Aβ Degradation Research Articles

A Nanoformulation-Mediated Multifunctional Stem Cell Therapy with Improved Beta-Amyloid Clearance and Neural Regeneration for Alzheimer's Disease.

Alzheimer's disease (AD) is a common dementia that is currently incurable. The existing treatments can only moderately relieve the symptoms of ADto slow down its progress. How to achieve effective neural regeneration to ameliorate cognitive impairments is a major challenge for current ADtreatment. Here, the therapeutic potential of a nanoformulation-mediated neural stem cell (NSC) therapy capable of simultaneous Aβ clearance and neural regeneration is investigated in a murine model. Genetically engineered NSCs capable of stably and continuously expressing neprilysin (NEP) are developed to enhance Aβdegradation and NSC survival in the brain. A PBAE-PLGA-Ag2 S-RA-siSOX9 (PPAR-siSOX9) nanoformulation with high gene/drug deliverability is synthesized to overcome AD microenvironment-associated adverse effects and to promote neuronal differentiation of the NEP-expressing NSCs. For achieving accurate stereotactic transplantation, Ag2 S quantum-dot-based fluorescence imaging is used to guide NSC transplantation in real time. This strategy shows numerous benefits, including efficient and long-lasting Aβ degradation, improved neural regeneration, and accurate cell transplantation. It is shown that a single administration of this therapy achieves long-term efficacy (6months) with respect to memory reversal and improvement of learning deficits.

CART mitigates oxidative stress and DNA damage in memory deficits of APP/PS1 mice via upregulating β‑amyloid metabolism‑associated enzymes.

Alzheimer's disease (AD) is the most common form of dementia that is primarily characterized by progressive cognitive deficits. The toxicity of amyloid β-protein (Aβ) serves an important role in the progression of AD, resulting in neuronal loss via a number of possible mechanisms, including oxidative stress, mitochondrial dysfunction, energy depletion, apoptosis and neuroinflammation. Previous studies have reported that cocaine amphetamine regulated transcript (CART) treatment improves memory and synaptic structure in APP/PS1 mice. Therefore, the present study aimed to investigate whether CART served a protective role against memory deficits in AD. APP/PS1 mice were treated with CART or PBS. Spatial memory was assessed using the Morris water maze. Oxidative stress and DNA damage were compared among wild-type, APP/PS1 and CART-treated APP/PS1 mice. The mRNA and protein expression levels of Aβ metabolism-associated enzymes, including neprilysin (NEP), insulin-degrading enzyme (IDE), receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptor-related protein 1 (LRP-1), in the hippocampus were measured via reverse transcription-quantitative PCR and western blotting, respectively. CART improved the memory impairment of APP/PS1 mice by reducing oxidative stress, inhibiting DNA damage and protecting against mitochondrial dysfunction in the cerebral cortex and hippocampus. CART also reduced cell senescence and oxidative stress in Aβ1-42-exposed primary cortical neurons in APP/PS1 mice. Moreover, CART promoted Aβ degradation via modulating Aβ metabolism-associated enzymes, including IDE, NEP, LRP-1 and RAGE. Collectively, the present study indicated that CART improved the learning and memory capacity of APP/PS mice, thus may have potential to serve as a novel therapeutic agent for AD.

Deletion of LRP1 From Astrocytes Modifies Neuronal Network Activity in an in vitro Model of the Tripartite Synapse.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor that binds over 40 potential ligands and is involved in processes such as cell differentiation, proliferation, and survival. LRP1 is ubiquitously expressed in the organism and enriched among others in blood vessels, liver, and the central nervous system (CNS). There, it is strongly expressed by neurons, microglia, immature oligodendrocytes, and astrocytes. The constitutive LRP1 knockout leads to embryonic lethality. Therefore, previous studies focused on conditional LRP1-knockout strategies and revealed that the deletion of LRP1 causes an increased differentiation of neural stem and precursor cells into astrocytes. Furthermore, astrocytic LRP1 is necessary for the degradation of Aβ and the reduced accumulation of amyloid plaques in Alzheimer’s disease. Although the role of LRP1 in neurons has intensely been investigated, the function of LRP1 with regard to the differentiation and maturation of astrocytes and their functionality is still unknown. To address this question, we generated an inducible conditional transgenic mouse model, where LRP1 is specifically deleted from GLAST-positive astrocyte precursor cells. The recombination with resulting knockout events was visualized by the simultaneous expression of the fluorescent reporter tdTomato. We observed a significantly increased number of GLT-1 expressing astrocytes in LRP1-depleted astrocytic cultures in comparison to control astrocytes. Furthermore, we investigated the influence of astrocytic LRP1 on neuronal activity and synaptogenesis using the co-culture of hippocampal neurons with control or LRP1-depleted astrocytes. These analyses revealed that the LRP1-deficient astrocytes caused a decreased number of single action potentials as well as a negatively influenced neuronal network activity. Moreover, the proportion of pre- and postsynaptic structures was significantly altered in neurons co-cultured with LPR1-depleted astrocytes. However, the number of structural synapses was not affected. Additionally, the supernatant of hippocampal neurons co-cultured with LRP1-deficient astrocytes showed an altered set of cytokines in comparison to the control condition, which potentially contributed to the altered neuronal transmission and synaptogenesis. Our results suggest astrocytic LRP1 as a modulator of synaptic transmission and synaptogenesis by altering the expression of the glutamate transporter on the cell surface on astrocytes and the release of cytokines in vitro.

The Role of Cathepsin B in the Degradation of Aβ and in the Production of Aβ Peptides Starting With Ala2 in Cultured Astrocytes.

Astrocytes may not only be involved in the clearance of Amyloid beta peptides (Aβ) in Alzheimer's disease (AD), but appear to produce N-terminally truncated Aβ (Aβn−x) independently of BACE1, which generates the N-Terminus of Aβ starting with Asp1 (Aβ1−x). A candidate protease for the generation of Aβn−x is cathepsin B (CatB), especially since CatB has also been reported to degrade Aβ, which could explain the opposite roles of astrocytes in AD. In this study, we investigated the influence of CatB inhibitors and the deletion of the gene encoding CatB (CTSB) using CRISPR/Cas9 technology on Aβ2−x and Aβ1−x levels in cell culture supernatants by one- and two-dimensional Urea-SDS-PAGE followed by immunoblot. While the cell-permeant inhibitors E64d and CA-074 Me did not significantly affect the Aβ1−x levels in supernatants of cultured chicken and human astrocytes, they did reduce the Aβ2−x levels. In the glioma-derived cell line H4, the Aβ2−x levels were likewise decreased in supernatants by treatment with the more specific, but cell-impermeant CatB-inhibitor CA-074, by CA-074 Me treatment, and by CTSB gene deletion. Additionally, a more than 2-fold increase in secreted Aβ1−x was observed under the latter two conditions. The CA-074 Me-mediated increase of Aβ1−x, but not the decrease of Aβ2−x, was influenced by concomitant treatment with the vacuolar H+-ATPase inhibitor Bafilomycin A1. This indicated that non-lysosomal CatB mediated the production of Aβ2−x in astrocytes, while the degradation of Aβ1−x seemed to be dependent on lysosomal CatB in H4 cells, but not in primary astrocytes. These findings highlight the importance of considering organelle targeting in drug development to promote Aβ degradation.

Ultrasound-mediated augmented exosome release from astrocytes alleviates amyloid-β-induced neurotoxicity.

Background: Extracellular vesicles, including exosomes, are secreted by a variety of cell types in the central nervous system. Exosomes play a role in removing intracellular materials from the endosomal system. Alzheimer's disease (AD) is caused by an overproduction or reduced amyloid-beta (Aβ) peptide clearance. Increased Aβ levels in the brain may impair the exosome-mediated Aβ clearance pathway. Therapeutic ultrasound stimulation demonstrated its potential for promoting Aβ degradation efficiency in clinical trials. However, the underlying mechanism of ultrasound stimulation is still unclear.Methods: In this study, astrocytes, the most abundant glial cells in the brain, were used for exosome production. Post insonation, exosomes from ultrasound-stimulated HA cells (US-HA-Exo) were collected, nanoparticle tracking analysis and protein analysis were used to measure and characterize exosomes. Neuroprotective effect of US-HA-Exo in oligomeric Aβ42 toxicated SH-SY5Y cells was tested. Cellular uptake and distribution of exosomes were observed by flow cytometry and confocal laser scanning microscopy. Focused ultrasound (FUS) with microbubbles was employed for blood-brain-barrier opening to achieve brain-targeted exosome delivery. After US-HA-Exo/FUS treatment, amyloid-β plaque in APP/PS1 mice were evaluated by Aβ immunostaining and thioflavin-S staining.Results: We showed that ultrasound resulted in an almost 5-fold increase in the exosome release from human astrocytes. Exosomes were rapidly internalized in SH-SY5Y cells, and colocalized with FITC-Aβ42, causing a decreased uptake of FITC-Aβ42. CCk-8 test results showed that US-HA-Exo could mitigate Aβ toxicity to neurons in vitro. The therapeutic potential of US-HA-Exo/FUS delivery was demonstrated by a decrease in thioflavin-S-positive amyloid plaques and Aβ immuno-staining, a therapeutic target for AD in APP/PS1 transgenic mice. The iTRAQ-based proteomic quantification was performed to gain mechanistic insight into the ultrasound effect on astrocyte-derived exosomes and their ability to alleviate Aβ neurotoxicity.Conclusion: Our results imply that US-HA-Exo have the potential to provide neuroprotective effects to reverse oligomeric amyloid-β-induced cytotoxicity in vitro and, when combined with FUS-induced BBB opening, enable the clearance of amyloid-β plaques in vivo.

Multifunctional peptide-assembled micelles for simultaneously reducing amyloid-β and reactive oxygen species.

The excessive production and deposition of amyloid-β (Aβ) is one of the most important etiologies of Alzheimer's disease (AD). The interaction between Aβ and metal ions produces aberrant reactive oxygen species (ROS), which induce oxidative stress and accelerate the progression of AD. To reduce Aβ plaques and ROS to maintain their homeostasis is an emerging and ingenious strategy for effective treatment of AD. Herein, we report the rational design of multifunctional micelles (MPGLT) based on a polymer-grafted peptide to simultaneously clear Aβ and ROS for AD therapy. The MPGLT integrating three functional peptides as a ROS scavenger (tk-GSH), β-sheet breaker (LP) and an autophagy activator (TK) respectively, could capture and degrade Aβ. Meanwhile, the tk-GSH on the surface of MPGLT effectively scavenges the intracellular ROS. Consequently, MPGLT reduced the cytotoxicity of Aβ and ROS. In vivo animal studies using an AD mouse model further showed that MPGLT could transport across the blood–brain barrier for decreasing the Aβ plaque and eliminating ROS in vivo. This peptide micelle-based synergistic strategy may provide novel insight for AD therapy.

Activating AhR alleviates cognitive deficits of Alzheimer's disease model mice by upregulating endogenous Aβ catabolic enzyme Neprilysin.

Rationale: Neprilysin (NEP) is a major endogenous catabolic enzyme of amyloid β (Aβ). Previous studies have suggested that increasing NEP expression in animal models of Alzheimer's disease had an ameliorative effect. However, the underlying signaling pathway that regulates NEP expression remains unclear. The aryl hydrocarbon receptor (AhR) is a ligand-activated cytoplasmic receptor and transcription factor. Recent studies have shown that AhR plays essential roles in the central nervous system (CNS), but its physiological and pathological roles in regulating NEP are not entirely known.Methods: Western blotting, immunofluorescence, quantitative RT-PCR and enzyme activity assay were used to verify the effects of AhR agonists on NEP in a cell model (N2a) and a mouse model (APP/PS1). Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were conducted to investigate the roles of AhR in regulating NEP transcription. Object recognition test and the Morris water maze task were performed to assess the cognitive capacity of the mice.Results: Activating AhR by the endogenous ligand L-Kynurenine (L-KN) or FICZ, or by the exogenous ligand diosmin or indole-3-carbinol (I3C) significantly increases NEP expression and enzyme activity in N2a cells and APP/PS1 mice. We also found that AhR is a direct transcription factor of NEP. Diosmin treatment effectively ameliorated the cognitive disorder and memory deficit of APP/PS1 transgenic mice. By knocking down AhR or using a small molecular inhibitor targeting AhR or NEP, we found that diosmin enhanced Aβ degradation through activated AhR and increased NEP expression.Conclusions: These results indicate a novel pathway for regulating NEP expression in neurons and that AhR may be a potential therapeutic target for the treatment of Alzheimer's disease.

Antiallergic drug desloratadine as a selective antagonist of 5HT2A receptor ameliorates pathology of Alzheimer's disease model mice by improving microglial dysfunction.

Alzheimer's disease (AD) is a progressively neurodegenerative disease characterized by cognitive deficits and alteration of personality and behavior. As yet, there is no efficient treatment for AD. 5HT2A receptor (5HT2AR) is a subtype of 5HT2 receptor belonging to the serotonin receptor family, and its antagonists have been clinically used as antipsychotics to relieve psychopathy. Here, we discovered that clinically first‐line antiallergic drug desloratadine (DLT) functioned as a selective antagonist of 5HT2AR and efficiently ameliorated pathology of APP/PS1 mice. The underlying mechanism has been intensively investigated by assay against APP/PS1 mice with selective 5HT2AR knockdown in the brain treated by adeno‐associated virus (AAV)‐ePHP‐si‐5HT2AR. DLT reduced amyloid plaque deposition by promoting microglial Aβ phagocytosis and degradation, and ameliorated innate immune response by polarizing microglia to an anti‐inflammatory phenotype. It stimulated autophagy process and repressed neuroinflammation through 5HT2AR/cAMP/PKA/CREB/Sirt1 pathway, and activated glucocorticoid receptor (GR) nuclear translocation to upregulate the transcriptions of phagocytic receptors TLR2 and TLR4 in response to microglial phagocytosis stimulation. Together, our work has highly supported that 5HT2AR antagonism might be a promising therapeutic strategy for AD and highlighted the potential of DLT in the treatment of this disease.

Gender dimorphism in serotonin dopamine and norepinephrine neurons of isodendritc core in postmortem human and nonhuman primate control subjects

AbstractBackgroundComorbidity of depression in bMets, NPSs and NDDs is resource demanding clinical area considering treatment challenges and patient care. NPSs represent prodromal symptoms of dementia results of neurobiological changes in specific subcortical region. Dynamic cascade of Aβ shifts from nonamyloidogenic is implicated in early cognitive impairment and depressive symptoms. Serotonergic system is implicated in Aβ pathology and NE is important to compensatory in Aβ degradation. Although the role of DA is not investigated in AD pathology. Overall neuromodulation in IDC of midbrain is imperative and intriguing aspect of monoaminergic association in AD and other NDDs pathology.Here we present genetic dimorphism of key area of the brain (5HT: dorsal raphe, DA:substantia nigra, Noradrenaline: locus ceruleous).Method4males and 4 females midbrain of control subject were collected from Neurobiobank (NIH) and ONPRC. Brain sections were collected 20‐micron sections on cover glass plain slides. 4mm tissue punches were used to collect RNA from tissues. Enough tissues used to generate 10ug‐20 ug average quality RNA. 4ug RNA from Raphe, substantia nigra and locus ceruleous was isolated using trizol isolation and sent to UTSW microarray core. The RNA was hybridized to Clariom D Human chip and CEL chip data obtained from UTSW core. CEL files were uploaded in Transcriptome Analysis Console (TAC4.0). Statistically significant data in Excel file is uploaded in Ingenuity Pathway analysis software to find biological pathways dominate female and male brains. Microarray analysis of adult serotonin neurons in human and NHP dorsal raphe, Substantia nigra and Locus ceruleous (DR/SN/LC) Chip has expression of 500.000 known and unknown RNA species transcript encoding 135,000 RNAResultHuman DR/SNC/LC have 6643/13475/9194 number of probe set and 850/896//501 pathways, and Rhesus DR/SNC/LC have 7261/2157/1938 probe sets and 872/218/198 pathways altered respectively with one or more genes. Major dominated pathways: electron transport chain. Oxidative phosphorylation, miRNA, mitochondria dysfunction, apoptosis signaling, will be presented in the posterConclusionMajor energy metabolism pathway dominates in all three area in male DR/SN/LC area clearly state female brain having lower energy metabolism may results in reduce homeostasis in Isodendritc core midbrain area. Although compensatory mechanism may still yet investigate.

Regulation of amyloid‐beta pathology by CNS amylin receptors in APP/PS1 mice

AbstractBackgroundAmylin is a pancreatic amyloid protein, known to regulate glucose homeostasis and satiety through its ability to sensitize insulin and leptin. Importantly, the amylin receptor (AMYR) is expressed throughout the brain, including areas important in both cognition and Alzheimer’s disease (AD) pathology, like the hippocampus. We and others have previously shown that an analogue of amylin, Pramlintide (PRAM), reduces Aβ plaque burden and recuses hippocampal‐dependent cognitive decline in AD‐mouse models. However, the mechanisms underlying the therapeutic benefits of PRAM are not known.MethodAPP/PS1 mice were treated with PRAM, or saline, chronically via a subcutaneous pump for eight weeks. Additionally, a second subcutaneous pump connected to an ICV cannula delivered an AMYR antagonist, AC187 or aCSF during the last four weeks of peripheral treatment. Radial Arm Water Maze was performed at the end of study to measure spatial memory. Soluble‐Aβ1‐42 hippocampal levels were measured via ELISA and APP processing enzymes were evaluated by Western Blot.ResultPreliminary data suggests central blockade of amylin receptors may exacerbate Aβ burden and prevent learning of spatial‐memory tasks; while PRAM treatment showed increased protein expression of certain Aβ degradation enzymes.ConclusionEvaluation of AMYR blockade in AD‐modeled mice has supported a deeper knowledge of amylin signaling in the brain.

Ablation of astrocytes affects Aβ degradation, microglia activation and synaptic connectivity in an ex vivo model of Alzheimer’s disease

AbstractBackgroundAstrocytes provide vital support to neurons in normal and pathological conditions. In Alzheimer's disease (AD) brains, reactive astrocytes have been found surrounding amyloid plaques, forming an astrocytic scar. However, their role and potential mechanisms through which they affect neuroinflammation, amyloid pathology and synaptic density in AD remain unclear.MethodTo explore the role of astrocytes on amyloid pathology and neuroinflammatory markers, we pharmacologically ablated astrocytes in organotypic brain culture slices (OBCSs) generated from the 5XFAD mouse model of amyloidosis with selective astrocytic toxin L‐alphaaminoadipate (L‐AAA). To examine the effects on synaptic circuitry, we measured dendritic spine number and size in OBCSs from Thy‐1‐GFP transgenic mice incubated with synthetic amyloid‐beta (Aβ)42, or double transgenics Thy‐1‐GFP/5XFAD mice treated with L‐AAA or vehicle for 24h.ResultTreatment of OBCSs with L‐AAA resulted in an increased expression of proinflammatory cytokines, such as TNF‐α in conditioned media, without changes in microglial density. In addition, pharmacological ablation of astrocytes led to an increase in Aβ levels in homogenates of OBCS from 5XFAD mice compared with vehicle controls, associated with reduced expression of Neprilysin and Apolipoprotein E, which are involved in Aβ clearance. In addition, OBSCs from wild‐type mice treated with L‐AAA and synthetic amyloid presented 56% higher levels of Aβ in culture media compared to sections treated with Aβ alone, concomitant with reduced expression of the Aβ degrading enzyme IDE in culture medium, suggesting that astrocytes contribute to Aβ clearance and degradation. Quantification of hippocampal dendritic spines revealed a reduction in their density following L‐AAA treatment in all groups analysed. In addition, pharmacological ablation of astrocytes resulted in a decrease in spine size in 5XFAD OBCSs but not in OBCSs from WT treated with synthetic Aβ compared to vehicle control.ConclusionAstrocytes play a protective role in AD by aiding Aβ clearance and supporting synaptic plasticity.

Association of Genes Involved in the Metabolic Pathways of Amyloid-β and Tau Proteins With Sporadic Late-Onset Alzheimer's Disease in the Southern Han Chinese Population.

The genes involved in the metabolic pathways of amyloid-β (Aβ) and tau proteins significantly influence the etiology of Alzheimer’s disease (AD). Various studies have explored the associations between some of these genes and AD in the Caucasian population; however, researches regarding these associations remain limited in the Chinese population. To systematically evaluate the associations of these genes with AD, we investigated 19 genes involved in the metabolism of Aβ and tau based on previous studies selected using the PubMed database. This study included 372 patients with sporadic late-onset AD (sLOAD) and 345 cognitively healthy individuals from southern China. The results were replicated in the International Genomics of Alzheimer’s Project (IGAP). Protein–protein interactions were determined using the STRING v11 database. We found that a single-nucleotide polymorphism, rs11682128, of BIN1 conferred susceptibility to sLOAD after adjusting for age, sex, and APOE ε4 status and performing the Bonferroni correction {corrected P = 0.000153, odds ratio (OR) [95% confidence interval (CI)] = 1.403 (1.079–1.824)}, which was replicated in the IGAP. Protein–protein interactions indicated that BIN1 was correlated with MAPT. Moreover, rare variants of NEP and FERMT2 (0.0026 < corrected P < 0.05), and the Aβ degradation, tau pathology, and tau phosphatase pathways (0.01 < corrected P < 0.05), were nominally significantly associated with sLOAD. This study suggested that the genes involved in the metabolic pathways of Aβ and tau contributed to the etiology of sLOAD in the southern Han Chinese population.

Microglia prevent beta-amyloid plaque formation in the early stage of an Alzheimer\u2019s disease mouse model with suppression of glymphatic clearance

BackgroundSoluble beta-amyloid (Aβ) can be cleared from the brain through various mechanisms including enzymatic degradation, glial cell phagocytosis, transport across the blood-brain barrier, and glymphatic clearance. However, the relative contribution of each clearance system and their compensatory effects in delaying the pathological process of Alzheimer’s disease (AD) are currently unknown.MethodsFluorescent trace, immunofluorescence, and Western blot analyses were performed to compare glymphatic clearance ability and Aβ accumulation among 3-month-old APP695/PS1-dE9 transgenic (APP/PS1) mice, wild-type mice, aquaporin 4 knock out (AQP4−/−) mice, and AQP4−/−/APP/PS1 mice. The consequence of selectively eliminating microglial cells, or downregulating apolipoprotein E (apoE) expression, on Aβ burden, was also investigated in the frontal cortex of AQP4−/−/APP/PS1 mice and APP/PS1 mice.ResultsAQP4 deletion in APP/PS1 mice significantly exaggerated glymphatic clearance dysfunction, and intraneuronal accumulation of Aβ and apoE, although it did not lead to Aβ plaque deposition. Notably, microglia, but not astrocytes, increased activation and phagocytosis of Aβ in the cerebral cortex of AQP4−/−/APP/PS1 mice, compared with APP/PS1 mice. Selectively eliminating microglia in the frontal cortex via local injection of clodronate liposomes resulted in deposition of Aβ plaques in AQP4−/−/APP/PS1 mice, but not APP/PS1 mice. Moreover, knockdown of apoE reduced intraneuronal Aβ levels in both APP/PS1 mice and AQP4−/−/APP/PS1 mice, indicating an inhibitory effect of apoE on Aβ clearance.ConclusionThe above results suggest that the glymphatic system mediated Aβ and apoE clearance and microglia mediated Aβ degradation synergistically prevent Aβ plague formation in the early stages of the AD mouse model. Protecting one or both of them might be beneficial to delaying the onset of AD.

Hyaluronan-carnosine conjugates inhibit A\u03b2 aggregation and toxicity

Alzheimer’s disease is the most common neurodegenerative disorder. Finding a pharmacological approach that cures and/or prevents the onset of this devastating disease represents an important challenge for researchers. According to the amyloid cascade hypothesis, increases in extracellular amyloid-β (Aβ) levels give rise to different aggregated species, such as protofibrils, fibrils and oligomers, with oligomers being the more toxic species for cells. Many efforts have recently been focused on multi-target ligands to address the multiple events that occur concurrently with toxic aggregation at the onset of the disease. Moreover, investigating the effect of endogenous compounds or a combination thereof is a promising approach to prevent the side effects of entirely synthetic drugs. In this work, we report the synthesis, structural characterization and Aβ antiaggregant ability of new derivatives of hyaluronic acid (Hy, 200 and 700 kDa) functionalized with carnosine (Car), a multi-functional natural dipeptide. The bioactive substances (HyCar) inhibit the formation of amyloid-type aggregates of Aβ42 more than the parent compounds; this effect is proportional to Car loading. Furthermore, the HyCar derivatives are able to dissolve the amyloid fibrils and to reduce Aβ-induced toxicity in vitro. The enzymatic degradation of Aβ is also affected by the interaction with HyCar.

Age, Sex Hormones, and Circadian Rhythm Regulate the Expression of Amyloid-Beta Scavengers at the Choroid Plexus.

Accumulation of amyloid-beta (Aβ) in the brain is thought to derive from the impairment of Aβ clearance mechanisms rather than from its overproduction, which consequently contributes to the development of Alzheimer’s disease. The choroid plexus epithelial cells constitute an important clearance route for Aβ, either by facilitating its transport from the cerebrospinal fluid to the blood, or by synthesizing and secreting various proteins involved in Aβ degradation. Impaired choroid plexus synthesis, secretion, and transport of these Aβ-metabolizing enzymes have been therefore associated with the disruption of Aβ homeostasis and amyloid load. Factors such as aging, female gender, and circadian rhythm disturbances are related to the decline of choroid plexus functions that may be involved in the modulation of Aβ-clearance mechanisms. In this study, we investigated the impact of age, sex hormones, and circadian rhythm on the expression of Aβ scavengers such as apolipoprotein J, gelsolin, and transthyretin at the rat choroid plexus. Our results demonstrated that mRNA expression and both intracellular and secreted protein levels of the studied Aβ scavengers are age-, sex-, and circadian-dependent. These data suggest that the Aβ-degradation and clearance pathways at the choroid plexus, mediated by the presence of Aβ scavengers, might be compromised as a consequence of aging and circadian disturbances. These are important findings that enhance the understanding of Aβ-clearance-regulating mechanisms at the blood–cerebrospinal fluid barrier.

Melatonin Induction of APP Intracellular Domain 50 SUMOylation Alleviates AD through Enhanced Transcriptional Activation and Aβ Degradation

The amyloid precursor protein (APP) intracellular domain (AICD) is implicated in the pathogenesis of Alzheimer’s disease (AD), but post-translational modification of AICD has rarely been studied and its role in AD is unknown. In this study, we examined the role and molecular mechanism of AICD SUMOylation in the pathogenesis of AD. We found that AICD is SUMO-modified by the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in the hippocampus at Lys-43 predominantly, and that knockdown of PIAS1 decreases endogenous AICD SUMOylation. AICD SUMOylation increases AICD association with its binding protein Fe65 and increases AICD nuclear translocation. Furthermore, AICD SUMOylation increases AICD association with cyclic AMP-responsive element binding protein (CREB) and p65 and their DNA binding for transcriptional activation of neprilysin (NEP) and transthyretin (TTR), two major Aβ-degrading enzymes, respectively. Consequently, AICD SUMOylation decreases the Aβ level, Aβ oligomerization, and amyloid plaque deposits. It also rescues spatial memory deficits in APP/PS1 mice. Conversely, blockade of AICD SUMOylation at Lys-43 produces the opposite effects. Melatonin is identified as an endogenous stimulus that induces AICD SUMOylation. It also decreases the Aβ level and rescues reduction of PIAS1, NEP, and TTR expression in APP/PS1 mice. In this study, we demonstrate that AICD SUMOylation functions as a novel endogenous defense mechanism to combat AD.

The novel DYRK1A inhibitor KVN93 regulates cognitive function, amyloid-beta pathology, and neuroinflammation

Regulating amyloid beta (Aβ) pathology and neuroinflammatory responses holds promise for the treatment of Alzheimer's disease (AD) and other neurodegenerative and/or neuroinflammation-related diseases. In this study, the effects of KVN93, an inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), on cognitive function and Aβ plaque levels and the underlying mechanism of action were evaluated in 5x FAD mice (a mouse model of AD). KVN93 treatment significantly improved long-term memory by enhancing dendritic synaptic function. In addition, KVN93 significantly reduced Aβ plaque levels in 5x FAD mice by regulating levels of the Aβ degradation enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE). Moreover, Aβ-induced microglial and astrocyte activation were significantly suppressed in the KVN-treated 5xFAD mice. KVN93 altered neuroinflammation induced by LPS in microglial cells but not primary astrocytes by regulating TLR4/AKT/STAT3 signaling, and in wild-type mice injected with LPS, KVN93 treatment reduced microglial and astrocyte activation. Overall, these results suggest that the novel DYRK1A inhibitor KVN93 is a potential therapeutic drug for regulating cognitive/synaptic function, Aβ plaque load, and neuroinflammatory responses in the brain.

Cathepsin D regulates cerebral A\u03b242/40 ratios via differential degradation of A\u03b242 and A\u03b240

BackgroundCathepsin D (CatD) is a lysosomal protease that degrades both the amyloid β-protein (Aβ) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aβ proteostasis in vivo and to more completely characterize the degradation of Aβ42 and Aβ40 by CatD.MethodsWe quantified Aβ degradation rates and levels of endogenous Aβ42 and Aβ40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aβ42 vs. Aβ40, and the degradation of aggregated Aβ42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aβ42 and Aβ40, as well as corresponding p3 fragments.ResultsGenetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aβ42 and Aβ40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aβ42—all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aβ42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aβ42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aβ42 vis-à-vis Aβ40. Specifically, Aβ42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aβ42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aβ42 vs. Aβ40 also extend to p3 fragments ending at positions 42 vs. 40.ConclusionsOur findings identify CatD as the principal intracellular Aβ-degrading protease identified to date, one that regulates Aβ42/40 ratios via differential degradation of Aβ42 vs. Aβ40. The finding that Aβ42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aβ42 and downstream pathological sequelae in AD.

Screening of Antibiotics Against β-amyloid as Anti-amyloidogenic Agents: A Drug Repurposing Approach.

β-amyloid (Aβ) production and aggregation are the main culprits of Alzheimer's disease (AD). There is no treatment available for halting the disease progression. Antibiotics are used not only to treat infections but also to some of the non-contagious diseases and have found active as anti-amyloidogenic agents. The aim of this work is to investigate anti-amyloidogenic activity of antibiotics as repurposing agents via inhibiting Aβ aggregation and fibril formation employing in silico and in vitro approaches. In silico screening was designed with receptor and ligand preparation, grid formation, docking simulation and its analysis. Thioflavin T-amyloid binding and protease-digestion studies were intended as in vitro assays. These methods assessed the pharmacological potential of antibiotics as anti-amyloidogenic agents. Paromomycin and Neomycin were identified with a higher order of estimated free energy of binding in in silico experiments. In in vitro screening, paromomycin significantly (p<0.01) reduced the fluorescence intensity and resistance to tryptic degradation of Aβ(1-42) peptides while neomycin had no or little effect (p<0.01) when compared to control. Results from docking and wet lab studies were found in correlation. Paromomycin exhibited higher anti-Aβ aggregating and defibrillogenic activity than neomycin and left an indication for further in vivo testing and could be a future promising antiamyloidal candidate for the treatment of several amyloidoses.

Omega-3 fatty acids increase OXPHOS energy for immune therapy of Alzheimer disease patients.

Sporadic late-onset Alzheimer disease (LOAD) preceded by mild cognitive impairment (MCI) is the most common type of dementia. Long-term studies of immunity to pathogenic amyloid-β (Aβ) in LOAD are lacking. Innate immunity of LOAD patients is malfunctioning in phagocytosis and degradation of Aβ and LOAD patients' macrophage transcriptome and metabolome are deregulated. We previously showed omega-3 fatty acid (ω-3)-mediated repair of unfolded protein response and here we show much broader transcriptomic effects. ω-3 treatment in vitro and ω-3 supplementation by the drink Smartfish (SMF) in vivo increased the transcripts of the genes and pathways of immunity, glycolysis, tricarboxylic acid cycle, OX-PHOS, nicotinamide dinucleotide (NAD+ ) synthesis, and reversed the defects in Aβ phagocytosis. In both peripheral blood mononuclear cells (PBMC) and macrophages, ω-3 increased ATP-linked oxygen consumption rate (OCR) and ω-3 with carnitine was superior to ω-3. ω-3 treatment in vitro and supplementation by the ω-3 drink SMF in vivo rescued macrophage phagocytosis when glycolysis or glycosylation were blocked. ω-3 provide flexible energy for immune clearance of the brain throughout the diurnal cycle, even in hypo- or hyper-glycemia. In certain LOAD patients, ω-3 may delay progression to dementia.