5XFAD Transgenic Mice Research Articles

NXP031 restores memory function by dual effects degrading Aβ accumulation and facilitating antioxidant pathway in Alzheimer's disease models

Alzheimer’s disease (AD) is a representative neurodegenerative disease that is characterized by the overaccumulation of amyloid beta (Aβ) proteins. Since AD is accompanied by excessive oxidative stress, which aggravates neurological pathologies, the use of antioxidants has been considered to prevent disease development. NXP031, a combination of vitamin C (VitC) and an optimized aptamer that binds to VitC and stabilizes the reactivity of VitC, was designed. This study aimed to evaluate the effects of NXP031 on AD pathology, including Aβ accumulation, Aβ-induced oxidative stress, neuronal damage, and neuroinflammation. When NXP031 was administered to 5xFAD transgenic mice, NXP031 exerted a strong inhibitory action on Aβ accumulation, superior to that of VitC, by inducing an increase in Aβ-degrading endopeptidase expression. NXP031 diminished lipid peroxidation levels, activated Nrf2-mediated antioxidant pathways, and suppressed overactivated neuroinflammation. An in vitro study using Neuro2a cells revealed that NXP031 protects the cells against oxidative stress by regulating the MAPK signaling pathway-mediated apoptosis. Additionally, the neuroprotective effects of NXP031 were confirmed in a dose-dependent manner when administered to intrahippocampal Aβ-injected mice, as NXP031 attenuated memory decline, neuronal apoptosis, synaptic degeneration, and excessive glial activation, and reduced NOX-2 expression in the hippocampus. Taken together, NXP031 reduced the Aβ burden by regulating Aβ-degeneration and attenuated memory impairment, neuronal death, synaptic degeneration, and neuroinflammation induced by Aβ toxicity. These results suggest the potential of NXP031 as a therapeutic agent for AD.

Abnormal EEG Effects of Acute Apomorphine Injection in 5xFAD Transgenic Mice Are Partially Normalized in Those Chronically Pretreated with Apomorphine: The Time–Frequency Clustering of EEG Spectra

Background: In experimental and clinical studies of pharmacological treatments for Alzheimer’s disease (AD), the electroencephalogram (EEG) frequency spectrum approach has demonstrated its efficacy in determining the characteristics of pathological changes in the functioning of different cerebral structures, interconnections between them, and disturbances in the brain neurotransmitter systems. The main results have been obtained in frames of traditionally used so-called “classical” EEG frequency bands: delta, theta, alpha, and beta. Objective: This unified approach simplifies comparing data from different studies but loses the dynamic peculiarities of the effects because of their time-dependent transition through the borders of the “classical” bands. Methods: In this study on non-narcotized freely moving 5xFAD transgenic mice, a model of AD, chronically pretreated with a non-selective dopamine (DA) receptor agonist, apomorphine (APO), we analyze the transitory EEG effects of acute APO injection in different brain areas by use of our “time–frequency” clustering program. The acute injection of APO was used to compare DA receptor sensitivity in 5xFAD mice pretreated with either APO or saline vs. wild-type (WT) mice pretreated with saline. Results: After acute APO injection, the clusters of enhanced EEG activity centered in the theta–alpha frequency range observed in WT mice disappeared in 5xFAD mice pretreated with saline and practically recovered in 5xFAD mice pretreated with APO. Conclusions: In 5xFAD mice pretreated with saline, the sensitivity of DA receptors was disturbed; chronic APO pretreatment mainly recovered this characteristic in 5xFAD mice. The “clustering” of pharmacological EEG effects and their time-dependent transition between classical frequency bands is a new effective approach for analyzing cerebral neurotransmission in neurodegenerative pathologies.

Safety, tolerability, pharmacokinetics and pharmacodynamics of a single intravenous dose of SHR-1707 in healthy adult subjects: two randomized, double-blind, single-ascending-dose, phase 1 studies

BackgroundSHR-1707 is a novel humanized anti-Aβ IgG1 monoclonal antibody that binds to Aβ fibrils and monomers to block the formation of Aβ plaques or to promote the microglial phagocytosis of Aβ. Preclinical studies showed that SHR-1707 reduced brain Aβ deposition in 5xFAD transgenic mice. Herein, we conducted two phase 1 studies to evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of a single intravenous dose of SHR-1707 in healthy adult subjects.MethodsTwo randomized, double-blind, single-ascending-dose, phase 1 studies were conducted in China (Study CHN) and Australia (Study AUS). Study CHN consisted of 2 parts. In Part 1, eligible healthy young adults (18–45 years) were sequentially randomized 8:2 to receive SHR-1707 (five cohorts: 2, 6, 20, 40, and 60 mg/kg) or placebo in each cohort; in Part 2, elderly subjects (55–80 years) were randomized 8:4 to receive SHR-1707 (20 mg/kg) or placebo. A similar design was used in Study AUS, but with only healthy young adults enrolled across three dosing cohorts (2, 20, and 60 mg/kg).ResultsSixty-two (part 1/2, n = 50/12; age range, 18–42/55–63 years) and 30 subjects (age range, 18–42 years) received SHR-1707 or placebo in Study CHN and Study AUS, respectively. In Study CHN, all treatment-related adverse events (TRAEs) were mild, with the most common being transient laboratory abnormalities. In Study AUS, TRAEs were mostly mild (1 moderate event each with SHR-1707/placebo); the most common TRAEs with SHR-1707 were dysgeusia and fatigue (8.3% each). In both studies, the exposure of SHR-1707 increased in a slightly greater than dose-proportional manner over the dose range of 2–60 mg/kg in young adults; there was a dose-dependent increase in plasma Aβ42 concentration following SHR-1707 administration compared with the placebo group. The safety and PK and PD profiles of SHR-1707 in the elderly subjects were consistent with the younger counterpart at the same dose level. No ethnic difference in safety, PK and PD of SHR-1707 was observed.ConclusionsA single intravenous dose of SHR-1707 at 2–60 mg/kg was safe and well tolerated in healthy young adult and elderly subjects. The PK and PD profiles are supportive for further clinical development.Trial registrationNCT04973189 (retrospectively registered on Jul.21, 2021) and NCT04745104 (registered on Feb.6, 2021) on clinicaltrials.gov.

Novel Insights Into DLAT's Role in Alzheimer's Disease-Related Copper Toxicity Through Microglial Exosome Dynamics.

Alzheimer's disease (AD) is a complex neurodegenerative disorder, with recent research emphasizing the roles of microglia and their secreted extracellular vesicles in AD pathology. However, the involvement of specific molecular pathways contributing to neuronal death in the context of copper toxicity remains largely unexplored. This study investigates the interaction between pyruvate kinase M2 (PKM2) and dihydrolipoamide S-acetyltransferase (DLAT), particularly focusing on copper-induced neuronal death in Alzheimer's disease. Gene expression datasets were analyzed to identify key factors involved in AD-related copper toxicity. The role of DLAT was validated using 5xFAD transgenic mice, while invitro experiments were conducted to assess the impact of microglial exosomes on neuronal PKM2 transfer and DLAT expression. The effects of inhibiting the PKM2 transfer via microglial exosomes on DLAT expression and copper-induced neuronal death were also evaluated. DLAT was identified as a critical factor in the pathology of AD, particularly in copper toxicity. In 5xFAD mice, increased DLAT expression was linked to hippocampal damage and cognitive decline. Invitro, microglial exosomes were shown to facilitate the transfer of PKM2 to neurons, leading to upregulation of DLAT expression and increased copper-induced neuronal death. Inhibition of PKM2 transfer via exosomes resulted in a significant reduction in DLAT expression, mitigating neuronal death and slowing AD progression. This study uncovers a novel pathway involving microglial exosomes and the PKM2-DLAT interaction in copper-induced neuronal death, providing potential therapeutic targets for Alzheimer's disease. Blocking PKM2 transfer could offer new strategies for reducing neuronal damage and slowing disease progression in AD.

Activation of the muscle-to-brain axis ameliorates neurocognitive deficits in an Alzheimer's disease mouse model via enhancing neurotrophic and synaptic signaling.

Skeletal muscle regulates central nervous system (CNS) function and health, activating the muscle-to-brain axis through the secretion of skeletal muscle-originating factors ("myokines") with neuroprotective properties. However, the precise mechanisms underlying these benefits in the context of Alzheimer's disease (AD) remain poorly understood. To investigate muscle-to-brain axis signaling in response to amyloid β (Aβ)-induced toxicity, we generated 5xFAD transgenic female mice with enhanced skeletal muscle function (5xFAD;cTFEB;HSACre) at prodromal (4-months old) and late (8-months old) symptomatic stages. Skeletal muscle TFEB overexpression reduced Aβ plaque accumulation in the cortex and hippocampus at both ages and rescued behavioral neurocognitive deficits in 8-month-old 5xFAD mice. These changes were associated with transcriptional and protein remodeling of neurotrophic signaling and synaptic integrity, partially due to the CNS-targeting myokine prosaposin (PSAP). Our findings implicate the muscle-to-brain axis as a novel neuroprotective pathway against amyloid pathogenesis in AD.

The Association of Brain Insulin Resistance with Anesthesia/Surgery-Induced Cognitive Deterioration Is Female-Specific in 5XFAD Transgenic Mice.

Our previous studies indicated that anesthesia/surgery could aggravate cognitive impairment and tau pathology in female 5XFAD transgenic (Tg) mice. However, it is unknown whether there are sex differences in the susceptibility of developing postoperative cognitive dysfunction in 5XFAD Tg mice. In this study, we aim to determine whether anesthesia/surgery can have different effects on female and male 5XFAD Tg mice, and to explore the underpinning mechanisms. The mice received abdominal surgery under isoflurane anesthesia. Morris water maze was used to assess the cognitive function. Hippocampal levels of p-tau (AT8), p-IRS1 (Ser612), IRS1, p-GSK3β (Tyr216), and p-GSK3β (Ser9) at postoperative day 1 were evaluated by western blot assays. Anesthesia/surgery exaggerated cognitive impairment and tau pathology in female, but not male 5XFAD Tg mice. The anesthesia/surgery led to elevated hippocampus protein levels of p-IRS1 (Ser612)/IRS1 ratio and p-GSK3β (Tyr216) and reduced hippocampus protein levels of p-GSK3β (Ser9) in female, but not male 5XFAD Tg mice. This study demonstrated that female 5XFAD Tg mice were more susceptible to anesthesia/surgery-induced cognitive deterioration and tau pathology aggravation, potentially due to female-specific brain insulin resistance.

The impacts of sex and the 5xFAD model of Alzheimer's disease on the sleep and spatial learning responses to feeding time.

The relationships between the feeding rhythm, sleep and cognition in Alzheimer's disease (AD) are incompletely understood, but meal time could provide an easy-to-implement method of curtailing disease-associated disruptions in sleep and cognition. Furthermore, known sex differences in AD incidence could relate to sex differences in circadian rhythm/sleep/cognition interactions. The 5xFAD transgenic mouse model of AD and non-transgenic wild-type controls were studied. Both female and male mice were used. Food access was restricted each day to either the 12-h light phase (light-fed groups) or the 12-h dark phase (dark-fed groups). Sleep (electroencephalographic/electromyographic) recording and cognitive behavior measures were collected. The 5xFAD genotype reduces NREM and REM as well as the number of sleep spindles. In wild-type mice, light-fed groups had disrupted vigilance state amounts, characteristics, and rhythms relative to dark-fed groups. These feeding time differences were reduced in 5xFAD mice. Sex modulates these effects. 5xFAD mice display poorer spatial memory that, in female mice, is curtailed by dark phase feeding. Similarly, female 5xFAD mice have decreased anxiety-associated behavior. These emotional and cognitive measures are correlated with REM amount. Our study demonstrates that the timing of feeding can alter many aspects of wake, NREM and REM. Unexpectedly, 5xFAD mice are less sensitive to these feeding time effects. 5xFAD mice demonstrate deficits in cognition which are correlated with REM, suggesting that this circadian-timed aspect of sleep may link feeding time and cognition. Sex plays an important role in regulating the impact of feeding time on sleep and cognition in both wild-type and 5xFAD mice, with females showing a greater cognitive response to feeding time than males.

Synbiotic therapy with Clostridium sporogenes and xylan promotes gut-derived indole-3-propionic acid and improves cognitive impairments in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized primarily by cognitive impairment. Recent investigations have highlighted the potential of nutritional interventions that target the gut-brain axis, such as probiotics and prebiotics, in forestalling the onset of AD. In this study, whole-genome sequencing was employed to identify xylan as the optimal carbon source for the tryptophan metabolism regulating probiotic Clostridium sporogenes (C. sporogenes). Subsequent in vivo studies demonstrated that administration of a synbiotic formulation comprising C. sporogenes (1 × 1010 CFU per day) and xylan (1%, w/w) over a duration of 30 days markedly enhanced cognitive performance and spatial memory faculties in the 5xFAD transgenic AD mouse model. The synbiotic treatment significantly reduced amyloid-β (Aβ) accumulation in the cortex and hippocampus of the brain. Importantly, synbiotic therapy substantially restored the synaptic ultrastructure in AD mice and suppressed neuroinflammatory responses. Moreover, the intervention escalated levels of the microbial metabolite indole-3-propionic acid (IPA) and augmented the relative prevalence of IPA-synthesizing bacteria, Lachnospira and Clostridium, while reducing the dominant bacteria in AD, such as Aquabacterium, Corynebacterium, and Romboutsia. Notably, synbiotic treatment also prevented the disruption of gut barrier integrity. Correlation analysis indicated a strong positive association between gut microbiota-generated IPA levels and behavioral changes. In conclusion, this study demonstrates that synbiotic supplementation significantly improves cognitive and intellectual deficits in 5xFAD mice, which could be partly attributed to enhanced IPA production by gut microbiota. These findings provide a theoretical basis for considering synbiotic therapy as a novel microbiota-targeted approach for the treatment of metabolic and neurodegenerative diseases.

Integrated Genome-Wide Analysis of DNA Methylation and Gene Expression in the Hippocampi of 5xFAD Alzheimer's Disease Mouse Model.

DNA methylation forms 5-methylcytosine and its regulation in the hippocampus is critical for learning and memory. Indeed, dysregulation of DNA methylation is associated with neurological diseases. Alzheimer's disease (AD) is the predominant of dementia and a neurodegenerative disorder. We examined the learning and memory function in 3- and 9-month-old wild-type and 5xfamiliar Alzheimer's disease (5xFAD) transgenic mice by performing the object recognition memory and Y-maze tests, and identified the hippocampal amyloid beta burden. To investigate the epigenetically regulated genes involved in the development or neuropathology of AD, we performed genome-wide DNA methylation sequencing and RNA sequencing analyses in the hippocampus of 9-month-old wild-type and 5xFAD tg mice. To validate the genes inversely regulated by epigenetics, we confirmed their methylation status and mRNA levels. At 9 months of age, 5xFAD tg mice showed significant cognitive impairment and amyloid-beta plaques in the hippocampus. DNA methylation sequencing identified a total of 13,777 differentially methylated regions, including 4484 of hyper- and 9293 of hypomethylated regions, that are associated with several gene ontology (GO) terms including 'nervous system development' and 'axon guidance'. In RNA sequencing analysis, we confirmed a total of 101 differentially expressed genes, including 52 up- and 49 downregulated genes, associated with GO functions such as 'positive regulation of synaptic transmission, glutamatergic' and 'actin filament organization'. Through further integrated analysis of DNA methylation and RNA sequencing, three epigenetically regulated genes were selected: thymus cell antigen 1, theta (Thy1), myosin VI (Myo6), and filamin A-interacting protein 1-like (Filip1l). The methylation level of Thy1 decreased and its mRNA levels increased, whereas that of Myo6 and Filip1l increased and their mRNA levels decreased. The common functions of these three genes may be associated with the neural cytoskeleton and synaptic plasticity. We suggest that the candidate genes epigenetically play a role in AD-associated neuropathology (i.e., amyloid-beta plaques) and memory deficit by influencing neural structure and synaptic plasticity. Furthermore, counteracting dysregulated epigenetic changes may delay or ameliorate AD onset or symptoms.

18F]Flotaza for Aβ Plaque Diagnostic Imaging: Evaluation in Postmortem Human Alzheimer's Disease Brain Hippocampus and PET/CT Imaging in 5xFAD Transgenic Mice.

The diagnostic value of imaging Aβ plaques in Alzheimer's disease (AD) has accelerated the development of fluorine-18 labeled radiotracers with a longer half-life for easier translation to clinical use. We have developed [18F]flotaza, which shows high binding to Aβ plaques in postmortem human AD brain slices with low white matter binding. We report the binding of [18F]flotaza in postmortem AD hippocampus compared to cognitively normal (CN) brains and the evaluation of [18F]flotaza in transgenic 5xFAD mice expressing Aβ plaques. [18F]Flotaza binding was assessed in well-characterized human postmortem brain tissue sections consisting of HP CA1-subiculum (HP CA1-SUB) regions in AD (n = 28; 13 male and 15 female) and CN subjects (n = 32; 16 male and 16 female). Adjacent slices were immunostained with anti-Aβ and analyzed using QuPath. In vitro and in vivo [18F]flotaza PET/CT studies were carried out in 5xFAD mice. Post-mortem human brain slices from all AD subjects were positively IHC stained with anti-Aβ. High [18F]flotaza binding was measured in the HP CA1-SUB grey matter (GM) regions compared to white matter (WM) of AD subjects with GM/WM > 100 in some subjects. The majority of CN subjects had no decipherable binding. Male AD exhibited greater WM than AD females (AD WM♂/WM♀ > 5; p < 0.001) but no difference amongst CN WM. In vitro studies in 5xFAD mice brain slices exhibited high binding [18F]flotaza ratios (>50 versus cerebellum) in the cortex, HP, and thalamus. In vivo, PET [18F]flotaza exhibited binding to Aβ plaques in 5xFAD mice with SUVR~1.4. [18F]Flotaza is a new Aβ plaque PET imaging agent that exhibited high binding to Aβ plaques in postmortem human AD. Along with the promising results in 5xFAD mice, the translation of [18F]flotaza to human PET studies may be worthwhile.

Herpes simplex virus 1 accelerates the progression of Alzheimer’s disease by modulating microglial phagocytosis and activating NLRP3 pathway

Accumulating evidence implicates that herpes simplex virus type 1 (HSV-1) has been linked to the development and progression of Alzheimer’s disease (AD). HSV-1 infection induces β-amyloid (Aβ) deposition in vitro and in vivo, but the effect and precise mechanism remain elusive. Here, we show that HSV-1 infection of the brains of transgenic 5xFAD mice resulted in accelerated Aβ deposition, gliosis, and cognitive dysfunction. We demonstrate that HSV-1 infection induced the recruitment of microglia to the viral core to trigger microglial phagocytosis of HSV-GFP-positive neuronal cells. In addition, we reveal that the NLRP3 inflammasome pathway induced by HSV-1 infection played a crucial role in Aβ deposition and the progression of AD caused by HSV-1 infection. Blockade of the NLRP3 inflammasome signaling reduces Aβ deposition and alleviates cognitive decline in 5xFAD mice after HSV-1 infection. Our findings support the notion that HSV-1 infection is a key factor in the etiology of AD, demonstrating that NLRP3 inflammasome activation functions in the interface of HSV-1 infection and Aβ deposition in AD.

A new neuroprotective candidate TJ1 targeting amyloidogenesis in 5xFAD Alzheimer’s disease mice

As one of the main pathmechanisms of Alzheimer’s disease (AD), amyloid-β (Aβ) is widely considered to be the prime target for the development of AD therapy. Recently, imidazolylacetophenone oxime ethers or esters (IOEs) have shown neuroprotective effects against neuronal cells damage, suggesting their potential use in the prevention and treatment of AD. Thirty IOEs compounds from our lab in-house library were constructed and screened for the inhibitory effects on Aβ42-induced cytotoxicity. Among them, TJ1, as a new IOEs hit, preliminarily showed the effect on inhibiting Aβ42-induced cytotoxicity. Furthermore, the inhibitory effects of TJ1 on Aβ42 aggregation were tested by ThT assays and TEM. The neuroprotective effects of TJ1 were evaluated in Aβ42-stimulated SH-SY5Y cells, LPS-stimulated BV-2 cells, and H2O2– and RSL3-stimulated PC12 cells. The cognitive improvement of TJ1 was assessed in 5xFAD (C57BL/6J) transgenic mouse. These results showed that TJ1 had strong neuroprotective effects and high blood–brain barrier (BBB) permeability without obvious cytotoxicity. TJ1 impeded the self-accumulation process of Aβ42 by acting on Aβ oligomerization and fibrilization. Besides, TJ1 reversed Aβ-, H2O2– and RSL3-induced neuronal cell damage and decreased neuroinflammation. In 5xFAD mice, TJ1 improved cognitive impairment, increased GSH level, reduced the level of Aβ42 and Aβ plaques, and attenuated the glia reactivation and inflammatory response in the brain,. Taken together, our results demonstrate that TJ1 improves cognitive impairments as a new neuroprotective candidate via targeting amyloidogenesis, which suggests the potential of TJ1 as a treatment for AD.

Behaviour Hallmarks in Alzheimer's Disease 5xFAD Mouse Model.

The 5xFAD transgenic mouse model widely used in Alzheimer's disease (AD) research recapitulates many AD-related phenotypes with a relatively early onset and aggressive age-dependent progression. Besides developing amyloid peptide deposits alongside neuroinflammation by the age of 2 months, as well as exhibiting neuronal decline by the age of 4 months that intensifies by the age of 9 months, these mice manifest a broad spectrum of behavioural impairments. In this review, we present the extensive repertoire of behavioural dysfunctions in 5xFAD mice, organised into four categories: motor skills, sensory function, learning and memory abilities, and neuropsychiatric-like symptoms. The motor problems, associated with agility and reflex movements, as well as balance and coordination, and skeletal muscle function, typically arise by the time mice reach 9 months of age. The sensory function (such as taste, smell, hearing, and vision) starts to deteriorate when amyloid peptide buildups and neuroinflammation spread into related anatomical structures. The cognitive functions, encompassing learning and memory abilities, such as visual recognition, associative, spatial working, reference learning, and memory show signs of decline from 4 to 6 months of age. Concerning neuropsychiatric-like symptoms, comprising apathy, anxiety and depression, and the willingness for exploratory behaviour, it is believed that motivational changes emerge by approximately 6 months of age. Unfortunately, numerous studies from different laboratories are often contradictory on the conclusions drawn and the identification of onset age, making preclinical studies in rodent models not easily translatable to humans. This variability is likely due to a range of factors associated with animals themselves, housing and husbandry conditions, and experimental settings. In the forthcoming studies, greater clarity in experimental details when conducting behavioural testing in 5xFAD transgenic mice could minimise the inconsistencies and could ensure the reliability and the reproducibility of the results.

Chemically Driven Clearance of Amyloid Aggregates by Polyfunctionalized Furo[2,3-b:4,5-b']dipyridine-Chalcone Hybrids to Ameliorate Memory in an Alzheimer Mouse Model.

The aberrant assembly of amyloid-β (Aβ) is implicated in Alzheimer's disease (AD). Recent clinical outcomes of Aβ-targeted immunotherapy reinforce the notion that clearing Aβ burden is a potential therapeutic approach for AD. Herein, to develop drug candidates for chemically driven clearance of Aβ aggregates, we synthesized 51 novel polyfunctionalized furo[2,3-b:4,5-b']dipyridine-chalcone hybrid compounds. After conducting two types of cell-free anti-Aβ functional assays, Aβ aggregation prevention and Aβ aggregate clearance, we selected YIAD-0336, (E)-8-((1H-pyrrol-2-yl)methylene)-10-(4-chlorophenyl)-2,4-dimethyl-7,8-dihydropyrido[3',2':4,5]furo[3,2-b]quinolin-9(6H)-one, for further in vivo investigations. As YIAD-0336 exhibited a low blood-brain barrier penetration profile, it was injected along with aggregated Aβ directly into the intracerebroventricular region of ICR mice and ameliorated spatial memory in Y-maze tests. Next, YIAD-0336 was orally administered to 5XFAD transgenic mice with intravenous injections of mannitol, and YIAD-0336 significantly removed Aβ plaques from the brains of 5XFAD mice. Collectively, YIAD-0336 dissociated toxic aggregates in the mouse brain and hence alleviated cognitive deterioration. Our findings indicate that chemically driven clearance of Aβ aggregates is a promising therapeutic approach for AD.

Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer’s disease pathology progression

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

Temporal-Focusing Multiphoton Excitation Single-Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores.

Single-molecule localization microscopy (SMLM) based on temporal-focusing multiphoton excitation (TFMPE) and single-wavelength excitation is used to visualize the three-dimensional (3D) distribution of spontaneously blinking fluorophore-labeled subcellular structures in a thick specimen with a nanoscale-level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out-of-focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single-wavelength TFMPE achieves wide-field and axially confined two-photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two-dimensional TFMPE-SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE-SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid-beta peptide deposits.

Temporal‐Focusing Multiphoton Excitation Single‐Molecule Localization Microscopy Using Spontaneously Blinking Fluorophores

AbstractSingle‐molecule localization microscopy (SMLM) based on temporal‐focusing multiphoton excitation (TFMPE) and single‐wavelength excitation is used to visualize the three‐dimensional (3D) distribution of spontaneously blinking fluorophore‐labeled subcellular structures in a thick specimen with a nanoscale‐level spatial resolution. To eliminate the photobleaching effect of unlocalized molecules in out‐of‐focus regions for improving the utilization rate of the photon budget in 3D SMLM imaging, SMLM with single‐wavelength TFMPE achieves wide‐field and axially confined two‐photon excitation (TPE) of spontaneously blinking fluorophores. TPE spectral measurement of blinking fluorophores is then conducted through TFMPE imaging at a tunable excitation wavelength, yielding the optimal TPE wavelength for increasing the number of detected photons from a single blinking event during SMLM. Subsequently, the TPE fluorescence of blinking fluorophores is recorded to obtain a two‐dimensional TFMPE‐SMLM image of the microtubules in cancer cells with a localization precision of 18±6 nm and an overall imaging resolution of approximately 51 nm, which is estimated based on the contribution of Nyquist resolution and localization precision. Combined with astigmatic imaging, the system is capable of 3D TFMPE‐SMLM imaging of brain tissue section of a 5XFAD transgenic mouse with the pathological features of Alzheimer's disease, revealing the distribution of neurotoxic amyloid‐beta peptide deposits.

Peripherally activated brain CD8+ T cells promote AD in 5xFAD mice

Abstract The role of the adaptive immunity in Alzheimer’s Disease (AD) remains poorly understood. Recent findings from our group, which link B cells to manifestation AD 1, and others, which show increase of CD8+ T cells in the AD brain 2, suggest that the adaptive immunity is an important but overlooked player in this progressive neurodegenerative disease. Here, our aim was to determine the role of CD8+ T cells in the brain in transgenic 5xFAD mice, which develop human AD-like symptoms. We manipulated brain CD8+ T cells in AD mice by depleting circulating CD8+ T cells with anti-CD8 mAb or expanding antigen-specific CD8+ T cells with Aβ-CoreS (encodes Ab1-11) RNA immunization. We found that while the CD8+ T cell depletion decreases Aβ plaques and disease-associated microglia in the hippocampus, and improves cognition, Aβ-CoreS exacerbates AD symptoms via increasing brain CD8+ T cells with cytotoxic and exhausted phenotype. Overall, our results indicate the AD manifestation requires brain antigen-specific CD8+ T cells, which upon peripheral induction/expansion infiltrate the brain and exacerbate AD symptoms. We propose that CD8+ T cells may similarly exacerbate AD in humans, as we detected their significant increase in the brain of deceased people with AD. 1. Kim et al. Nat Commun. 2021;12(1):2185. 2. Gate et al. (2020). Nature 577, 399-404. This research was supported by the Intramural Research Program of the National Institute on Aging, NIH

Interaction of exogenous acetylcholinesterase and butyrylcholinesterase with amyloid-β plaques in human brain tissue

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are associated with amyloid-β (Aβ) plaques and exhibit altered biochemical properties in human Alzheimer's disease (AD), as well as in the transgenic 5XFAD mouse model of AD amyloidosis. In the brains of the 5XFAD mouse model devoid of BChE enzyme (5XFAD/BChE-KO), incubation of tissue sections with exogenous BChE purified from human plasma (pl-BChE) leads to its association with Aβ plaques and its biochemical properties are comparable to those reported for endogenous BChE associated with plaques in both human AD and in 5XFAD mouse brain tissue. We sought to determine whether these observations in 5XFAD/BChE-KO mice also apply to human brain tissues. To do so, endogenous ChE activity in human AD brain tissue sections was quenched with 50 % aqueous acetonitrile (MeCNaq) leaving the tissue suitable for further studies. Quenched sections were then incubated with recombinant AChE (r-AChE) or pl-BChE and stained for each enzymes' activity. Exogenous r-AChE or pl-BChE became associated with Aβ plaques, and when bound, had properties that were comparable to the endogenous ChE enzymes associated with plaques in AD brain tissues without acetonitrile treatment. These findings in human AD brain tissue extend previous observations in the 5XFAD/BChE-KO mouse model and demonstrate that exogenously applied r-AChE and pl-BChE have high affinity for Aβ plaques in human brain tissues. This association alters the biochemical properties of these enzymes, most likely due a conformational change. If incorporation of AChE and BChE in Aβ plaques facilitates AD pathogenesis, blocking this association could lead to disease-modifying approaches to AD. This work provides a method to study the mechanism of AChE and BChE interaction with Aβ plaque pathology in post-mortem human brain tissue.

BIN1K358R suppresses glial response to plaques in mouse model of Alzheimer's disease.

The BIN1 coding variant rs138047593 (K358R) is linked to Late-Onset Alzheimer's Disease (LOAD) via targeted exome sequencing. To elucidate the functional consequences of this rare coding variant on brain amyloidosis and neuroinflammation, we generated BIN1K358R knock-in mice using CRISPR/Cas9 technology. These mice were subsequently bred with 5xFAD transgenic mice, which serve as a model for Alzheimer's pathology. The presence of the BIN1K358R variant leads to increased cerebral amyloid deposition, with a dampened response of astrocytes and oligodendrocytes, but not microglia, at both the cellular and transcriptional levels. This correlates with decreased neurofilament light chain in both plasma and brain tissue. Synaptic densities are significantly increased in both wild-type and 5xFAD backgrounds homozygous for the BIN1K358R variant. The BIN1 K358R variant modulates amyloid pathology in 5xFAD mice, attenuates the astrocytic and oligodendrocytic responses to amyloid plaques, decreases damage markers, and elevates synaptic densities. BIN1 rs138047593 (K358R) coding variant is associated with increased risk of LOAD. BIN1 K358R variant increases amyloid plaque load in 12-month-old 5xFAD mice. BIN1 K358R variant dampens astrocytic and oligodendrocytic response to plaques. BIN1 K358R variant decreases neuronal damage in 5xFAD mice. BIN1 K358R upregulates synaptic densities and modulates synaptic transmission.