A novel PDHK inhibitor restored cognitive dysfunction and limited neurodegeneration without affecting amyloid pathology in 5xFAD mouse, a model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia. Although drugs focusing on reducing amyloid ß slow progression, they fail to improve cognitive function. Deficits in glucose metabolism are reflected in FDG-PET and parallel the neurodegeneration and synaptic marker loss closely preceding cognitive decline, but the role of metabolic deficits as a cause or consequence of neurodegeneration is unclear. Pyruvate dehydrogenase (PDH) is lost in AD and an important enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle by converting pyruvate into acetyl-CoA. It is negatively regulated by pyruvate dehydrogenase kinase (PDHK) through phosphorylation. METHODS: In the present study, we assessed the in vitro/ in vivo pharmacological profile of the novel PDHK inhibitor that we discovered, Compound A. We also assessed the effects of Compound A on AD-related phenotypes including neuron loss and cognitive impairment using 5xFAD model mice. RESULTS: Compound A inhibited human PDHK1, 2 and 3 but had no inhibitory activity on PDHK4. In primary neurons, Compound A enhanced pyruvate and lactate utilization, but did not change glucose levels. In contrast, in primary astrocytes, Compound A enhanced pyruvate and glucose utilization and enhanced lactate production. In an efficacy study using 5xFAD mice, Compound A ameliorated the cognitive dysfunction in the novel object recognition test and Morris water maze. Moreover, Compound A prevented neuron loss in the hippocampus and cerebral cortex of 5xFAD without affecting amyloid ß deposits. CONCLUSIONS: These results suggest ameliorating metabolic deficits by activating PDH by Compound A can limit neurodegeneration and is a promising therapeutic strategy for treating AD.

Spatiotemporal Dysregulation of Neuron-Glia Related Genes and Pro-/Anti-Inflammatory miRNAs in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD), the leading cause of dementia, is a multifactorial disease influenced by aging, genetics, and environmental factors. miRNAs are crucial regulators of gene expression and play significant roles in AD onset and progression. This exploratory study analyzed the expression levels of 28 genes and 5 miRNAs (miR-124-3p, miR-125b-5p, miR-21-5p, miR-146a-5p, and miR-155-5p) related to AD pathology and neuroimmune responses using RT-qPCR. Analyses were conducted in the prefrontal cortex (PFC) and the hippocampus (HPC) of the 5xFAD mouse AD model at 6 and 9 months old. Data highlighted upregulated genes encoding for glial fibrillary acidic protein (Gfap), triggering receptor expressed on myeloid cells (Trem2) and cystatin F (Cst7), in the 5xFAD mice at both regions and ages highlighting their roles as critical disease players and potential biomarkers. Overexpression of genes encoding for CCAAT enhancer-binding protein alpha (Cebpa) and myelin proteolipid protein (Plp) in the PFC, as well as for BCL2 apoptosis regulator (Bcl2) and purinergic receptor P2Y12 (P2yr12) in the HPC, together with upregulated microRNA(miR)-146a-5p in the PFC, prevailed in 9-month-old animals. miR-155 positively correlated with miR-146a and miR-21 in the PFC, and miR-125b positively correlated with miR-155, miR-21, while miR-146a in the HPC. Correlations between genes and miRNAs were dynamic, varying by genotype, region, and age, suggesting an intricate, disease-modulated interaction between miRNAs and target pathways. These findings contribute to our understanding of miRNAs as therapeutic targets for AD, given their multifaceted effects on neurons and glial cells.

Association of GLOD4 with Alzheimer's Disease in Humans and Mice.

Background: Glyoxalase domain containing protein 4 (GLOD4), a protein of an unknown function, is associated with Alzheimer's disease (AD). Three GLOD4 isoforms are known. The mechanism underlying GLOD4's association with AD was unknown. Objective: To assess GLOD4's role in the central nervous system by studying GLOD4 isoforms expression in human frontal cerebral cortical tissues from AD patients and in brains of Blmh-/-5xFAD mouse AD model of AD. Methods: GLOD4 protein and mRNA were quantified in human and mouse brains by western blotting and RT-qPCR, respectively. Mouse brain amyloid-ß (Aß) was quantified by western blotting. Behavioral assessments of mice were performed by cognitive/neuromotor testing. Glod4 gene in mouse neuroblastoma N2a-APPswe cells was silenced by RNA interference and Glod4 protein/mRNA, Aß precursor protein (Aßpp)/mRNA, Atg5, p62, and Lc3 mRNAs were quantified. Results: GLOD4 mRNA and protein isoforms were downregulated in cortical tissues from AD patients compared to non-AD controls. Glod4 mRNA was downregulated in brains of Blmh-/-5xFAD mice compared to Blmh+/+5xFAD sibling controls, but not in Blmh-/- mice without the 5xFAD transgene compared to Blmh+/+ sibling controls. The 5xFAD transgene downregulated Glod4 mRNA in Blmh-/- mice of both sexes and in Blmh+/+ males but not females. Attenuated Glod4 was associated with elevated Aß and worsened memory/sensorimotor performance in Blmh-/-5xFAD mice. Glod4 depletion in N2a-APPswe cells upregulated AßPP, and downregulated autophagy-related Atg5, p62, and Lc3 genes. Conclusions: These findings suggest that GLOD4 interacts with AßPP and the autophagy pathway, and that disruption of these interactions leads to Aß accumulation and cognitive/neurosensory deficits.

Toxoplasma gondii Infection of Alzheimer's Disease Mice Reduces Brain Amyloid Density Globally and Regionally.

BACKGROUND: Toxoplasma gondii infection of Alzheimer's disease model mice decreases amyloid ß plaques. We aimed to determine if there is a brain regional difference in amyloid ß reduction in the brains of T. gondii-infected compared to control mice. METHOD: Three-month-old 5xFAD (AD model) mice were injected with T. gondii or with phosphate-buffered saline as a control. Intact brains were harvested at 6 weeks postinfection, optically cleared using iDISCO+, and brain-wide amyloid burden was visualized using volumetric light-sheet imaging. Amyloid signal was quantified across each brain and computationally mapped to the Allen Institute Brain Reference Atlas to determine amyloid density in each region. RESULTS: A brain-wide analysis of amyloid in control and T. gondii-infected 5xFAD mice revealed that T. gondii infection decreased amyloid burden in the brain globally as well as in the cortex and hippocampus, and many daughter regions. Daughter regions that showed reduced amyloid burden included the prelimbic cortex, visual cortex, and retrosplenial cortex. The olfactory tubercle, a region known to have increased monocytes following T. gondii infection, also showed reduced amyloid after infection. CONCLUSIONS: T. gondii infection of AD mice reduces amyloid burden in a brain region-specific manner that overlaps with known regions of T. gondii infection and peripheral immune cell infiltration.

Fluvoxamine maleate alleviates amyloid-beta load and neuroinflammation in 5XFAD mice to ameliorate Alzheimer disease pathology.

Introduction: Alzheimer pathology (AD) is characterized by the deposition of amyloid beta (Aß) and chronic neuroinflammation, with the NLRP3 inflammasome playing a significant role. This study demonstrated that the OCD drug fluvoxamine maleate (FXN) can potently ameliorate AD pathology in 5XFAD mice by promoting autophagy-mediated clearance of Aß and inhibiting the NLRP3 inflammasome. Methods: We used mice primary astrocytes to establish the mechanism of action of FXN against NLRP3 inflammasome by using various techniques like ELISA, Western blotting, confocal microscopy, Immunofluorescence, etc. The anti-AD activity of FXN was validated in transgenic 5XFAD mice following two months of treatment. This was followed by behavior analysis, examination of inflammatory and autophagy proteins and immunohistochemistry analysis for Aß load in the hippocampi. Results: Our data showed that FXN, at a low concentration of 78 nM, induces autophagy to inhibit NF-κB and the NLRP3 inflammasome, apart from directly inhibiting NLRP3 inflammasome in primary astrocytes. FXN activated the PRKAA2 pathway through CAMKK2 signaling, leading to autophagy induction. It inhibited the ATP-mediated NLRP3 inflammasome activation by promoting the autophagic degradation of NF-κB, resulting in the downregulation of pro-IL-1ß and NLRP3. The anti-NLRP3 inflammasome effect of FXN was reversed when autophagy was inhibited by either genetic knockdown of the PRKAA2 pathway or pharmacological inhibition with bafilomycin A1. Furthermore, FXN treatment led to improved AD pathology in 5XFAD mice, resulting in significant improvements in various behavioral parameters such as working memory and neuromuscular coordination, making their behavior more similar to that of wild-type animals. FXN improved behavior in 5XFAD mice by clearing the Aß deposits from the hippocampi and significantly reducing multiple inflammatory proteins, including NF-κB, GFAP, IBA1, IL-1ß, TNF-α, and IL-6, which are associated with NF-κB and NLRP3 inflammasome in the brain. Moreover, these changes were accompanied by increased expression of autophagic proteins. Discussion: Our data suggest that FXN ameliorates AD pathology, by simultaneously targeting two key pathological features: Aß deposits and neuroinflammation. As an already approved drug, FXN holds potential as a candidate for human studies against AD.

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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

O-demethyl galantamine alters protein expression in cerebellum of 5xFAD mice.

Background/aim: Alzheimer's disease (AD), one of the most common health issues, is characterized by memory loss, severe behavioral disorders, and eventually death. Despite many studies, there are still no drugs that can treat AD or stop it from progressing. Previous in vitro tests showed that O-demethyl galantamine (ODG) might have therapeutic potential owing to its 10 times higher acetylcholinesterase inhibitory activity than galantamine (GAL). Materials and methods: We aimed to assess the effect of ODG at the molecular level in a 12-month-old 5xFAD Alzheimer's mouse model. To this end, following the administrations of ODG and GAL (used as a positive control), protein alterations were investigated in the cortex, hippocampus, and cerebellum regions of the brain. Surprisingly, GAL altered proteins prominently in the cortex, while ODG exclusively exerted its effect on the cerebellum. Results: GNB1, GNB2, NDUFS6, PAK2, and RhoA proteins were identified as the top 5 hub proteins in the cerebellum of ODG-treated mice. Reregulation of these proteins through Ras signaling and retrograde endocannabinoid signaling pathways, which were found to be enriched, might contribute to reversing AD-induced molecular changes. Conclusion: We suggest that, since it targets specifically the cerebellum, ODG may be further evaluated for combination therapies for AD.

Investigating the impact of environmental enrichment on proteome and neurotransmitter-related profiles in an animal model of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with behavioral and cognitive impairments. Unfortunately, the drugs the Food and Drug Administration currently approved for AD have shown low effectiveness in delaying the progression of the disease. The focus has shifted to non-pharmacological interventions (NPIs) because of the challenges associated with pharmacological treatments for AD. One such intervention is environmental enrichment (EE), which has been reported to restore cognitive decline associated with AD effectively. However, the therapeutic mechanisms by which EE improves symptoms associated with AD remain unclear. Therefore, this study aimed to reveal the mechanisms underlying the alleviating effects of EE on AD symptoms using histological, proteomic, and neurotransmitter-related analyses. Wild-type (WT) and 5XFAD mice were maintained in standard housing or EE conditions for 4 weeks. First, we confirmed the mitigating effects of EE on cognitive impairment in an AD animal model. Then, histological analysis revealed that EE reduced Aß accumulation, neuroinflammation, neuronal death, and synaptic loss in the AD brain. Moreover, proteomic analysis by liquid chromatography-tandem mass spectrometry showed that EE enhanced synapse- and neurotransmitter-related networks and upregulated synapse- and neurotransmitter-related proteins in the AD brain. Furthermore, neurotransmitter-related analyses showed an increase in acetylcholine and serotonin concentrations as well as a decrease in polyamine concentration in the frontal cortex and hippocampus of 5XFAD mice raised under EE conditions. Our findings demonstrate that EE restores cognitive impairment by alleviating AD pathology and regulating synapse-related proteins and neurotransmitters. Our study provided neurological evidence for the application of NPIs in treating AD.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Early spatial recognition memory deficits in 5XFAD female mice are associated with disruption of prefrontal parvalbumin neurons.

Women have a two-fold increased risk of developing Alzheimer's disease (AD) than men, yet the underlying mechanisms of this sex-specific vulnerability remain unknown. Here, we aimed at determining in the 5XFAD mouse model whether deficits in prefrontal-dependent cognitive functions, which are impacted in the preclinical stages of AD, appear earlier in females, and whether these cognitive deficits are associated with alterations in the activity of prefrontal parvalbumin (PV)-neurons that regulate prefrontal circuits activity. We observed that 3.5-month-old 5XFAD females, but not males, display impairments in spatial short-term recognition memory, a function that relies on the integrity of the prefrontal cortex. Hippocampal-dependent cognitive functions were intact in both sexes. We then observed that 5XFAD females have more prefrontal PV neurons expressing the marker of chronic activity FosB; this was inversely correlated with prefrontal-dependent cognitive performances. Our findings show for the first time sex-specific, early deregulation of prefrontal PV neurons activity, which is associated with early appearance of prefrontal-dependent cognitive functions in 5XFAD females providing a potential novel mechanism to the increased risk to AD in females.

Platycodon grandiflorum root extract inhibits Aß deposition by breaking the vicious circle linking oxidative stress and neuroinflammation in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease accompanied by irreversible cognitive impairment. A deleterious feedback loop between oxidative stress and neuroinflammation in early AD exacerbates AD-related pathology. Platycodon grandiflorum root extract (PGE) has antioxidant and anti-inflammatory effects in several organs. However, the mechanisms underlying the effects of PGE in the brain remain unclear, particularly regarding its impact on oxidative/inflammatory damage and Aß deposition. Thus, we aim to identify the mechanism through which PGE inhibits Aß deposition and oxidative stress in the brain by conducting biochemical and histological analyses. First, to explore the antioxidant mechanism of PGE in the brain, we induced oxidative stress in mice injected with scopolamine and investigated the effect of PGE on cognitive decline and oxidative damage. We also assessed the effect of PGE on reactive oxygen species (ROS) generation and the expressions of antioxidant enzymes and neurotrophic factor in H2O2- and Aß-treated HT22 hippocampal cells. Next, we investigated whether PGE, which showed antioxidant effects, could reduce Aß deposition by mitigating neuroinflammation, especially microglial phagocytosis. We directly verified the effect of PGE on microglial phagocytosis, microglial activation markers, and pro-inflammatory cytokines in Aß-treated BV2 microglial cells. Moreover, we examined the effect of PGE on neuroinflammation, inducing microglial responses in Aß-overexpressing 5XFAD transgenic mice. PGE exerts antioxidant effects in the brain, enhances microglial phagocytosis of Aß, and inhibits neuroinflammation and Aß deposition, ultimately preventing neuronal cell death in AD. Taken together, our findings indicate that the therapeutic potential of PGE in AD is mediated by its targeting of multiple pathological processes.

[18F]Flotaza for Aß 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 Aß 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 Aß 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 Aß 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-Aß 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-Aß. 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 Aß plaques in 5xFAD mice with SUVR~1.4. [18F]Flotaza is a new Aß plaque PET imaging agent that exhibited high binding to Aß 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.

A clathrin mediated endocytosis scaffolding protein, Intersectin 1, changes in an isoform, brain region, and sex specific manner in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia and is characterized by the accumulation of amyloid-beta (Aß) plaques and neurofibrillary Tau tangles in the brain. We previously identified a set of candidate AD microRNAs (miRNAs) in human cerebrospinal fluid (CSF) and used a target prediction pipeline to identify mRNAs and pathways that could potentially be regulated by the miRNAs. Of these pathways, clathrin mediated endocytosis (CME) was selected for further investigation. CME is altered in multiple brain cell types in AD and is implicated in early cellular phenotypes such as enlarged early endosomes and pathogenic processing of Aß. However, a comprehensive evaluation of major CME hub proteins in humans with AD across multiple brain regions is lacking. Thus, we used immunoblots to evaluate human post-mortem AD and control (CTL) frontal cortex (FC; AD n = 22, CTL n = 23) and hippocampus (HP; AD n = 34, CTL n = 22) for changes in Intersectin 1 (ITSN1), Phosphatidylinositol Binding Clathrin Assembly Protein gene (PICALM), Clathrin Light Chain (CLT), FCH and Mu Domain Containing Endocytic Adaptor 1 (FCHO1), Adaptor Related Protein Complex 2 (AP2) Subunit Alpha 1 (AP2A1), and Dynamin 2 (DNM2). Of these, we found that in AD, ITSN1-long (ITSN1-L) was decreased in the FC of males and HP of females, while ITSN1-short was increased in the HP of both males and females. We further evaluated ITSN1-L levels in cortex (CTX) and HP of the 5xFAD mouse model of Aß pathology at different timepoints during aging and disease progression by immunoblot (n = 5-8 per group). At 3 months, female 5xFAD exhibited an increase of ITSN1-L in CTX but a decrease at 6 and 9 months. Additionally, immunofluorescent staining of 5xFAD primary HP neurons showed an increase of ITSN1-L in matured 5xFAD neurons at 21 and 28 days in vitro. Together, our studies show that in AD, isoforms of ITSN1 change in a brain region-and sex-dependent manner. Further, changes in ITSN1-L are transient with levels increasing during early Aß accumulation and decreasing during later progression. These findings suggest that ITSN1 expression, and consequently CME activity, may change depending on the stage of disease progression.

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.

Apolipoprotein E4 interferes with lipid metabolism to exacerbate depression-like behaviors in 5xFAD mice.

BACKGROUND: Apolipoprotein E4 (ApoE4) allele is the strongest genetic risk factor for late-onset Alzheimer's disease, and it can aggravate depressive symptoms in non-AD patients. However, the impact of ApoE4 on AD-associated depression-like behaviors and its underlying pathogenic mechanisms remain unclear. METHODS: This study developed a 5xFAD mouse model overexpressing human ApoE4 (E4FAD). Behavioral assessments and synaptic function tests were conducted to explore the effects of ApoE4 on cognition and depression in 5xFAD mice. Changes in peripheral and central lipid metabolism, as well as the levels of serotonin (5-HT) and γ-aminobutyric acid (GABA) neurotransmitters in the prefrontal cortex, were examined. In addition, the protein levels of 24-dehydrocholesterol reductase/glycogen synthase kinase-3 beta/mammalian target of rapamycin (DHCR24/GSK3ß/mTOR) and postsynaptic density protein 95/calmodulin-dependent protein kinase II/brain-derived neurotrophic factor (PSD95/CaMK-II/BDNF) were measured to investigate the molecular mechanism underlying the effects of ApoE4 on AD mice. RESULTS: Compared with 5xFAD mice, E4FAD mice exhibited more severe depression-like behaviors and cognitive impairments. These mice also exhibited increased amyloid-beta deposition in the hippocampus, increased astrocyte numbers, and decreased expression of depression-related neurotransmitters 5-HT and GABA in the prefrontal cortex. Furthermore, lipid metabolism disorders were observed in E4FAD, manifesting as elevated low-density lipoprotein cholesterol and reduced high-density lipoprotein cholesterol in peripheral blood, decreased cholesterol level in the prefrontal cortex, and reduced expression of key enzymes and proteins related to cholesterol synthesis and homeostasis. Abnormal expression of proteins related to the DHCR24/GSK3ß/mTOR and PSD95/CaMK-II/BDNF pathways was also observed. CONCLUSION: This study found that ApoE4 overexpression exacerbates depression-like behaviors in 5xFAD mice and confirmed that ApoE4 reduces cognitive function in these mice. The mechanism may involve the induction of central and peripheral lipid metabolism disorders. Therefore, modulating ApoE expression or function to restore cellular lipid homeostasis may be a promising therapeutic target for AD comorbid with depression. This study also provided a better animal model for studying AD comorbid with depression.

Methionine Sulfoxide Speciation in Mouse Hippocampus Revealed by Global Proteomics Exhibits Age- and Alzheimer's Disease-Dependent Changes Targeted to Mitochondrial and Glycolytic Pathways.

Methionine oxidation to the sulfoxide form (MSox) is a poorly understood post-translational modification of proteins associated with non-specific chemical oxidation from reactive oxygen species (ROS), whose chemistries are linked to various disease pathologies, including neurodegeneration. Emerging evidence shows MSox site occupancy is, in some cases, under enzymatic regulatory control, mediating cellular signaling, including phosphorylation and/or calcium signaling, and raising questions as to the speciation and functional nature of MSox across the proteome. The 5XFAD lineage of the C57BL/6 mouse has well-defined Alzheimer's and aging states. Using this model, we analyzed age-, sex-, and disease-dependent MSox speciation in the mouse hippocampus. In addition, we explored the chemical stability and statistical variance of oxidized peptide signals to understand the needed power for MSox-based proteome studies. Our results identify mitochondrial and glycolytic pathway targets with increases in MSox with age as well as neuroinflammatory targets accumulating MSox with AD in proteome studies of the mouse hippocampus. Further, this paper establishes a foundation for reproducible and rigorous experimental MSox-omics appropriate for novel target identification in biological discovery and for biomarker analysis in ROS and other oxidation-linked diseases.

APOE Christchurch enhances a disease-associated microglial response to plaque but suppresses response to tau pathology.

Background: Apolipoprotein E ε4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD). A recent case report identified a rare variant in APOE, APOE3-R136S (Christchurch), proposed to confer resistance to autosomal dominant Alzheimer's Disease (AD). However, it remains unclear whether and how this variant exerts its protective effects. Methods: We introduced the R136S variant into mouse Apoe (ApoeCh) and investigated its effect on the development of AD-related pathology using the 5xFAD model of amyloidosis and the PS19 model of tauopathy. We used immunohistochemical and biochemical analysis along with single-cell spatial transcriptomics and proteomics to explore the impact of the ApoeCh variant on AD pathological development and the brain's response to plaques and tau. Results: In 5xFAD mice, ApoeCh enhances a Disease-Associated Microglia (DAM) phenotype in microglia surrounding plaques, and reduces plaque load, dystrophic neurites, and plasma neurofilament light chain. By contrast, in PS19 mice, ApoeCh suppresses the microglial and astrocytic responses to tau-laden neurons and does not reduce tau accumulation or phosphorylation, but partially rescues tau-induced synaptic and myelin loss. We compared how microglia responses differ between the two mouse models to elucidate the distinct DAM signatures induced by ApoeCh. We identified upregulation of antigen presentation-related genes in the DAM response in a PS19 compared to a 5xFAD background, suggesting a differential response to amyloid versus tau pathology that is modulated by the presence of ApoeCh. Conclusions: These findings highlight the ability of the ApoeCh variant to modulate microglial responses based on the type of pathology, enhancing DAM reactivity in amyloid models and dampening neuroinflammation to promote protection in tau models. This suggests that the Christchurch variant's protective effects likely involve multiple mechanisms, including changes in receptor binding and microglial programming.

Amyloid-ß neuropathology induces bone loss in male mice by suppressing bone formation and enhancing bone resorption.

Alzheimer's disease (AD) and osteoporosis often coexist in the elderly. Although observational studies suggest an association between these two diseases, the pathophysiologic link between AD and skeletal health has been poorly defined. We examined the skeletal phenotype of 5xFAD mice, an AD model with accelerated neuron-specific amyloid-ß accumulation causing full-blown AD phenotype by the age of 8 months. Micro-computed tomography indicated significantly lower trabecular and cortical bone parameters in 8-month-old male, but not female, 5xFAD mice than sex-matched wild-type littermates. Dynamic histomorphometry revealed reduced bone formation and increased bone resorption, and quantitative RT-PCR showed elevated skeletal RANKL gene expression in 5xFAD males. These mice also had diminished body fat percentage with unaltered lean mass, as determined by dual-energy X-ray absorptiometry (DXA), and elevated Ucp1 mRNA levels in brown adipose tissue, consistent with increased sympathetic tone, which may contribute to the osteopenia observed in 5xFAD males. Nevertheless, no significant changes could be detected between male 5xFAD and wild-type littermates regarding the serum and skeletal concentrations of norepinephrine. Thus, brain-specific amyloid-ß pathology is associated with osteopenia and appears to affect both bone formation and bone resorption. Our findings shed new light on the pathophysiologic link between Alzheimer's disease and osteoporosis.

Intranasal insulin treatment ameliorates spatial memory, muscular strength, and frailty deficits in 5xFAD mice.

The 5xFAD mouse model shows age-related weight loss as well as cognitive and motor deficits. Metabolic dysregulation, especially impaired insulin signaling, is also present in AD. This study examined whether intranasal delivery of insulin (INI) at low (0.875 U) or high (1.750 U) doses would ameliorate these deficits compared to saline in 10-month-old female 5xFAD and B6SJL wildtype (WT) mice. INI increased forelimb grip strength in the wire hang test in 5xFAD mice in a dose-dependent manner but did not improve the performance of 5xFAD mice on the balance beam. High INI doses reduced frailty scores in 5xFAD mice and improved spatial memory in both acquisition and reversal probe trials in the Morris water maze. INI increased swim speed in 5xFAD mice but had no effect on object recognition memory or working memory in the spontaneous alternation task, nor did it improve memory in the contextual or cued fear memory tasks. High doses of insulin increased the liver, spleen, and kidney weights and reduced brown adipose tissue weights. P-Akt signaling in the hippocampus was increased by insulin in a dose-dependent manner. Altogether, INI increased strength, reduced frailty scores, and improved visual spatial memory. Hypoglycemia was not present after INI, however alterations in tissue and organ weights were present. These results are novel and important as they indicate that intra-nasal insulin can reverse cognitive, motor and frailty deficits found in this mouse model of AD.

Metabolomics profiling reveals distinct, sex-specific signatures in serum and brain metabolomes in mouse models of Alzheimer's disease.

INTRODUCTION: Increasing evidence suggests that metabolic impairments contribute to early Alzheimer's disease (AD) mechanisms and subsequent dementia. Signals in metabolic pathways conserved across species can facilitate translation. METHODS: We investigated differences in serum and brain metabolites between the early-onset 5XFAD and late-onset LOAD1 (APOE4.Trem2*R47H) mouse models of AD to C57BL/6J controls at 6 months of age. RESULTS: We identified sex differences for several classes of metabolites, such as glycerophospholipids, sphingolipids, and amino acids. Metabolic signatures were notably different between brain and serum in both mouse models. The 5XFAD mice exhibited stronger differences in brain metabolites, whereas LOAD1 mice showed more pronounced differences in serum. DISCUSSION: Several of our findings were consistent with results in humans, showing glycerophospholipids reduction in serum of apolipoprotein E (apoE) ε4 carriers and replicating the serum metabolic imprint of the APOE ε4 genotype. Our work thus represents a significant step toward translating metabolic dysregulation from model organisms to human AD. HIGHLIGHTS: This was a metabolomic assessment of two mouse models relevant to Alzheimer's disease. Mouse models exhibit broad sex-specific metabolic differences, similar to human study cohorts. The early-onset 5XFAD mouse model primarily alters brain metabolites while the late-onset LOAD1 model primarily changes serum metabolites. Apolipoprotein E (apoE) ε4 mice recapitulate glycerophospolipid signatures of human APOE ε4 carriers in both brain and serum.