Human Amyloid Precursor Protein Research Articles

Associations of amyloid-β oligomers and plaques with neuropathology in the AppNL-G-F mouse

Abstract Amyloid-β pathology and neurofibrillary tangles lead to glial activation and neurodegeneration in Alzheimer’s disease. Here we have investigated relationships between the levels of amyloid-β oligomers, amyloid-β plaques, glial activation and markers related to neurodegeneration in the AppNL-G-F triple mutation mouse line and in a knock-in line homozygous for the common human amyloid precursor protein (Apphu mouse). Relationships between neuropathological features were characterized with immunohistochemistry and imaging mass cytometry. Markers assessing human amyloid-β proteins, microglial and astrocytic activation, and neuronal and synaptic densities were used in mice between 2.5 and 12 months of age. We found that amyloid-β oligomers were abundant in the brains of Apphu mice in the absence of classical amyloid-β plaques. These brains showed morphological changes consistent with astrocyte activation but no evidence of microglial activation or synaptic or neuronal pathology. By contrast, both high levels of amyloid-β oligomers and numerous plaques accumulated in AppNL-G-F mice in association with substantial astrocytic and microglial activation. The increase in amyloid-β oligomers over time were more strongly correlated with astrocytic than with microglia activation. Spatial analyses suggested that activated microglia were more closely associated with amyloid-β oligomers than with amyloid-β plaques in AppNL-G-F mice, which also showed age-dependent decreases in neuronal and synaptic density markers. Comparative study of the two models highlights the dependence of glial and neuronal pathology on the nature and aggregation state of the amyloid-β peptide. Astrocyte activation and neuronal pathology appeared to be more strongly associated with amyloid-β oligomers than amyloid-β plaques, although amyloid-β plaques were associated with microglia activation.

The Association of Circulating Glucagon-Like Peptide-1 with Cognitive Functions and Biomarkers in Alzheimer's Disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disease that is clinically characterized by progressive cognitive decline. Glucagon-like peptide-1 (GLP-1) is a hormone that belongs to the incretin family and is released in response to nutrient intake. It plays a role in maintaining metabolic homeostasis and has been suggested to be involved in maintaining the brain microenvironment. However, the role of GLP-1 in AD pathogenesis has not been fully illustrated. This study aims to investigate the clinical relevance of GLP-1 in AD and the effects of GLP-1 in amyloid-β (Aβ) metabolism in vitro. In this study, 39 AD patients and 120 cognitively intact controls were included. Plasma levels of GLP-1 were measured using ELISA. SH-SY5Y cells overexpressing human amyloid precursor protein (APP) were treated with GLP-1. Western blot analysis was used to assess the effects of GLP-1 on the metabolism of Aβ. Plasma GLP-1 levels were decreased with aging. Plasma GLP-1 levels were lower in AD patients in comparison with healthy older adults. Plasma GLP-1 levels were positively associated with Mini-Mental State Examination scores but negatively associated with plasma pTau181 levels. GLP-1 dose-dependently increased the area fraction of mitochondrial staining in vitro. Furthermore, GLP-1 dose-dependently promoted the α-cleavage of APP, thus reducing the generation of Aβ. GLP-1 has neuroprotective effects in AD, and therefore the decrease in GLP-1 levels during aging might contribute to the development of AD.

Box-Behnken Design-Based Optimization of Phytochemical Extraction from Diplazium esculentum (Retz.) Sw. Associated with Its Antioxidant and Anti-Alzheimer's Properties.

A previous study reported that the ethanolic extract of the edible fern, Diplazium esculentum (Retz.) Sw. (DE), obtained from a non-optimized extraction condition exhibited anti-Alzheimer's disease (AD) properties through the inhibition of a rate-limiting enzyme in amyloid peptide formation, β-secretase-1 (BACE-1). Nevertheless, a non-optimized or suboptimal extraction may lead to several issues, such as a reduction in extraction efficiency and increased time and plant materials. In this study, extraction of the DE was optimized to obtain appropriate BACE-1 inhibition using a Box-Behnken design (BBD) and response surface methodology (RSM). Data revealed that the optimal extraction condition was 70% (v/v) aqueous ethanol, 50 min extraction time, 30 °C extraction temperature, and 1:30 g/mL solid/liquid ratio, giving BACE-1 inhibition at 56.33%. In addition, the extract also exhibited significant antioxidant activities compared to the non-optimized extraction. Metabolomic phytochemical profiles and targeted phytochemical analyses showed that kaempferol, quercetin, and their derivatives as well as rosmarinic acid were abundant in the extract. The optimized DE extract also acted synergistically with donepezil, an AD drug suppressing BACE-1 activities. Data received from Drosophila-expressing human amyloid precursor proteins (APPs) and BACE-1, representing the amyloid hypothesis, showed that the optimized DE extract penetrated the fly brains, suppressed BACE-1 activities, and improved locomotor functions. The extract quenched the expression of glutathione S transferase D1 (GSTD1), inositol-requiring enzyme (IRE-1), and molecular chaperone-binding immunoglobulin (Bip), while donepezil suppressed these genes and other genes involved in antioxidant and endoplasmic reticulum (ER) stress response, including superoxide dismutase type 1 (SOD1), activating transcription factor 6 (ATF-6), and protein kinase R-like endoplasmic reticulum kinase (PERK). To sum up, the optimized extraction condition reduced extraction time while resulting in higher phytochemicals, antioxidants, and BACE-1 inhibitors.

Persistent ∆FosB expression limits recurrent seizure activity and provides neuroprotection in the dentate gyrus of APP mice

Recurrent seizures lead to accumulation of the activity-dependent transcription factor ∆FosB in hippocampal dentate granule cells in both mouse models of epilepsy and mouse models of Alzheimer’s disease (AD), which is also associated with increased incidence of seizures. In patients with AD and related mouse models, the degree of ∆FosB accumulation corresponds with increasing severity of cognitive deficits. We previously found that ∆FosB impairs spatial memory in mice by epigenetically regulating expression of target genes such as calbindin that are involved in synaptic plasticity. However, the suppression of calbindin in conditions of neuronal hyperexcitability has been demonstrated to provide neuroprotection to dentate granule cells, indicating that ∆FosB may act over long timescales to coordinate neuroprotective pathways. To test this hypothesis, we used viral-mediated expression of ∆JunD to interfere with ∆FosB signaling over the course of several months in transgenic mice expressing mutant human amyloid precursor protein (APP), which exhibit spontaneous seizures and develop AD-related neuropathology and cognitive deficits. Our results demonstrate that persistent ∆FosB activity acts through discrete modes of hippocampal target gene regulation to modulate neuronal excitability, limit recurrent seizure activity, and provide neuroprotection to hippocampal dentate granule cells in APP mice.

Prominent tauopathy and intracellular β-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis

BackgroundCathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aβ pathology and tauopathy in vivo.MethodsCatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau.ResultsDeletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aβ, manifesting as intense, exclusively intracellular aggregates; extracellular Aβ deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aβ42.ConclusionsOur findings support a major role for CatD in the proteostasis of both Aβ and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aβ accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aβ42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.

Conclusive demonstration of iatrogenic Alzheimer’s disease transmission in a model of stem cell transplantation

The risk of iatrogenic disease is often underestimated as a concern in contemporary medical procedures, encompassing tissue and organ transplantation, stem cell therapies, blood transfusions, and the administration of blood-derived products. In this context, despite the prevailing belief that Alzheimer's disease (AD) manifests primarily in familial and sporadic forms, our investigation reveals an unexpected transplantable variant of AD in a preclinical context, potentially indicating iatrogenic transmission in AD patients. Through adoptive transplantation of donor bone marrow stem cells carrying a mutant human amyloid precursor protein (APP) transgene into either APP-deficient knockout or normal recipient animals, we observed rapid development of AD pathological hallmarks. These pathological features were significantly accelerated and emerged within 6-9months post transplantation and included compromised blood-brain barrier integrity, heightened cerebral vascular neoangiogenesis, elevated brain-associated β-amyloid levels, and cognitive impairment. Furthermore, our findings underscore the contribution of β-amyloid burden originating outside of the central nervous system to AD pathogenesis within the brain. We conclude that stem cell transplantation from donors harboring a pathogenic mutant allele can effectively transfer central nervous system diseases to healthy recipients, mirroring the pathogenesis observed in the donor. Consequently, our observations advocate for genomic sequencing of donor specimens prior to tissue, organ, or stem cell transplantation therapies, as well as blood transfusions and blood-derived product administration, to mitigate the risk of iatrogenic diseases.

A novel mouse model for N-terminal truncated Aβ2-x generation through meprin β overexpression in astrocytes

Neurotoxic amyloid-β (Aβ) peptides cause neurodegeneration in Alzheimer’s disease (AD) patients’ brains. They are released upon proteolytic processing of the amyloid precursor protein (APP) extracellularly at the β-secretase site and intramembranously at the γ-secretase site. Several AD mouse models were developed to conduct respective research in vivo. Most of these classical models overexpress human APP with mutations driving AD-associated pathogenic APP processing. However, the resulting pattern of Aβ species in the mouse brains differs from those observed in AD patients’ brains. Particularly mutations proximal to the β-secretase cleavage site (e.g., the so-called Swedish APP (APPswe) fostering Aβ1-x formation) lead to artificial Aβ production, as N-terminally truncated Aβ peptides are hardly present in these mouse brains. Meprin β is an alternative β-secretase upregulated in brains of AD patients and capable of generating N-terminally truncated Aβ2-x peptides. Therefore, we aimed to generate a mouse model for the production of so far underestimated Aβ2-x peptides by conditionally overexpressing meprin β in astrocytes. We chose astrocytes as meprin β was detected in this cell type in close proximity to Aβ plaques in AD patients’ brains. The meprin β-overexpressing mice showed elevated amyloidogenic APP processing detected with a newly generated neo-epitope-specific antibody. Furthermore, we observed elevated Aβ production from endogenous APP as well as AD-related behavior changes (hyperlocomotion and deficits in spatial memory). The novel mouse model as well as the established tools and methods will be helpful to further characterize APP cleavage and the impact of different Aβ species in future studies.

Altered amyloid-β structure markedly reduces gliosis in the brain of mice harboring the Uppsala APP deletion

Deposition of amyloid beta (Aβ) into plaques is a major hallmark of Alzheimer’s disease (AD). Different amyloid precursor protein (APP) mutations cause early-onset AD by altering the production or aggregation properties of Aβ. We recently identified the Uppsala APP mutation (APPUpp), which causes Aβ pathology by a triple mechanism: increased β-secretase and altered α-secretase APP cleavage, leading to increased formation of a unique Aβ conformer that rapidly aggregates and deposits in the brain. The aim of this study was to further explore the effects of APPUpp in a transgenic mouse model (tg-UppSwe), expressing human APP with the APPUpp mutation together with the APPSwe mutation. Aβ pathology was studied in tg-UppSwe brains at different ages, using ELISA and immunohistochemistry. In vivo PET imaging with three different PET radioligands was conducted in aged tg-UppSwe mice and two other mouse models; tg-ArcSwe and tg-Swe. Finally, glial responses to Aβ pathology were studied in cell culture models and mouse brain tissue, using ELISA and immunohistochemistry. Tg-UppSwe mice displayed increased β-secretase cleavage and suppressed α-secretase cleavage, resulting in AβUpp42 dominated diffuse plaque pathology appearing from the age of 5–6 months. The γ-secretase cleavage was not affected. Contrary to tg-ArcSwe and tg-Swe mice, tg-UppSwe mice were [11C]PiB-PET negative. Antibody-based PET with the 3D6 ligand visualized Aβ pathology in all models, whereas the Aβ protofibril selective mAb158 ligand did not give any signals in tg-UppSwe mice. Moreover, unlike the other two models, tg-UppSwe mice displayed a very faint glial response to the Aβ pathology. The tg-UppSwe mouse model thus recapitulates several pathological features of the Uppsala APP mutation carriers. The presumed unique structural features of AβUpp42 aggregates were found to affect their interaction with anti-Aβ antibodies and profoundly modify the Aβ-mediated glial response, which may be important aspects to consider for further development of AD therapies.

Abstract WP291: Cerebral Amyloid Angiopathy Induces Liver Inflammation in Aged Mice

Introduction: Cerebral amyloid angiopathy (CAA) is a chronic age-related neurodegenerative disease, identified by the presence of amyloid-beta (Aβ) deposition within the vasculature of the brain, leading to stroke and progressive cognitive decline. The liver is a key peripheral organ involved in the clearance of excess Aβ in the circulation. However, the role of the brain-liver axis in CAA is unknown. Here, we investigate inflammatory changes in the liver in a murine model of CAA. Methods: Aged (>23 months) male and female Tg-SwDI (CAA) mice harboring Swedish, Dutch, and Iowa mutations of the human amyloid precursor protein were used as a mouse model of CAA along with age-matched wild-type (WT) controls (n=3-7/grp). Hepatic inflammation was analyzed with histological and immunofluorescence imaging. Single cells from liver tissue were isolated and analyzed using flow cytometry. Total RNA was isolated to examine gene expression in the liver using real-time quantitative PCR. Results: Our data showed that CAA increases infiltration of immune cells proximal to liver sinusoids across both sexes compared to WT mice. We found increased macrophages (i.e., F4/80 + cells) in the liver of CAA mice compared to WT controls. Flow cytometry on the liver revealed that female CAA mice had a significant increase in dendritic cells (i.e., CD80 + CD45 + CD11c + cells, *** P <0.001) compared to WT females. Further, we found that Oatp2 , a membrane transporter which mediates hepatocytic uptake of toxic molecules (e.g., Aβ), is associated with sex-specific CAA pathophysiology in the liver. Conclusions: In this study, we found that sex differences may exist in CAA-induced liver inflammation. Further investigations are needed to study how vascular amyloidosis shapes the immune responses and peripheral Aβ clearance in the context of aging and CAA.

Abstract TP28: The Effects of Social Isolation on Cerebral Amyloid Angiopathy

Introduction: Cerebral amyloid angiopathy (CAA) is characterized by amyloid β (Aβ) deposition in the cortical and leptomeningeal vessels, and it is a largely untreatable cause of intracerebral hemorrhage and dementia. Studies estimate a 10-50% prevalence of CAA in the general elderly population, positioning it as one of the strongest vascular contributors to age-related cognitive impairment. With aging, the prevalence of social isolation (SI) also increases. Isolated individuals have an increased risk of Aβ-related cognitive decline and heightened inflammatory response to stress. Yet, whether and how SI exacerbates CAA-induced cognitive decline is unknown. Methods: TgSwDI mice harboring Swedish, Dutch, and Iowa mutations of human amyloid precursor protein were used as a mouse model of CAA. Male and female C57BL6 wildtype (WT) and CAA mice were randomly assigned to paired housing (PH) or SI at 3 months. Neurobehaviors were measured at baseline and every 3 months using the novel object recognition test (NORT), tail suspension test (TST), and fear conditioning (FC). At 12 months, microglia phenotype, vascular structure, and Aβ burden were assessed using flow cytometry and immunostaining. Results: Both WT and CAA isolated animals displayed significantly greater immobility in TST at 9 months post-SI, suggesting a depression-like behavior (n=10-15/grp, p<0.0001). Isolated CAA mice displayed exacerbated cognitive deficits in NORT and FC while WT mice showed intact cognition (n=10-15/grp, p<0.05). Specifically, SI-induced cognitive deficits were more prominent in the female CAA mice compared to males (n=5-8/grp, p<0.01). Sex differences were also observed in CAA mice in frequencies of Aβ-containing endothelial cells (CD31 + ) and vessel-associated microglia (VAM, CD45 int CD11b + CD31 + ) as well as in microglial phagocytosis of Aβ (n=3/grp, p<0.05). SI increased the frequency of Aβ-containing microglia in isolated CAA mice compared to their PH littermates. Conclusion: SI induced behavioral deficits in both WT and CAA mice but exacerbated cognitive decline only in the CAA SI mice. SI also influences microglial phagocytosis of Aβ. Further research is needed to determine the cellular and molecular mechanism underlying SI-induced changes in CAA.

Abstract 20: Axonal Neuropathy and Inflammatory Response in Peripheral Nerves of a Murine Cerebral Amyloid Angiopathy Model

Introduction: Cerebral amyloid angiopathy (CAA) induces intracerebral hemorrhages, small vessel disease, and cognitive decline in elderly individuals. CAA is also associated with slower walking speed and abnormal gait rhythms compared to healthy controls. Little is known regarding the impact of peripheral nervous system abnormalities on CAA-associated gait disturbance. Furthermore, it is unknown whether peripheral nerves pose extracerebral targets of amyloid-related endoneurial inflammation. Hypothesis: Gait disturbance in a CAA model is associated with peripheral neuropathy through endoneurial inflammation related to amyloid accumulation. Methods: Aged (19-24 months) Tg-SwDI mice (CAA mice) of both sexes harboring mutations of human amyloid precursor protein (APP) were used as a CAA model. Age-matched C57BL/6 wild-type (WT) mice were used as controls (n=6-11/grp). Gait disturbance and sensorimotor function were assessed via DigiGait and grip strength testing. Flow cytometry was used to analyze immune cells in the sciatic nerve. Tibial nerve morphometry was performed to assess for neuropathy. Results: CAA mice had weaker grip strength ( CAA: 97.7 ± 17.6 g, WT 111.5 ± 8.1 g, p=0.039 ) and evidence of neuropathic gait disturbances, reflected by increased paw angles ( CAA: 21.1 ± 4.8°, WT 11.7 ± 3.4°, p=0.0002 ) and reduced paw areas ( CAA: 0.4 ± 0.05 cm 2 , WT 0.5 ± 0.06 cm 2 , p=0.013 ) impacting the ground when walking compared to WT mice. Tibial nerve morphometry showed a reduced number of myelinated nerve fibers ( CAA: 1023 ± 230.1, WT 1318 ± 45.8, p=0.0047 ) suggesting potential axonal degeneration in CAA mice. Flow cytometry showed an increase in the count of macrophage (both M1 and M2 subtypes) lineage cells ( M1 CAA: 102.3 ± 29.2, WT 57.9 ± 36.3, p=0.009 ) as well as M1 macrophage associated pro-inflammatory cytokine TNF-α ( CAA: 99.7 ± 28.3, WT 53.8 ± 12.2, p=0.009 ) in sciatic nerves of CAA mice compared to WT mice. Conclusions: CAA mice display an enhanced inflammatory response within the endoneurium which may contribute to observed axonal degeneration, ultimately impairing sensorimotor function and gait. Further studies are needed to unravel the mechanistic interplay between amyloid pathology, endoneurial inflammation, and axonal neuropathy in this CAA model.

Temporal Alterations in White Matter in An App Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia and results in neurodegeneration and cognitive impairment. White matter (WM) is affected in AD and has implications for neural circuitry and cognitive function. The trajectory of these changes across age, however, is still not well understood, especially at earlier stages in life. To address this, we used the AppNL-G-F/NL-G-F knock-in (APPKI) mouse model that harbors a single copy knock-in of the human amyloid precursor protein (APP) gene with three familial AD mutations. We performed in vivo diffusion tensor imaging (DTI) to study how the structural properties of the brain change across age in the context of AD. In late age APPKI mice, we observed reduced fractional anisotropy (FA), a proxy of WM integrity, in multiple brain regions, including the hippocampus, anterior commissure (AC), neocortex, and hypothalamus. At the cellular level, we observed greater numbers of oligodendrocytes in middle age (prior to observations in DTI) in both the AC, a major interhemispheric WM tract, and the hippocampus, which is involved in memory and heavily affected in AD, prior to observations in DTI. Proteomics analysis of the hippocampus also revealed altered expression of oligodendrocyte-related proteins with age and in APPKI mice. Together, these results help to improve our understanding of the development of AD pathology with age, and imply that middle age may be an important temporal window for potential therapeutic intervention.

Silica Nanoparticle Exposure Implicates β-Amyloid (1-42) Inbound and the Accelerating Alzheimer's Disease Progression in Mice Overexpressing Mutated Forms of Human Amyloid Precursor Protein and Presenilin 1 Genes.

The increasing nanoparticle (NP) applications in the biomedical field have become an emerging concern regarding human health. NP exposure may play a role in the accelerating Alzheimer's disease (AD) progression; however, the etiology of this disorder is complex and remains largely unclear. Here, we identified that intravenous injection of silica NPs (SiNPs) caused the blood-brain barrier breakdown via downregulating tight junction-related gene expressions. Meanwhile, SiNPs upregulate the transport receptor for advanced glycation end products (RAGE) that govern the β-amyloid (Aβ) influx to the brain; however, low-density lipoprotein receptor-related protein 1 (LRP1) that controls the efflux of Aβ from the brain was not affected. Consequently, an increase in Aβ burden in the brain of SiNP-challenged APP/PS1 mice was found. Intriguingly, plasma apolipoprotein E (ApoE) adsorbed on the surface of SiNPs partially relieves this effect. Using ApoE knockout (ApoE-/-) mice, we confirmed that SiNPs covered with serum without ApoE showed further elevated AD symptoms. Together, this study offered a compilation of data to support the potential risk factors of NP exposure and AD pathology.

Translocator protein (TSPO) ligands attenuate mitophagy deficits in the SH-SY5Y cellular model of Alzheimer's disease via the autophagy adaptor P62

Mitochondrial dysfunction has been widely implicated in the pathogenesis of Alzheimer's disease (AD), with accumulation of damaged and dysfunctional mitochondria occurring early in the disease. Mitophagy, which governs mitochondrial turnover and quality control, is impaired in the AD brain, and strategies aimed at enhancing mitophagy have been identified as promising therapeutic targets. The translocator protein (TSPO) is an outer mitochondrial membrane protein that is upregulated in AD, and ligands targeting TSPO have been shown to exert neuroprotective effects in mouse models of AD. However, whether TSPO ligands modulate mitophagy in AD has not been explored. Here, we provide evidence that the TSPO-specific ligands Ro5-4864 and XBD173 attenuate mitophagy deficits and mitochondrial fragmentation in a cellular model of AD overexpressing the human amyloid precursor protein (APP). Ro5-4864 and XBD173 appear to enhance mitophagy via modulation of the autophagic cargo receptor P62/SQSTM1, in the absence of an effect on PARK2, PINK1, or LC3 level. Taken together, these findings indicate that TSPO ligands may be promising therapeutic agents for ameliorating mitophagy deficits in AD.

Flavonoids and fibrate modulate apoE4-induced processing of amyloid precursor protein in neuroblastoma cells.

Apolipoprotein (apo) E4, being a major genetic risk factor for Alzheimer's disease (AD), is actively involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid β (Aβ) peptide, the principle constituent of amyloid plaques in Alzheimer Disease (AD) patients. ApoE4 is believed to affect APP processing through intracellular cholesterol homeostasis, whereas lowering the cholesterol level by pharmacological agents has been suggested to reduce Aβ production. This study has investigated the effects of hypolipidemic agents fenofibrate, and the flavonoids-naringenin and diosmetin-on apoE4-induced APP processing in rat neuroblastoma cells stably transfected with human wild-type APP 695 (B103-hAPP695wt). B103-hAPP695wt cells were pretreated with different doses of flavonoids and fenofibrate for 1 h prior to apoE4 exposure for 24 h. ApoE4-induced production of intra- and extracellular Aβ peptides has been reduced with fenofibrate, naringenin, and diosmetin treatments. Pretreatment with diosmetin has significantly reduced apoE4-induced full-length APP (fl- APP) expression, whereas naringenin and fenofibrate had no effect on it. In addition, the increase in the apoE4-induced secretion of sAPPtotal and sAPPα has been dose-dependently reduced with drug pretreatment. On the other hand, the decrease in the expression of both APP-carboxy terminal fragments (CTF)-α and -β (generated by the α- or β-secretase cleavage of APP) by apoE4 was dose-dependently increased in cells pretreated with fenofibrate and naringenin but not diosmetin. Thus, we suggest that fenofibrate, naringenin, and diosmetin treatments can reduce apoE4- induced Aβ production by distinct mechanisms that may prove useful in developing drugs for AD patients.

Exercise combined with postbiotics treatment results in synergistic improvement of mitochondrial function in the brain of male transgenic mice for Alzheimer’s disease

BackgroundIt has been suggested that exercise training and postbiotic supplement could decelerate the progress of functional and biochemical deterioration in double transgenic mice overexpresses mutated forms of the genes for human amyloid precursor protein (APPsw) and presenilin 1 (m146L) (APP/PS1TG). Our earlier published data indicated that the mice performed better than controls on the Morris Maze Test parallel with decreased occurrence of amyloid-β plaques in the hippocampus. We investigated the neuroprotective and therapeutic effects of high-intensity training and postbiotic supplementation.MethodsThirty-two adult APP/PS1TG mice were randomly divided into four groups: (1) control, (2) high-intensity training (3) postbiotic, (4) combined (training and postbiotic) treatment for 20 weeks. In this study, the whole hemibrain without hippocampus was used to find molecular traits explaining improved brain function. We applied qualitative RT-PCR for gene expression, Western blot for protein level, and Zymography for LONP1 activity. Disaggregation analysis of Aβ-40 was performed in the presence of Lactobacillus acidophilus and Bifidobacterium longum lysate.ResultsWe found that exercise training decreased Alzheimer’s Disease (AD)-related gene expression (NF-kB) that was not affected by postbiotic treatment. The preparation used for postbiotic treatment is composed of tyndallized Bifidobacterium longum and Lactobacillus acidophilus. Both of the postbiotics effectively disaggregated amyloid-β/Aβ-40 aggregates by chelating Zn2+ and Cu2+ ions. The postbiotic treatment decreased endogenous human APPTG protein expression and mouse APP gene expression in the hemibrains. In addition, the postbiotic treatment elevated mitochondrial LONP1 activity as well.ConclusionOur findings revealed distinct mechanisms behind improved memory performance in the whole brain: while exercise training modulates NF-kB signaling pathway regulating immune response until postbiotic diminishes APP gene expression, disaggregates pre-existing amyloid-β plaques and activates mitochondrial protein quality control in the region of brain out of hippocampus. Using the above treatments complements and efficiently slows down the development of AD.

Amyloid precursor protein facilitates SARS-CoV-2 virus entry into cells and enhances amyloid-β-associated pathology in APP/PS1 mouse model of Alzheimer’s disease

Although there are indications of a trend towards less severe acute respiratory symptoms and a decline in overall lethality from the novel Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), more and more attention has been paid to the long COVID, including the increased risk of Alzheimer’s disease (AD) in COVID-19 patients. In this study, we aim to investigate the involvement of N-terminal amyloid precursor protein (APP) in SARS-CoV-2-induced amyloid-β (Aβ) pathology. Utilizing both in vitro and in vivo methodologies, we first investigated the interaction between the spike protein of SARS-CoV-2 and N-terminal APP via LSPR and CoIP assays. The in vitro impacts of APP overexpression on virus infection were further evaluated in HEK293T/ACE2 cells, SH-SY5Y cells, and Vero cells. We also analyzed the pseudovirus infection in vivo in a mouse model overexpressing human wild-type APP. Finally, we evaluated the impact of APP on pseudovirus infection within human brain organoids and assessed the chronic effects of pseudovirus infection on Aβ levels. We reported here for the first time that APP, the precursor of the Aβ of AD, interacts with the Spike protein of SARS-CoV-2. Moreover, both in vivo and in vitro data further indicated that APP promotes the cellular entry of the virus, and exacerbates Aβ-associated pathology in the APP/PS1 mouse model of AD, which can be ameliorated by N-terminal APP blockage. Our findings provide experimental evidence to interpret APP-related mechanisms underlying AD-like neuropathology in COVID-19 patients and may pave the way to help inform risk management and therapeutic strategies against diseases accordingly.

Distinct immune signatures associated with sex and genotype in the 12m novel hAPP hAPOE mouse risk model of Alzheimer’s disease

AbstractBackgroundUnderstanding the biology of model systems is critically important in the identification of biomarkers to delay or prevent Alzheimer’s disease. Previous findings in our group demonstrate sex differences in immune transcripts in the AD brain and in the humanized apolipoprotein E (APOE) mouse risk model. With this study, we extend these discoveries to further investigate the effect of APOE genotype using a novel risk mouse model with humanized amyloid precursor protein (APP) and humanized‐APOE transgenes (Jackson Laboratory). We test the hypothesis that during midlife aging, hAPP hAPOE ε4/4 genotype, and female sex will show increased risk profiles for Aβ 42:40 ratio and immunophenotypes associated with Alzheimer’s disease.MethodTo identify midlife (12 months; human correlate 44 years) immunophenotypes that may contribute to AD 3 different risk genotypes were studied: hAPP (6 male /6 female), hAPP hAPOE ε3/3 (16 male/16 female), and hAPP hAPOE ε4/4 (16 male /16 female). The left hemisphere of the brain was collected and prepared for multi‐color flow cytometry. Microglial reactivity (MHCII, CD68), phagocytosis (pHrodo), oxidative stress (CellRox), and the presence of lymphocytes (CD3, CD4, CD8, CD19) were determined. Peripheral blood was collected and assessed for monocytes and lymphocytes while plasma was isolated and ELISA was used to detect the presence of Aβ 40‐42, and levels of pro‐inflammatory cytokines. Female animals were monitored daily by lavage to determine midlife endocrine status.ResulthAPP hAPOE ε4/4 animals demonstrated accelerated endocrine aging compared to hAPP hAPOE ε3/3 animals. Plasma beta‐amyloid 42:40 ratio, and levels of IL‐1B, and IL‐6 showed a significant interaction between sex and hAPP hAPOE genotype (p<0.05, p<0.05, p<0.05). Plasma levels of IL‐5 and CXCL1 trended toward genotype significance with p = 0.0544 and p = 0.0556, respectively. Percentage of MHCII+ activated microglia was significantly impacted by sex (p = 0.0005) and by genotype (p<0.0001).ConclusionThe interaction between hAPP and hAPOE genotype, in coordination with biological sex significantly impacts immunophenotypes of the CNS and periphery during midlife aging. Precision interventions targeting sex and genotype‐dependent prodromal immune biomarkers may serve as an opportunity to support homeostatic immune milieu or to modulate harmful inflammatory signaling and reduce risk of AD or delay AD onset.

Distinct immune signatures associated with sex and genotype in the 15m novel hAPP+hAPOE mouse risk model of Alzheimer’s disease

AbstractBackgroundAlzheimer’s disease research continues to struggle with the translatability of animal models to human patients. Exploring whole systems is integral to early AD biomarker discovery in the pursuit of therapeutics to delay or prevent AD. Previous finds in our lab indicate sex differences in immune transcription levels in human AD brain and in the humanized apolipoprotein E (APOE) mouse model. Herein we investigated the impact of the APOE genotype in a novel mouse model, humanized amyloid precursor protein (APP), and humanized APOE transgenes (Jackson Laboratory).MethodsTo identify immunophenotypes at late‐midlife (15 months: late 40s – early 50s human correlative) a novel hAPP+hAPOE mouse model was utilized: hAPP (5M:5F), hAPP+hAPOE ε3/3 (16M:16F), & hAPP+hAPOE ε4/4 (16M:16F). The brain was hemisected. The left side was processed for multi‐color flow cytometry analysis. Peripheral blood was taken via cardiac puncture. The plasma was isolated and ELISA was used to detect levels of Aβ 40‐42 and pro‐inflammatory cytokines. PBMCs were isolated from the peripheral blood for multi‐color flow cytometry analysis. Microglia (CD45, CD11b), microglia reactivity (MHCII, CD68), phagocytosis (pHrodo), and oxidative stress (CellRox) levels were determined in the brain. Lymphocytes (CD3, CD4, CD8, CD19), lymphocyte activity (CD69), and oxidative stress (CellRox) levels were determined in both the brain and peripheral blood. Midlife endocrine status was determined for the female mice by daily lavage.ResultshAPP+hAPOE ε4/4 animals exhibited accelerated endocrine aging evidence by increased proportion of irregular cycling mice, compared to hAPP hAPOE ε3/3 animals. Microglial activation marker by MHCII was significantly impacted by sex (p<0.0001) and less so by genotype (p = 0.0551). Microglial function measured by pHrodo was impacted by sex (p<0.0001) with hAPP+hAPOE ε3/3 males exhibiting greater phagocytosis in MHCII+ microglia relative to their female counterpart.ConclusionThe interaction between the genotypes APP and APOE in combination with sex has a significant impact on immune phenotypes in the late‐midlife brain. These indications offer unique insights for future targeted therapeutics. Our findings support further investigation into neuroimmune activation in mid‐life to delay or prevent AD.

Investigating the role of mitochondrial calcium uniporter beta in Alzheimer’s disease

AbstractBackgroundWe have recently shown that mitochondrial calcium (mCa2+) overload significantly contributes to the development of Alzheimer’s disease (AD). We found that either genetic inhibition of the neuronal mitochondrial calcium uniporter (MCU, the primary route for mCa2+ influx)1 or enhancement of mitochondrial Na+/Ca2+ exchanger (NCLX, the primary route for mCa2+ efflux)2 in the AD mouse model reduces AD‐associated phenotype and mitochondrial permeability transition pore (MPTP)‐induced cell death. However, blocking either the mCa2+ influx or enhancing mCa2+ efflux can also shut down the bioenergetics. Thus, it is vital to find a regulator of mCa2+ homeostasis that will positively impact Ca2+‐dependent physiological functions.MethodsTo define the new molecular regulatory mechanism to limit mCa2+ overload in AD that will maintain bioenergetics and prevent cell death, we have examined the role of Mitochondrial Calcium Uniporter Beta (MCUB), a paralog of MCU that acts as a negative regulator of mCa2+ uptake3. This study used AD patient samples, mice, and cell lines to examine MCUB expression. A neuroblastoma cell line stably expressing the human Swedish mutant amyloid precursor protein (N2a/APPswe) with adenovirus encoding MCUB was examined for mitochondrial alterations in: Ca2+ handling, ROS generation, permeability transition pore activation, oxidative phosphorylation (OxPHOS) and cell death.Results5xFAD mice and human AD brain samples displayed significant reductions in the expression of MCUB. Studies performed in N2a/APPswe neurons discovered that mCa2 handling, redox, and bioenergetics was severely impaired in AD cells. Rescue of MCUB via adenoviral expression enhanced the clearance of pathogenic mCa2+, enhanced OxPHOS, and prevented ROS and cell death.ConclusionsOur data suggest that MCUB modulation has the therapeutic potential to preserve bioenergetics and limit cell death in AD. This project will further utilize neuronal‐specific knockouts and overexpression of MCUB to increase or decrease mCa2+ uptake, respectively, in 5xFAD mouse models and will explore whether and how MCUB‐mediated changes in uptake are able to preserve metabolism, mCa2+ dynamics, and AD‐phenotype in vivo.