × Tg‐AD mice, and switched the activated microglia phenotype promoting their phagocytic ability. The present findings are the first to demonstrate that selective COX‐1 inhibition reduces neuroinflammation, neuropathology, and improves cognitive function in 3 × Tg‐AD mice. Thus, selective COX‐1 inhibition should be further investigated as a potential therapeutic approach for AD.     

Exploration of multi‐target effects of 3‐benzoyl‐5‐hydroxychromen‐2‐one in Alzheimer’s disease cell and mouse models

Microtubule‐associated protein Tau, abundant in the central nervous system (CNS), plays crucial roles in microtubule assembly and stabilization. Abnormal Tau phosphorylation and aggregation are a common pathogenic hallmark in Alzheimer's disease (AD). Hyperphosphorylation of Tau could change its conformation and result in self‐aggregation, increased oxidative stress, and neuronal death. In this study, we examined the potential of licochalcone A (a natural chalcone) and five synthetic derivatives (LM compounds) for inhibiting Tau misfolding, scavenging reactive oxygen species (ROS) and providing neuroprotection in human cells expressing proaggregant ΔK280 Tau_RD‐DsRed. All test compounds were soluble up to 100 μM in cell culture media and predicted to be orally bioavailable and CNS‐active. Among them, licochalcone A and LM‐031 markedly reduced Tau misfolding and associated ROS, promoted neurite outgrowth, and inhibited caspase 3 activity in ΔK280 Tau_RD‐DsRed 293 and SH‐SY5Y cells. Mechanistic studies showed that LM‐031 upregulates HSPB1 chaperone, NRF2/NQO1/GCLC pathway, and CREB‐dependent BDNF/AKT/ERK/BCL2 pathway in ΔK280 Tau_RD‐DsRed SH‐SY5Y cells. Decreased neurite outgrowth upon induction of ΔK280 Tau_RD‐DsRed was rescued by LM‐031, which was counteracted by knockdown of NRF2 or CREB. LM‐031 further rescued the downregulated NRF2 and pCREB, reduced Aβ and Tau levels in hippocampus and cortex, and ameliorated cognitive deficits in streptozocin‐induced hyperglycemic 3 × Tg‐AD mice. Our findings strongly indicate the potential of LM‐031 for modifying AD progression by targeting HSPB1 to reduce Tau misfolding and activating NRF2 and CREB pathways to suppress apoptosis and promote neuron survival, thereby offering a new drug development avenue for AD treatment.     

Design of nonapeptide LVFFARKHH: A bifunctional agent against Cu2+‐mediated amyloid β‐protein aggregation and cytotoxicity

Dysfunctional accumulation of amyloid β‐protein (Aβ) mediated by Cu²⁺ exhibits higher neurotoxicity and accelerates the progress of Alzheimer's disease, so inhibition of Cu²⁺‐mediated Aβ aggregation and cytotoxicity has been considered as a therapeutic strategy for the disease. Herein, a nonapeptide was designed by linking HH to the C‐terminus of a peptide inhibitor of Aβ aggregation, LVFFARK (LK7). We found that the nonapeptide, LK7‐HH, possessed dual functionality, including enhanced inhibition capability on Aβ aggregation as compared to LK7, and chelating Cu²⁺ with a dissociation constant of 5.50 μM. This enabled LK7‐HH to arrest the generation of reactive oxygen species catalyzed by Cu²⁺ or Cu²⁺‐Aβ complex, and to inhibit Cu²⁺‐induced Aβ aggregation. Moreover, in contrast with the cytotoxicity of LK7 aggregates, LK7‐HH was biocompatible because HH conjugation made its aggregation behavior different from LK7. Thus, LK7‐HH efficiently suppressed Cu²⁺‐mediated Aβ aggregation and cytotoxicity. An equimolar concentration of LK7‐HH increased cell viability from 50% to 90% when treating Aβ₄₀‐Cu²⁺ complexes. The results provided insights into the roles of HH in enhancing the inhibition of Aβ and Cu²⁺‐induced Aβ aggregations, in eliminating Cu²⁺‐induced cytotoxicities by arresting generation of reactive oxygen species, and in making the peptide biocompatible. Therefore, this work would contribute to the design of potent peptide‐based inhibitors of Cu²⁺‐mediated Aβ aggregation and cytotoxicity.