Therapeutic Strategy For Alzheimer's Disease Research Articles

Novel systemic delivery of a peptide‐mediated anti‐sense oligonucleotide to reduce α‐synuclein in a mouse model of Alzheimer’s Disease

AbstractBackgroundNeurodegenerative disorders of aging are characterized by the progressive accumulation of proteins such as α‐synuclein (α‐syn) and amyloid beta (Aβ). Misfolded and aggregated α‐syn has been implicated in neurological disorders such as Parkinson’s disease (PD), and Dementia with Lewy Bodies (DLB), but less so in Alzheimer’s Disease (AD). However, accumulation of α‐syn has been confirmed in over 50% of postmortem brains of AD patients. To date, no therapeutic strategy has consistently downregulated α‐syn in AD.MethodHere we tested the hypothesis that using a peptide (ApoB11) that binds a modified antisense oligonucleotide (2’‐O‐methyl si α‐syn or 2’‐OMe) for transport across the blood‐brain barrier following systemic administration, would downregulate α‐syn expression in an AD mouse model and improve behavioral and neuropathologic phenotypes. We tested the efficacy of the ASO in knocking down α‐syn both in vitro, using neuronal cell lines, and in vivo by measuring α‐syn levels in brains of treated mice with 2mg/kg at 6, 7 and 8 months of age.ResultWe found that treatment of APP transgenic mice with systemic delivery of ApoB11:ASO‐α‐syn reduces expression of α‐synuclein in specific regions of hippocampi of 9 month‐old APP transgenic mice. Downregulation of α‐syn led to a significant reduction of Aβ plaques burden, rescued neuronal loss and prevented astrogliosis, as compared to untreated PSAPP mice. Importantly, we found that AD mice treated with the ASO‐syn had greatly improved hippocampal and spatial memory function as shown by Morris Water Maze tests.ConclusionCollectively, the data shown in this work supports the use of ApoB11:ASO α‐syn conjugates delivered systemically to downregulate α‐syn as a promising future therapeutic strategy in AD. Our work suggests that knocking down α‐syn could be necessary to prevent neuropathology and prevent spatial memory loss.

PSEN2 poison exon inclusion is common across brain regions in late‐onset Alzheimer’s disease

AbstractBackgroundPathogenic variants in APP, PSEN1 and PSEN2 can cause early‐onset familial Alzheimer’s disease (AD). The molecular events leading to AD in individuals with late‐onset sporadic AD are still unclear even though they share the same clinical and pathological features of AD.MethodUsing targeted PacBio long read isoform sequencing (Iso‐Seq), we recently reported the characterization of tens of thousands of complete PSEN1 and PSEN2 transcripts in the prefrontal cortex of individuals with sporadic AD, familial AD (carrying PSEN1 and PSEN2 pathogenic variants), and controls.ResultWe found several alternative splicing differences in PSEN2 that are significantly elevated in sporadic AD including inclusion of a human‐specific cryptic exon present in intron 9 of PSEN2 (termed exon 9B) present in 11.8% of full‐length reads in sporadic AD samples relative to 1.9% of controls (p = 0.002) and 3.4% of PSEN variant carriers (p = 0.005) as well as a 77bp intron retention product prior to exon 6. These PSEN2 splice products are predicted to generate a prematurely truncated PSEN2 protein and were confirmed in an independent RNA‐seq dataset of ∼80 AD cases and ∼80 controls from cerebellum tissue (Course et al., Brain 2022). To extend these findings we analyzed PSEN2 exon 9B isoform levels in parietal and temporal lobe tissue samples. We found exon 9B was significantly elevated in sporadic AD relative to controls in the parietal lobe (p = 0.02) as well as the temporal lobe (p = <0.001). However, levels of exon 9B were independent of TDP‐43 pathology or MSUT2 protein levels, which is intriguing given their roles as RNA binding proteins implicated in AD. We are working to establish the consequence of exon 9B inclusion in APP processing, Aβ and PSEN2 protein products in addition to possible cell autonomous roles for PSEN2 splice variants in microglia by leveraging single cell sequencing data.ConclusionCollectively, our findings expand our understanding of PSEN2 splice variants in the development of late‐onset sporadic AD, which can point to novel therapeutic strategies for AD.

Establishment and application of a microglial phagocytosis/cell health high content assay for Alzheimer’s disease drug discovery targeting microglia

AbstractBackgroundTargeting microglial phagocytosis has been proposed as a therapeutic strategy for Alzheimer’s disease drug discovery. A reliable cellular assay capable of measuring drug‐regulated phagocytosis activity while simultaneously measuring treatment‐caused cell stress and toxicity is needed for drug discovery research targeting microglia. This need motivated us to develop and establish a high content microglial phagocytosis/cell health assay to meet the challenge.MethodThe assay uses 384‐well cellular imaging plates, microglial cell lines (HMC3 and BV2) and mouse primary microglia, pHrodo‐myelin/membrane debris as ligand, and a high content imaging system for imaging and analysis. Assay procedures include 1) plate cells on day 1 and incubate cell plates overnight in 37oC; 2) treat cells with compounds on day 2; 3) add pHrodo‐myelin/membrane debris to cell plates on day 3 and incubate in 37oC for 20 hours; 4) stain cell nuclei with Hoechst‐33342 for 30 min before performing high content imaging and analysis on day 4. Three selected parameters are measured from cell images: 1) mean total fluorescence intensity per cell of pHrodo‐myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (assessing effects on proliferation and cell death); and 3) average nuclear intensity (detecting induced apoptosis).ResultVarious treatments targeting different mechanisms regulating microglial phagocytosis were tested; we observed that Cytochalasin D inhibited phagocytosis, LPS stimulated phagocytosis in BV2 cells but not HMC3 cells, Idelalisib (PI3Kd inhibitor) inhibited HMC3 cell phagocytosis, and Saracatinib (Src/abl family of kinases inhibitor) stimulated HMC3 phagocytosis. These results validated the assay for use in drug discovery targeting microglial phagocytosis. With the assay, we identified some INPP5D inhibitors and some PLCG2 activators stimulated phagocytosis in a concentration range without significant cell stress/toxicity with all three cell types tested.ConclusionA microglial phagocytosis/cell health assay was established. The simultaneous measurement of phagocytosis and cell health allows for the distinction of drug effects on regulation of phagocytosis from cellular stress/toxicity‐related changes, a distinguishing feature of the assay, which helped compound selection of drug discovery projects targeting INPP5D and PLCG2 respectively.

Clusterin is a Potential Therapeutic Target in Alzheimer's Disease.

In recent years, Clusterin, a glycosylated protein with multiple biological functions, has attracted extensive research attention. It is closely associated with the physiological and pathological states within the organism. Particularly in Alzheimer's disease (AD) research, Clusterin plays a significant role in the disease's occurrence and progression. Numerous studies have demonstrated a close association between Clusterin and AD. Firstly, the expression level of Clusterin in the brain tissue of AD patients is closely related to pathological progression. Secondly, Clusterin is involved in the deposition and formation of β-amyloid, which is a crucial process in AD development. Furthermore, Clusterin may affect the pathogenesis of AD through mechanisms such as regulating inflammation, controlling cell apoptosis, and clearing pathological proteins. Therefore, further research on the relationship between Clusterin and AD will contribute to a deeper understanding of the etiology of this neurodegenerative disease and provide a theoretical basis for developing early diagnostic and therapeutic strategies for AD. This also makes Clusterin one of the research focuses as a potential biomarker for AD diagnosis and treatment monitoring.

N6-methyladenosine-modified circRIMS2 mediates synaptic and memory impairments by activating GluN2B ubiquitination in Alzheimer's disease

BackgroundSynaptic degeneration occurs in the early stage of Alzheimer's disease (AD) before devastating symptoms, strongly correlated with cognitive decline. Circular RNAs (circRNAs) are abundantly enriched in neural tissues, and aberrant expression of circRNAs precedes AD symptoms, significantly correlated with clinical dementia severity. However, the direct relationship between circRNA dysregulation and synaptic impairment in the early stage of AD remains poorly understood.MethodsHippocampal whole-transcriptome sequencing was performed to identify dysregulated circRNAs and miRNAs in 4-month-old wild-type and APP/PS1 mice. RNA antisense purification and mass spectrometry were utilized to unveil interactions between circRIMS2 and methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3). The roles of circRIMS2/miR-3968 in synaptic targeting of UBE2K-mediated ubiquitination of GluN2B subunit of NMDA receptor were evaluated via numerous lentiviruses followed by morphological staining, co-immunoprecipitation and behavioral testing. Further, a membrane-permeable peptide was used to block the ubiquitination of K1082 on GluN2B in AD mice.ResultscircRIMS2 was significantly upregulated in 4-month-old APP/PS1 mice, which was mediated by METTL3-dependent N6-methyladenosine (m6A) modification. Overexpression of circRIMS2 led to synaptic and memory impairments in 4-month-old C57BL/6 mice. MiR-3968/UBE2K was validated as the downstream of circRIMS2. Elevated UBE2K induced synaptic dysfunction of AD through ubiquitinating K1082 on GluN2B. Silencing METTL3 or blocking the ubiquitination of K1082 on GluN2B with a short membrane-permeable peptide remarkably rescued synaptic dysfunction in AD mice.ConclusionsIn conclusion, our study demonstrated that m6A-modified circRIMS2 mediates the synaptic and memory impairments in AD by activating the UBE2K-dependent ubiquitination and degradation of GluN2B via sponging miR-3968, providing novel therapeutic strategies for AD.

Activation of neurogenesis in the hippocampus is a novel therapeutic target for Alzheimer's disease.

Highlights Targeting single mechanisms of physiological (aging) and pathological (neurodegeneration) loss of function in the brain may not be sufficient. Cell–cell interactions between transplanted adult stem cells and resident cells via gap junctions have the potential to support the aging or diseased brain. These cell–cell interactions can also increase hippocampal neurogenesis. This may be a novel therapeutic strategy for Alzheimer's disease and other neurodegenerative diseases that could be applied alongside any established treatments.

Effect of woohwangchungsimwon and donepezil co-treatment on cognitive function and serum metabolic profiles in a scopolamine-induced model of Alzheimer's disease

Ethnopharmacological relevanceWoohwangchungsimwon (WCW) is a traditional medicine used in East Asian countries to treat central nervous system disorders. Reported pharmacological properties include antioxidant effects, enhanced learning and memory, and protection against ischemic neuronal cell death, supporting its use in treating neurodegenerative diseases like Alzheimer's disease (AD). Aim of the studyThe study aims to assess the effects of co-treatment with WCW and donepezil on cognitive functions and serum metabolic profiles in a scopolamine-induced AD model. Materials and methodsCell viability and reactive oxygen species (ROS) levels were measured in amyloid β-peptide25–35 (Aβ25–35)-induced SH-SY5Y cells. An AD model was established in ICR mice by intraperitoneal scopolamine administration. Animals underwent the step-through passive avoidance test (PAT) and Morris water maze (MWM) test. Hippocampal tissues were collected to examine specific protein expression. Serum metabolic profiles were analyzed using nuclear magnetic resonance (NMR) spectroscopy. ResultsCo-treatment with WCW and donepezil increased cell viability and reduced ROS production in Aβ25–35-induced SH-SY5Y cells compared to that with donepezil treatment alone. Co-treatment improved cognitive functions and was comparable to donepezil treatment alone in the PAT and MWM tests. Pathways related to tyrosine, phenylalanine, and tryptophan biosynthesis, phenylalanine metabolism, and cysteine and methionine metabolism were altered by co-treatment. Levels of tyrosine and methionine, major serum metabolites in these pathways, were significantly reduced after co-treatment. ConclusionsCo-treatment with WCW and donepezil shows promise as a therapeutic strategy for AD and is comparable to donepezil alone in improving cognitive function. Reduced tyrosine and methionine levels after co-treatment may enhance cognitive function by mitigating hypertyrosinemia and hyperhomocysteinemia, known risk factors for AD. The serum metabolic profiles obtained in this study can serve as a foundation for developing other bioactive compounds using a scopolamine-induced mouse model.

Mechanistic Intimate Insights into the Role of Hydrogen Sulfide in Alzheimer's Disease: A Recent Systematic Review.

In the rapidly evolving field of Alzheimer's Disease (AD) research, the intricate role of Hydrogen Sulfide (H2S) has garnered critical attention for its diverse involvement in both pathological substrates and prospective therapeutic paradigms. While conventional pathophysiological models of AD have primarily emphasized the significance of amyloid-beta (Aβ) deposition and tau protein hyperphosphorylation, this targeted systematic review meticulously aggregates and rigorously appraises seminal contributions from the past year elucidating the complex mechanisms of H2S in AD pathogenesis. Current scholarly literature accentuates H2S's dual role, delineating its regulatory functions in critical cellular processes-such as neurotransmission, inflammation, and oxidative stress homeostasis-while concurrently highlighting its disruptive impact on quintessential AD biomarkers. Moreover, this review illuminates the nuanced mechanistic intimate interactions of H2S in cerebrovascular and cardiovascular pathology associated with AD, thereby exploring avant-garde therapeutic modalities, including sulfurous mineral water inhalations and mud therapy. By emphasizing the potential for therapeutic modulation of H2S via both donors and inhibitors, this review accentuates the imperative for future research endeavors to deepen our understanding, thereby potentially advancing novel diagnostic and therapeutic strategies in AD.

Proteasome inhibition protects blood–brain barrier P-glycoprotein and lowers Aβ brain levels in an Alzheimer’s disease model

BackgroundLoss of P-glycoprotein (P-gp) at the blood–brain barrier contributes to amyloid-β (Aβ) brain accumulation in Alzheimer’s disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aβ triggers P-gp loss by activating the ubiquitin–proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aβ accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood–brain barrier P-gp from degradation in hAPP mice in vivo.MethodsWe treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood–brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aβ plasma and brain levels with ELISA.ResultsWe found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood–brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aβ40 and Aβ42 brain levels compared to vehicle-treated hAPP mice.ConclusionsOur data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aβ brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aβ transport across the blood–brain barrier may prove an effective treatment for patients.

Neural stem cell-derived exosomes promote mitochondrial biogenesis and restore abnormal protein distribution in a mouse model of Alzheimer's disease.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00040/figure1/v/2023-11-20T171125Z/r/image-tiff Mitochondrial dysfunction is a hallmark of Alzheimer's disease. We previously showed that neural stem cell-derived extracellular vesicles improved mitochondrial function in the cortex of APP/PS1 mice. Because Alzheimer's disease affects the entire brain, further research is needed to elucidate alterations in mitochondrial metabolism in the brain as a whole. Here, we investigated the expression of several important mitochondrial biogenesis-related cytokines in multiple brain regions after treatment with neural stem cell-derived exosomes and used a combination of whole brain clearing, immunostaining, and lightsheet imaging to clarify their spatial distribution. Additionally, to clarify whether the sirtuin 1 (SIRT1)-related pathway plays a regulatory role in neural stem cell-derived exosomes interfering with mitochondrial functional changes, we generated a novel nervous system-SIRT1 conditional knockout APP/PS1 mouse model. Our findings demonstrate that neural stem cell-derived exosomes significantly increase SIRT1 levels, enhance the production of mitochondrial biogenesis-related factors, and inhibit astrocyte activation, but do not suppress amyloid-β production. Thus, neural stem cell-derived exosomes may be a useful therapeutic strategy for Alzheimer's disease that activates the SIRT1-PGC1α signaling pathway and increases NRF1 and COXIV synthesis to improve mitochondrial biogenesis. In addition, we showed that the spatial distribution of mitochondrial biogenesis-related factors is disrupted in Alzheimer's disease, and that neural stem cell-derived exosome treatment can reverse this effect, indicating that neural stem cell-derived exosomes promote mitochondrial biogenesis.

Methylene blue accelerates liquid-to-gel transition of tau condensates impacting tau function and pathology

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer’s disease and other tauopathies. Recently, liquid–liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid–liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Self-Destructive Nanoscavengers Capture and Clear Neurotoxic Soluble β-Amyloid Aggregates.

Cerebral soluble β-amyloid aggregates (sAβs) accumulation is one of the most important causes in Alzheimer's disease (AD) progression. In order to mitigate the neurotoxicity induced by sAβs and achieve enhanced AD therapeutic outcomes, robust sAβs clearance become an emerging task. Herein, a self-destructive nanoscavenger (SDNS) is reported based on multifunctional peptide-polymer complexes that can capture extracellular sAβs via hydrogen-bonding interactions and deliver them into microglial lysosomes. The internalized SDNS then occurs self-destruction within lysosomes and upregulates autophagy, thereby promoting the degradation of neurotoxic sAβs. Importantly, the enhanced autophagy also significantly suppresses the secretion of inflammatory factors by microglia, which is induced by internalized sAβs. Given that cerebral persistent inflammatory environment disturbs microglia-mediated phagocytosis and degradation, it is believed that this synergistic approach has valuable potential as a therapeutic strategy for AD.

Interplays between different secretases in the generation of Amyloid-[formula omitted] lead to the emergence of ‘Amyloid-[formula omitted] rise’

The abnormal accumulation of Amyloid-β (Aβ) initials the pathological progress of Alzheimer’s disease (AD). γ-secretase, which is a key secretase involving in the formation of Aβ, becomes one of candidate therapeutic targets for preventing the pathological progress of AD. In order to reduce the amount of Aβ in brain, different types of γ-secretase inhibitor were designed to limit the catalytic rate of γ-secretase. However, there is a paradoxical phenomenon called ‘Aβ rise’ can be found in experiment. In this phenomenon, the amount of Aβ will rise as the lower levels of γ-secretase inhibitor is added, which hinders the development of related drugs. In present paper, a mathematical model based on the generation of Aβ is proposed to explore the biological mechanism of ‘Aβ rise’. Through rigorous analysis about the dynamic behavior for this model, we firstly obtain the index for the emergence of ‘Aβ rise’. Furthermore, the analysis results show that both the inhibition of C-terminal fragment (C99) to α-secretase cleavage of amyloid precursor protein (APP) and the lower γ-secretase independent degradation for C99 are contributed to ‘Aβ rise’. Hence, enhancing the intensity of γ-secretase independent degradation of C99 can effectively block the appearance of ‘Aβ rise’. These findings will give new insights on the arise of ‘Aβ rise’ and therapeutic strategies for AD.

MALDI mass spectrometry imaging discloses the decline of sulfoglycosphingolipid and glycerophosphoinositol species in the brain regions related to cognition in a mouse model of Alzheimer's disease

Aging and neurodegenerative disease are accompanied by lipid perturbations in the brain. Understanding the changes in the contents and functional activity of lipids remains a challenge not only because of the many areas in which lipids perform bioactivities but also because of the technical limitations in identifying lipids and their metabolites. In the present study, we aimed to evaluate how brain lipids are altered in Alzheimer's disease (AD)-like pathology by using mass spectrometry imaging (MSI). The spatial distributions and relative abundances of lipids in the brains were compared between APP/PS1 mice and their age-matched wild-type (WT) mice by matrix-assisted laser desorption ionization (MALDI) MSI assays. The comparisons were correlated with the analysis using a spectrophotometric method to determine the relative contents of sulfatides in different brain regions. Significant changes of brain lipids between APP/PS1 and WT mice were identified: eight sulfoglycosphingolipid species, namely, sulfatides/sulfated hexosyl ceramides (ShexCer) and two glycerophosphoinositol (GroPIn) species, PI 36:4 and PI 38:4. The declines in the spatial distributions of these ShexCer and GroPIn species in the APP/PS1 mice brains were associated with learning- and memory-related brain regions. Compared with young WT mice, aged WT mice showed significant decreases in the levels of these ShexCer and GroPIn species. Our results provide technical clues for assessing the impact of brain lipid metabolism on the senescent and neurodegenerative brain. The decline in sulfatides and GroPIns may be crucial markers during brain senescence and AD pathology. Appropriate lipid complementation might be important potentials as a therapeutic strategy for AD.

Enhancement of neural regeneration as a therapeutic strategy for Alzheimer's disease (Review).

Alzheimer's disease (AD), the most common cause of dementia worldwide, has gradually become a global health concern for society and individuals with the process of global ageing. Although extensive research has been carried out on AD, the etiology and pathological mechanism of the disease are still unclear, and there is no specific drug to cure or delay AD progression. The exploration of enhancing nerve regeneration in AD has gradually attracted increasing attention. In the current review, the existing therapeutic strategies were summarized to induce nerve regeneration which can increase the number of neurons, and improve the survival of neurons, the plasticity of synapses and synaptic activity. The strategies include increasing neurotrophic expression (such as brain-derived neurotrophic factor and nerve growth factor), inhibiting acetylcholinesterase (such as donepezil, tacrine, rivastigmine and galanthamine), elevating histone deacetylase levels (such as RGFP-966, Tasquinimod, CM-414 and 44B), stimulating the brain by physiotherapy (such as near-infrared light, repetitive transcranial magnetic stimulation, and transcranial direct current stimulation) and transplanting exogenous neural stem cells. However, further evaluations need to be performed to determine the optimal treatment. The present study reviews recent interventions for enhancing adult neurogenesis and attempts to elucidate their mechanisms of action, which may provide a theoretical basis for inducing nerve regeneration to fight against AD.

The Sigma Receptors in Alzheimer's Disease: New Potential Targets for Diagnosis and Therapy.

Sigma (σ) receptors are a class of unique proteins with two subtypes: the sigma-1 (σ1) receptor which is situated at the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM), and the sigma-2 (σ2) receptor, located in the ER-resident membrane. Increasing evidence indicates the involvement of both σ1 and σ2 receptors in the pathogenesis of Alzheimer's disease (AD), and thus these receptors represent two potentially effective biomarkers for emerging AD therapies. The availability of optimal radioligands for positron emission tomography (PET) neuroimaging of the σ1 and σ2 receptors in humans will provide tools to monitor AD progression and treatment outcomes. In this review, we first summarize the significance of both receptors in the pathophysiology of AD and highlight AD therapeutic strategies related to the σ1 and σ2 receptors. We then survey the potential PET radioligands, with an emphasis on the requirements of optimal radioligands for imaging the σ1 or σ2 receptors in humans. Finally, we discuss current challenges in the development of PET radioligands for the σ1 or σ2 receptors, and the opportunities for neuroimaging to elucidate the σ1 and σ2 receptors as novel biomarkers for early AD diagnosis, and for monitoring of disease progression and AD drug efficacy.

Tight control of the APP-Mint1 interaction in regulating amyloid production

Generation of amyloid-β (Aβ) peptides through the proteolytic processing of the amyloid precursor protein (APP) is a pathogenic event in Alzheimer’s disease (AD). APP is a transmembrane protein and endocytosis of APP mediated by the YENPTY motif is a key step in Aβ generation. Mints, a family of cytosolic adaptor proteins, directly bind to the YENPTY motif of APP and facilitate APP trafficking and processing. Here, we generated and examined two Mint1 mutants, Tyr633Ala of Mint1 (Mint1Y633A) that enhanced APP binding, and Tyr549Ala and Phe610Ala mutant (Mint1Y549A/F610A), that reduced APP binding. We investigated how perturbing the APP-Mint1 interaction through these Mint1 mutants alter APP and Mint1 cellular dynamics and Mint1′s interaction with its other binding partners. We found that Mint1Y633A increased binding affinity specifically for APP and presenilin1 (catalytic subunit of γ-secretase), that subsequently enhanced APP endocytosis in primary murine neurons. Conversely, Mint1Y549A/F610A exhibited reduced APP affinity and Aβ secretion. The effect of Mint1Y549A/F610A on Aβ release was greater compared to knocking down all three Mint proteins supporting the APP-Mint1 interaction is a critical factor in Aβ production. Altogether, this study highlights the potential of targeting the APP-Mint1 interaction as a therapeutic strategy for AD.

The Evaluation of Rac1 Signaling as a Potential Therapeutic Target of Alzheimer's Disease.

The Small GTPase Rac1 is critical for various fundamental cellular processes, including cognitive functions. The cyclical activation and inactivation of Rac1, mediated by Rac guanine nucleotide exchange factors (RacGEFs) and Rac GTPase-activating proteins (RacGAPs), respectively, are essential for activating intracellular signaling pathways and controlling cellular processes. We have recently shown that the Alzheimer's disease (AD) therapeutic drug donepezil activates the Rac1-PAK pathway in the nucleus accumbens (NAc) for enhanced aversive learning. Also, PAK activation itself in the NAc enhances aversive learning. As aversive learning allows short-term preliminary AD drug screening, here we tested whether sustained Rac1 activation by RacGAP inhibition can be used as an AD therapeutic strategy for improving AD-learning deficits based on aversive learning. We found that the RacGAP domain of breakpoint cluster region protein (Bcr) (Bcr-GAP) efficiently inhibited Rac1 activity in a membrane ruffling assay. We also found that, in striatal/accumbal primary neurons, Bcr knockdown by microRNA mimic-expressing adeno-associated virus (AAV-miRNA mimic) activated Rac1-PAK signaling, while Bcr-GAP-expressing AAV inactivated it. Furthermore, conditional knockdown of Bcr in the NAc of wild-type adult mice enhanced aversive learning, while Bcr-GAP expression in the NAc inhibited it. The findings indicate that Rac1 activation by RacGAP inhibition enhances aversive learning, implying the AD therapeutic potential of Rac1 signaling.

Identification of tau-tubulin kinase 1 inhibitors by microfluidics-based mobility shift assay from a kinase inhibitor library

Tau tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that phosphorylates multiple residues in tau protein. Hyperphosphorylated tau is the main cause of tauopathy, such as Alzheimer's disease (AD). Therefore, preventing tau phosphorylation by inhibiting TTBK1 has been proposed as a therapeutic strategy for AD. However, few substrates of TTBK1 are reported for a biochemical assay and few inhibitors targeting TTBK1 have been reported so far. In this study, we identified a fluorescein amidite (FAM)-labeled peptide 15 from a small peptide library as the optimal peptide substrate for human TTBK1 (hTTBK1). We then developed and validated a microfluidics-based mobility shift assay (MMSA) with peptide 15. We further confirmed that peptide 15 could also be used in the ADP-Glo kinase assay. The established MMSA was applied for screening of a 427-compound kinase inhibitor library, yielding five compounds with IC50s of several micro molars against hTTBK1. Among them, three compounds, AZD5363, A-674,563 and GSK690693 inhibited hTTBK1 in an ATP competitive manner and molecular docking simulations revealed that they enter the ATP pocket and form one or two hydrogen bonds to the hinge region with hTTBK1. Another hit compound, piceatannol, showed non-ATP competitive inhibitory effect on hTTBK1 and may serve as a starting point to develop highly selective hTTBK1 inhibitors. Altogether, this study provided a new in vitro platform for the development of novel hTTBK1 inhibitors that might have potential applications in AD prevention.

Improving Blood Monocyte Energy Metabolism Enhances Its Ability to Phagocytose Amyloid-β and Prevents Alzheimer's Disease-Type Pathology and Cognitive Deficits.

Deficiencies in the clearance of peripheral amyloid β (Aβ) play a crucial role in the progression of Alzheimer's disease (AD). Previous studies have shown that the ability of blood monocytes to phagocytose Aβ is decreased in AD. However, the exact mechanism of Aβ clearance dysfunction in AD monocytes remains unclear. In the present study, we found that blood monocytes in AD mice exhibited decreases in energy metabolism, which was accompanied by cellular senescence, a senescence-associated secretory phenotype, and dysfunctional phagocytosis of Aβ. Improving energy metabolism rejuvenated monocytes and enhanced their ability to phagocytose Aβ in vivo and in vitro. Moreover, enhancing blood monocyte Aβ phagocytosis by improving energy metabolism alleviated brain Aβ deposition and neuroinflammation and eventually improved cognitive function in AD mice. This study reveals a new mechanism of impaired Aβ phagocytosis in monocytes and provides evidence that restoring their energy metabolism may be a novel therapeutic strategy for AD.