Amyloid Precursor Protein Processing Research Articles

Exercise training and BDNF injections alter APP processing enzymes and improve cognition.

Exercise reduces cognitive aging, neurodegeneration, and Alzheimer's disease (AD) risk. Acute exercise reduces the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), the rate limiting enzyme in the production of Aβ. However, mechanisms mediating these effects remain largely unknown. Work has implicated brain-derived neurotrophic factor (BDNF) in the processing of amyloid precursor protein (APP). BDNF is an exercise-induced neurotrophin known for its role in synaptic plasticity, neurite growth, and neuronal survival. Previously, our lab has shown using an ex vivo model that treatment of the prefrontal cortex with BDNF reduced BACE1 activity, highlighting a BDNF to BACE1 link. The purpose of this research was to examine whether BDNF treatments resulted in similar biochemical adaptations to APP processing as exercise training. Male C57BL6/J mice were assigned into one of four groups (n=12/group): 1) control; 2) exercise training (progressive treadmill training 5 days/week); 3) BDNF (0.5 mg/kg body mass subcutaneous injection 5 days/week); or 4) endurance training and BDNF, for an 8-week intervention. Recognition memory was measured with a novel object recognition test. Serum, the prefrontal cortex, and hippocampus were collected. BDNF improved recognition memory to a similar extent as endurance training. BDNF and exercise decreased BACE1 activity and increased ADAM10 activity in the prefrontal cortex, indicating a shift in APP processing. Our novel results indicate that BDNF exerts similar beneficial effects on cognition and APP processing as exercise. Future evidence-based preventative or therapeutic interventions that increase BDNF and reduce BACE1 will be valuable for populations at risk of AD.

Hydroxy‐docosahexaenoic acid (DHA‐H) and its immediate metabolite Heneicosapentaenoic acid (HPA) exert neuroprotection and prevent amyloidogenic APP processing

AbstractBackgroundDHA‐H (Hydroxy‐docosahexaenoic acid) is a molecule in development for Alzheimer’s disease (AD) treatment based on the concept of membrane lipid therapy (melitherapy). Once entered the cell, DHA‐H is metabolized via α‐oxidation to the fatty acid HPA (Heneicosapentaenoic acid). DHA‐H has been shown to reduce the amyloidogenic processing of Amyloid Precursor Protein (APP), as well as having a neuroprotective effect in cellular models. Although the mechanism of action of DHA‐H is not yet fully understood, results obtained in excitotoxicity models and lipid profile analysis suggest that HPA could be a DHA‐H effector.Methods: APP processing was assessed in cell cultures of HEK293 and N2a neuroblastoma cell lines and analyzed by western blot. Neuroprotective effect of DHA‐H and HPA was tested in neuron cells differentiated from SH‐SY5Y neuroblastoma cells that were stimulated with NMDA (N‐Methyl‐D‐Aspartate)/Ca to induce excitotoxicity. Lipid samples for lipid profile analysis were obtained from the same cultured cells by addition of chloroform/methanol. Fatty acids were transformed into methyl esters and analyzed by GC‐FID (Gas Chromatography‐Flame ionization detector).Results: DHA‐H administration decreases the levels of derivatives from the APP amyloidogenic processing. DHA‐H and HPA showed a neuroprotective effect in a neuronal model of NMDA‐induced excitotoxicity. DHA‐H administration does not significantly alter the main fatty acids but clearly increases HPA levels in the cells.ConclusionAll these results together demonstrate that DHA‐H exerts neuroprotection and prevents amyloidogenic processing possibly through its metabolic intermediate HPA. Therefore, HPA appears as a new promising molecule for Alzheimer’s therapy.

A systematic review of therapeutic strategies against amyloid‐β peptides in Alzheimer’s disease: Past, present, and future

AbstractBackgroundLargely recognized too late, Alzheimer’s disease (AD) affects around 46 million people worldwide. The etiology of AD is still puzzling the scientific community, and its drug development and clinical trials have had high failure rate. In the recent decade, numerous studies targeting amyloid‐β peptides (Aβ) and their metabolism have been tested in patients, raising additional questions regarding AD.MethodIn order to integrate recent (from 2014) findings and analyze their clinical potential, we conducted a systematic review of investigational studies, follow‐up studies and review articles, that focus on anti‐Aβ therapies. The records were required to investigate the safety, tolerability, pharmacokinetics, efficacy, or immunogenicity of anti‐amyloid treatments in human population, excluding short and interrupted studies (<10 days), and clinical trials concerning other pathologies or comorbidities.ResultThus far, experimental therapies directed against Aβ have not been able to significantly reduce the progress of AD and memory impairment. The majority of therapeutic strategies were focused on amyloid precursor protein processing, and active or passive immunotherapy. Fewer studies evaluated drugs affecting Aβ misfolding, aggregation or clearance. Targeting β‐ and γ‐secretase have had unwanted results, but the activators of α‐secretase and immunotherapy against Aβ oligomers are giving some promise. Recent FDA approval and EU refusal of aducanumab sparked hopes and controversies, since the latest trial data confirmed amyloid plaque removal, without clear cognitive benefits.ConclusionTargeting only Aβ was not able to significantly improve the cognition of patients or modify the AD progression. The research is slowly moving toward multifunctional drugs targeting factors that precede the Aβ aggregation process, i.e., pathologic positive feedback loop linking Aβ production and other factors, such as neuronal hyperexcitability, neuroinflammation, or oxidative stress. Further development of early detection biomarkers, antibodies and additional agents that would act on both amyloid and non‐amyloid targets, could be the key to slowing down this global epidemic.FundingThis work was supported by grants BFU2017‐82494‐P and PID2020‐115823GB‐I00 funded by MCIN/AEI/ 10.13039/501100011033 and by ERDF “A way of making Europe” by the European Union; both to LJ‐D and JDN‐L. DJ held a predoctoral scholarship from Plan Propio de Investigación–ESF Programme of University of Castilla‐La Mancha.

Similarities in Pathobiology of Alzheimer’s Disease and Type 2 Diabetes Mellitus: A Proof of Concept by a Case Study

AbstractBackgroundAlzheimer’s disease (AD) is the leading cause of dementia worldwide. Developing countries will have >3 times the prevalence of the developed countries by the year 2050. Our previous studies with a Pakistani population have suggested that type 2 diabetes mellitus (T2DM) is strongly associated with impaired cognitive functioning, and genetic pathways showed strong resemblance to known AD patterns. The present case studies investigated two important pathways, i.e., Amyloid Precursor Protein (APP) processing and Lipid Metabolism, using gene expression profiling arrays to compare and contrast AD and T2DM patients from Pakistan.MethodWe performed high‐throughput qRT‐PCR with preconfigured a TaqMan Low Density Profiling Array specific to Human Alzheimer’s Disease (ABI, CAT # 441887) to measure the altered gene expressions and their corresponding pathways (Ingenuity Pathway Analysis, IPA®). Both the T2DM (n = 2; M/F each) and AD (n = 2, M/F each) patients (cases) were recruited from outpatient departments of a regional hospital, and were compared with their respective matched, healthy controls.ResultOverall, we observed a differential gene expression profile in AD and T2DM participants compared to controls. CYP46A1, MAPT, CAPNS1, GAL, AGER, CAPN1, PSENEN, ST6GAL1, GRIN2B, GRIN2C, GRIN2A, and GAP43 was downregulated in both T2DM and AD patients. ACHE, CHRM3, GRIN2C and GAP43 significantly (p value <0.05) downregulated in all T2DM patients. CDC2, UCHL1, CTSG, CASP3, IDE, NAE1, CDK5R1, IL6, APBB2, GLS are the top 10 common genes that were upregulated in both AD and T2DM groups. The IPA analysis revealed important Canonical Pathways, that included, viz., Amyloid Processing, Neuroinflammation Signaling, ErB4 Signaling, nNOs Signaling. Molecular Mechanisms of Cancer. The top disease and biofunctions included Metabolic Disease, Neurological disease, Organismal Injury and Abnormalities, Psychological Disorders, Cardiovascular Disease, with the top upstream regulators PHLPP2, IL33, APP, CRP, TAC1.ConclusionThe differential gene expression patterns and associated disease pathways from this pilot study suggest that specific genes and pathways were common to both diseases and may suggest common underlying processes and causes. Information from such biochemical and genetic studies will be crucial to the development of therapeutic targets to treat AD.

Inhibition of ACAT as a Therapeutic Target for Alzheimer's Disease Is Independent of ApoE4 Lipidation.

APOE4, encoding apolipoprotein E4 (apoE4), is the greatest genetic risk factor for Alzheimer's disease (AD), compared to the common APOE3. While the mechanism(s) underlying APOE4-induced AD risk remains unclear, increasing the lipidation of apoE4 is an important therapeutic target as apoE4-lipoproteins are poorly lipidated compared to apoE3-lipoproteins. ACAT (acyl-CoA: cholesterol-acyltransferase) catalyzes the formation of intracellular cholesteryl-ester droplets, reducing the intracellular free cholesterol (FC) pool. Thus, inhibiting ACAT increases the FC pool and facilitates lipid secretion to extracellular apoE-containing lipoproteins. Previous studies using commercial ACAT inhibitors, including avasimibe (AVAS), as well as ACAT-knock out (KO) mice, exhibit reduced AD-like pathology and amyloid precursor protein (APP) processing in familial AD (FAD)-transgenic (Tg) mice. However, the effects of AVAS with human apoE4 remain unknown. In vitro, AVASinduced apoE efflux at concentrations of AVASmeasured in the brains of treated mice. AVAS treatment of male E4FAD-Tg mice (5xFAD+/-APOE4+/+) at6-8months had no effect on plasma cholesterol levels or distribution, the original mechanism forAVAS treatment of CVD. In the CNS, AVAS reduced intracellular lipid droplets, indirectly demonstrating target engagement. Surrogate efficacy was demonstrated by an increase in Morris water maze measures of memory and postsynaptic protein levels. Amyloid-beta peptide (Aβ) solubility/deposition and neuroinflammation were reduced, critical components of APOE4-modulated pathology. However, there was no increase in apoE4 levels or apoE4 lipidation, while amyloidogenic and non-amyloidogenic processing of APP were significantly reduced. This suggests that the AVAS-induced reduction in Aβ via reduced APP processing was sufficient to reduce AD pathology, as apoE4-lipoproteins remained poorly lipidated.

Protective Alzheimer's disease-associated APP A673T variant predominantly decreases sAPPβ levels in cerebrospinal fluid and 2D/3D cell culture models

The rare A673T variant was the first variant found within the amyloid precursor protein (APP) gene conferring protection against Alzheimer's disease (AD). Thereafter, different studies have discovered that the carriers of the APP A673T variant show reduced levels of amyloid beta (Aβ) in the plasma and better cognitive performance at high age. Here, we analyzed cerebrospinal fluid (CSF) and plasma of APP A673T carriers and control individuals using a mass spectrometry-based proteomics approach to identify differentially regulated targets in an unbiased manner. Furthermore, the APP A673T variant was introduced into 2D and 3D neuronal cell culture models together with the pathogenic APP Swedish and London mutations. Consequently, we now report for the first time the protective effects of the APP A673T variant against AD-related alterations in the CSF, plasma, and brain biopsy samples from the frontal cortex. The CSF levels of soluble APPβ (sAPPβ) and Aβ42 were significantly decreased on average 9–26% among three APP A673T carriers as compared to three well-matched controls not carrying the protective variant. Consistent with these CSF findings, immunohistochemical assessment of cortical biopsy samples from the same APP A673T carriers did not reveal Aβ, phospho-tau, or p62 pathologies. We identified differentially regulated targets involved in protein phosphorylation, inflammation, and mitochondrial function in the CSF and plasma samples of APP A673T carriers. Some of the identified targets showed inverse levels in AD brain tissue with respect to increased AD-associated neurofibrillary pathology. In 2D and 3D neuronal cell culture models expressing APP with the Swedish and London mutations, the introduction of the APP A673T variant resulted in lower sAPPβ levels. Concomitantly, the levels of sAPPα were increased, while decreased levels of CTFβ and Aβ42 were detected in some of these models. Our findings emphasize the important role of APP-derived peptides in the pathogenesis of AD and demonstrate the effectiveness of the protective APP A673T variant to shift APP processing towards the non-amyloidogenic pathway in vitro even in the presence of two pathogenic mutations.

APP and Bace1: Differential effect of cholesterol enrichment on processing and plasma membrane mobility

High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the β-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion.

The Labyrinthine Landscape of APP Processing: State of the Art and Possible Novel Soluble APP-Related Molecular Players in Traumatic Brain Injury and Neurodegeneration.

Amyloid Precursor Protein (APP) and its cleavage processes have been widely investigated in the past, in particular in the context of Alzheimer's Disease (AD). Evidence of an increased expression of APP and its amyloidogenic-related cleavage enzymes, β-secretase 1 (BACE1) and γ-secretase, at the hit axon terminals following Traumatic Brain Injury (TBI), firstly suggested a correlation between TBI and AD. Indeed, mild and severe TBI have been recognised as influential risk factors for different neurodegenerative diseases, including AD. In the present work, we describe the state of the art of APP proteolytic processing, underlining the different roles of its cleavage fragments in both physiological and pathological contexts. Considering the neuroprotective role of the soluble APP alpha (sAPPα) fragment, we hypothesised that sAPPα could modulate the expression of genes of interest for AD and TBI. Hence, we present preliminary experiments addressing sAPPα-mediated regulation of BACE1, Isthmin 2 (ISM2), Tetraspanin-3 (TSPAN3) and the Vascular Endothelial Growth Factor (VEGFA), each discussed from a biological and pharmacological point of view in AD and TBI. We finally propose a neuroprotective interaction network, in which the Receptor for Activated C Kinase 1 (RACK1) and the signalling cascade of PKCβII/nELAV/VEGF play hub roles, suggesting that vasculogenic-targeting therapies could be a feasible approach for vascular-related brain injuries typical of AD and TBI.

Shared Neurological and Cognitive Factors in COVID-19 and Alzheimer’s Disease: A Literature Review

Introduction: The neuropathological etiology of COVID-19 suggests infection may increase the risk of neurodegenerative disorders, most notably Alzheimer’s disease (AD). Despite some overlap between COVID-19 and AD, the current research on this relationship is developing and evidence has been mixed. This review aims to assess the literature on how the neurological and cognitive sequalae associated with COVID-19 infection affect risk for AD and its associated symptoms, and vice versa. Methods: Articles were found by searching through the PubMed database with the terms (sensory OR cognitive OR neurological OR neuroimaging) AND (Alzheimer’s disease OR Alzheimer’s OR dementia) AND (COVID-19 OR SARS-CoV-2 OR COVID). Search inclusion criteria required the papers be written in English, be published in 2020 or later, and pertain to COVID-19 and/or AD specifically. This process was supplemented by manual searching. The articles used for this review include meta-analyses, literature reviews, and prospective and retrospective empirical studies. Results: Various well-known AD risk factors and outcomes may be observed in COVID-19 patients, and vice versa. These include amyloid precursor protein (APP) buildup, tau hyperphosphorylation, the apolipoprotein E (APOE) e4e4 genotype, angiotensin converting enzyme 2 (ACE2) gene expression, cholinergic functioning, delirium, cognitive decline, and deleterious effects on brain structure and function. Discussion: COVID-19 may increase AD risk and development by increasing protein build-up and damaging AD-related brain regions, which may underlie sensory and cognitive deficits. Furthermore, COVID-19 and AD may interact in positive feedback loops to worsen the development of both. These interactions may be mediated by demyelination, inflammation, APOE e4e4 genotype, and ACE2 expression. Clinical implications for those with COVID-19 and/or AD include the possibility of treatments aimed at cholinergic functioning, as well as high flow oxygen therapy. Conclusion: COVID-19 may increase the risk for and development of AD. AD, in turn, may also increase risk for COVID-19 infection, acting in a positive feedback loop. Future directives include further research on tau pathology, delirium, and amyloid precursor protein processing. Additionally, studies could benefit from telemedicine. Lastly, assessment of AD risk due to COVID-19 could integrate delirium and subjective reports of “brain fog” as measures for underlying risk factors.

Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

Porphyromonas gingivalis-induced periodontitis could contribute to cognitive impairment in Sprague-Dawley rats via the P38 MAPK signaling pathway.

Periodontitis is one of the most common oral diseases and has been shown to be a risk factor for systemic diseases. Our aim was to investigate the relationship between periodontitis and cognitive impairment and to explore the role of the P38 MAPK signaling pathway in this process. We established a periodontitis model by ligating the first molars of SD rats with silk thread and injecting Porphyromonas gingivalis (P. gingivalis) or P. gingivalis plus the P38 MAPK inhibitor SB203580 at the same time for ten weeks. We assessed alveolar bone resorption and spatial learning and memory using microcomputed tomography and the Morris water maze test, respectively. We used transcriptome sequencing to explore the genetic differences between the groups. The gingival tissue, peripheral blood and hippocampal tissue were assessed for the cytokines TNF-α, IL-1β, IL-6, IL-8 and C reactive protein (CRP) with enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). We observed the presence of P. gingivalis in the hippocampus of rats by paraffin-fluorescence in situ hybridization (FISH). We determined the activation of microglia by immunofluorescence. Finally, Western blot analysis was employed to determine the expression of amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1) and P38MAPK pathway activation. We demonstrated that silk ligature-induced periodontitis plus injection of P. gingivalis into subgingival tissue could lead to memory and cognitive impairment. Transcriptome sequencing results suggested that there were neurodegenerative diseases in the P. gingivalis group, and the MWM test showed that periodontitis reduced the spatial learning and memory ability of mild cognitive impairment (MCI) model rats. We found high levels of inflammatory factors (TNF-α, IL-1β, IL-6, and IL-8) and CRP in the gingiva, peripheral blood and hippocampus, and the expression of APP and BACE1 was upregulated, as was the P38 MAPK pathway activation. Activated microglia and the presence of P. gingivalis were also found in the hippocampus. P38 MAPK inhibitors mitigated all of these changes. Our findings strongly suggest that topical application of P. gingivalis increases the inflammatory burden in the peripheral and central nervous systems (CNS) and that neuroinflammation induced by activation of P38 MAPK leads to impaired learning and memory in SD rats. It can also modulate APP processing. Therefore, P38 MAPK may serve as a linking pathway between periodontitis and cognitive impairment.

Acute interleukin-6 modulates key enzymes involved in prefrontal cortex and hippocampal amyloid precursor protein processing.

Exercise is beneficial for individuals with Alzheimer's disease (AD). In rodent models of AD, exercise decreases the amyloidogenic processing of the amyloid precursor protein (APP). Although, it remains unclear how exercise is promoting this shift away from pathological APP processing, there is emerging evidence that exercise-induced factors released from peripheral tissues may facilitate these alterations in brain APP processing. Interleukin-6 (IL-6) is released from multiple organs into peripheral circulation during exercise and is among the most characterized exerkines. The purpose of this study is to examine if acute IL-6 can modulate key enzymes responsible for APP processing, namely a disintegrin and metalloproteinase 10 (ADAM10) and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), which initiate the non-amyloidogenic and amyloidogenic cascades respectively. Male 10-week-old C57BL/6J mice underwent and acute treadmill exercise bout or were either injected with IL-6 or a PBS control 15 min prior to tissue collection. ADAM10 and BACE1 enzyme activity, mRNA, and protein expression, as well as downstream markers of both cascades, including sAPPa and sAPPβ, were examined. Exercise increased circulating IL-6 and brain IL-6 signaling (pSTAT3 and Socs3 mRNA). This occurred alongside a reduction in BACE1 activity and an increase in ADAM10 activity. IL-6 injection reduced BACE1 activity and increased sAPPa protein content in the prefrontal cortex. In the hippocampus, IL-6 injection decreased BACE1 activity and sAPPβ protein content. Our results show that acute IL-6 injection increases markers of the non-amyloidogenic cascade and decreases markers of the amyloidogenic cascade in the cortex and hippocampus of the brain.

Evaluation of therapeutic effects of tetramethylpyrazine nitrone in Alzheimer's disease mouse model and proteomics analysis.

The pathophysiology of Alzheimer's disease (AD) is multifactorial with characteristic extracellular accumulation of amyloid-beta (Aβ) and intraneuronal aggregation of hyperphosphorylated tau in the brain. Development of disease-modifying treatment for AD has been challenging. Recent studies suggest that deleterious alterations in neurovascular cells happens in parallel with Aβ accumulation, inducing tau pathology and necroptosis. Therefore, therapies targeting cellular Aβ and tau pathologies may provide a more effective strategy of disease intervention. Tetramethylpyrazine nitrone (TBN) is a nitrone derivative of tetramethylpyrazine, an active ingredient from Ligusticum wallichii Franchat (Chuanxiong). We previously showed that TBN is a potent scavenger of free radicals with multi-targeted neuroprotective effects in rat and monkey models of ischemic stroke. The present study aimed to investigate the anti-AD properties of TBN. We employed AD-related cellular model (N2a/APPswe) and transgenic mouse model (3×Tg-AD mouse) for mechanistic and behavioral studies. Our results showed that TBN markedly improved cognitive functions and reduced Aβ and hyperphosphorylated tau levels in mouse model. Further investigation of the underlying mechanisms revealed that TBN promoted non-amyloidogenic processing pathway of amyloid precursor protein (APP) in N2a/APPswe in vitro. Moreover, TBN preserved synapses from dendritic spine loss and upregulated synaptic protein expressions in 3×Tg-AD mice. Proteomic analysis of 3×Tg-AD mouse hippocampal and cortical tissues showed that TBN induced neuroprotective effects through modulating mitophagy, MAPK and mTOR pathways. In particular, TBN significantly upregulated PINK1, a key protein for mitochondrial homeostasis, implicating PINK1 as a potential therapeutic target for AD. In summary, TBN improved cognitive functions in AD-related mouse model, inhibited Aβ production and tau hyperphosphorylation, and rescued synaptic loss and neuronal damage. Multiple mechanisms underlie the anti-AD effects of TBN including the modulation of APP processing, mTOR signaling and PINK1-related mitophagy.

A Diet Containing Rutin Ameliorates Brain Intracellular Redox Homeostasis in a Mouse Model of Alzheimer’s Disease

Quercetin has been studied extensively for its anti-Alzheimer's disease (AD) and anti-aging effects. Our previous studies have found that quercetin and in its glycoside form, rutin, can modulate the proteasome function in neuroblastoma cells. We aimed to explore the effects of quercetin and rutin on intracellular redox homeostasis of the brain (reduced glutathione/oxidized glutathione, GSH/GSSG), its correlation with β-site APP cleaving enzyme 1 (BACE1) activity, and amyloid precursor protein (APP) expression in transgenic TgAPP mice (bearing human Swedish mutation APP transgene, APPswe). On the basis that BACE1 protein and APP processing are regulated by the ubiquitin-proteasome pathway and that supplementation with GSH protects neurons from proteasome inhibition, we investigated whether a diet containing quercetin or rutin (30 mg/kg/day, 4 weeks) diminishes several early signs of AD. Genotyping analyses of animals were carried out by PCR. In order to determine intracellular redox homeostasis, spectrofluorometric methods were adopted to quantify GSH and GSSG levels using o-phthalaldehyde and the GSH/GSSG ratio was ascertained. Levels of TBARS were determined as a marker of lipid peroxidation. Enzyme activities of SOD, CAT, GR, and GPx were determined in the cortex and hippocampus. ΒACE1 activity was measured by a secretase-specific substrate conjugated to two reporter molecules (EDANS and DABCYL). Gene expression of the main antioxidant enzymes: APP, BACE1, a Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), caspase-3, caspase-6, and inflammatory cytokines were determined by RT-PCR. First, overexpression of APPswe in TgAPP mice decreased GSH/GSSG ratio, increased malonaldehyde (MDA) levels, and, overall, decreased the main antioxidant enzyme activities in comparison to wild-type (WT) mice. Treatment of TgAPP mice with quercetin or rutin increased GSH/GSSG, diminished MDA levels, and favored the enzyme antioxidant capacity, particularly with rutin. Secondly, both APP expression and BACE1 activity were diminished with quercetin or rutin in TgAPP mice. Regarding ADAM10, it tended to increase in TgAPP mice with rutin treatment. As for caspase-3 expression, TgAPP displayed an increase which was the opposite with rutin. Finally, the increase in expression of the inflammatory markers IL-1β and IFN-γ in TgAPP mice was lowered by both quercetin and rutin. Collectively, these findings suggest that, of the two flavonoids, rutin may be included in a day-to-day diet as a form of adjuvant therapy in AD.

Age-Dependent Dysregulation of APP in Neuronal and Skin Cells from Fragile X Individuals.

Fragile X syndrome (FXS) is the most common form of monogenic intellectual disability and autism, caused by the absence of the functional fragile X messenger ribonucleoprotein 1 (FMRP). FXS features include increased and dysregulated protein synthesis, observed in both murine and human cells. Altered processing of the amyloid precursor protein (APP), consisting of an excess of soluble APPα (sAPPα), may contribute to this molecular phenotype in mice and human fibroblasts. Here we show an age-dependent dysregulation of APP processing in fibroblasts from FXS individuals, human neural precursor cells derived from induced pluripotent stem cells (iPSCs), and forebrain organoids. Moreover, FXS fibroblasts treated with a cell-permeable peptide that decreases the generation of sAPPα show restored levels of protein synthesis. Our findings suggest the possibility of using cell-based permeable peptides as a future therapeutic approach for FXS during a defined developmental window.

Interplay between APP and glypican-1 processing and α-synuclein aggregation in undifferentiated and differentiated human neural progenitor cells.

In Parkinson's disease, there is an accumulation of α-synuclein (SYN) aggregates in neurons, which is promoted by neuroinflammation. In neural cells, cytokine-induced SYN aggregation is modulated by heparan sulfate (HS) derived from glypican-1 (GPC1) by amyloid precursor protein (APP) and nitric oxide (NO)-dependent cleavage. We have explored possible interplay between APP, GPC1, and SYN in undifferentiated and differentiated neural progenitor cells (NPCs) by modulating APP and GPC1 processing. Effects were monitored by immunofluorescence microscopy and slot immunoblotting using antibodies recognizing APP degradation products, HS released from GPC1, and SYN aggregates (filamentous SYN [SYNfil]). Suppression of HS release from GPC1 by inhibition of β-secretase or by NO deprivation resulted in no or slight increase in SYNfil aggregation. Stimulation of HS release by ascorbate did not further increase SYNfil staining. Interleukin-6 (IL-6) induced increased APP and GPC1 processing and SYNfil formation, which was reduced when β-secretase was inhibited and when HS release was impeded by NO deprivation. Ascorbate restored APP and GPC1 processing but did not affect SYNfil formation. Ascorbate-dependent differentiation of NPC resulted in the expression of tyrosine hydroxylase (TH) which colocalized with SYNfil. Suppression of APP processing by inhibition of β-secretase greatly disturbed the differentiation process. IL-6 induced coclustering of APP-degradation products, TH, HS, and SYNfil, which could be reversed by stimulation of HS release from GPC1 by excess ascorbate. We suggest that continuous release of HS from GPC1 moderates SYN aggregation and supports differentiation of NPC to dopaminergic neurons.

Artificial neural network models driven novel virtual screening workflow for the identification and biological evaluation of BACE1 inhibitors.

Beta-site amyloid-β precursor protein-cleaving enzyme 1 (BACE1) is a transmembrane aspartic protease and has shown potential as a possible therapeutic target for Alzheimer's disease. This aggravating disease involves the aberrant production of β amyloid plaques by BACE1 which catalyzes the rate-limiting step by cleaving the amyloid precursor protein (APP), generating the neurotoxic amyloid β protein that aggregates to form plaques leading to neurodegeneration. Therefore, it is indispensable to inhibit BACE1, thus modulating the APP processing. In this study, we present a workflow that utilizes a multi-stage virtual screening protocol for identifying potential BACE1 inhibitors by employing multiple artificial neural network-based models. Collectively, all the hyperparameter tuned models were assigned a task to virtually screen Maybridge library, thus yielding a consensus of 41 hits. The majority of these hits exhibited optimal pharmacokinetic properties confirmed by high central nervous system multiparameter optimization (CNS-MPO) scores. Further shortlisting of 8 compounds by molecular docking into the active site of BACE1 and their subsequent in-vitro evaluation identified 4 compounds as potent BACE1 inhibitors with IC50 values falling in the range 0.028-0.052 μM and can be further optimized with medicinal chemistry efforts to improve their activity.

Patchouli alcohol attenuates the cognitive deficits in a transgenic mouse model of Alzheimer’s disease via modulating neuropathology and gut microbiota through suppressing C/EBPβ/AEP pathway

BackgroundAlzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive cognitive dysfunctions and behavioral impairments. Patchouli alcohol (PA), isolated from Pogostemonis Herba, exhibits multiple pharmacological properties, including neuroprotective effects. This study aimed to investigate the therapeutic effects of PA against AD using the TgCRND8 transgenic AD mouse model, and to explore the underlying mechanisms targeting CCAAT/enhancer-binding protein β/asparagine endopeptidase (C/EBPβ/AEP) signaling pathway.MethodsAfter genotyping to confirm the transgenicity, drug treatments were administered intragastrically once daily to 3-month-old TgCRND8 mice for 4 consecutive months. Several behavioral tests were applied to assess different aspects of neurological functions. Then the brain and colon tissues were harvested for in-depth mechanistic studies. To further verify whether PA exerts anti-AD effects via modulating C/EBPβ/AEP signaling pathway in TgCRND8 mice, adeno-associated virus (AAV) vectors encoding CEBP/β were bilaterally injected into the hippocampal CA1 region in TgCRND8 mice to overexpress C/EBPβ. Additionally, the fecal microbiota transplantation (FMT) experiment was performed to verify the potential role of gut microbiota on the anti-AD effects of PA.ResultsOur results showed that PA treatment significantly improved activities of daily living (ADL), ameliorated the anxiety-related behavioral deficits and cognitive impairments in TgCRND8 mice. PA modulated the amyloid precursor protein (APP) processing. PA also markedly reduced the levels of beta-amyloid (Aβ) 40 and Aβ42, suppressed Aβ plaque burdens, inhibited tau protein hyperphosphorylation at several sites and relieved neuroinflammation in the brains of TgCRND8 mice. Moreover, PA restored gut dysbiosis and inhibited the activation of the C/EBPβ/AEP signaling pathway in the brain and colon tissues of TgCRND8 mice. Interestingly, PA strikingly alleviated the AD-like pathologies induced by the overexpression of C/EBPβ in TgCRND8 mice. Additionally, the FMT of fecal microbiota from the PA-treated TgCRND8 mice significantly alleviated the cognitive impairments and AD-like pathologies in the germ-free TgCRND8 mice.ConclusionAll these findings amply demonstrated that PA could ameliorate the cognitive deficits in TgCRND8 mice via suppressing Aβ plaques deposition, hyperphosphorylation of tau protein, neuroinflammation and gut dysbiosis through inhibiting the activation of C/EBPβ/AEP pathway, suggesting that PA is a promising naturally occurring chemical worthy of further development into the pharmaceutical treatment of AD.

The Breakthroughs and Caveats of Using Human Pluripotent Stem Cells in Modeling Alzheimer’s Disease

Modeling Alzheimer's disease (AD) using human-induced pluripotent stem cells (iPSCs) is a field now spanning 15 years. Developments in the field have shown a shift in using simple 2D cortical neuron models to more advanced tri-cultures and 3D cerebral organoids that recapitulate more features of the disease. This is largely due to development and optimization of new cell protocols. In this review, we highlight recent major breakthroughs in the AD field and the implications this has in modeling AD using iPSCs (AD-iPSCs). To date, AD-iPSCs have been largely used to recapitulate and study impaired amyloid precursor protein (APP) processing and tau phosphorylation in both familial and sporadic AD. AD-iPSCs have also been studied for varying neuronal and glial dysfunctions. Moreover, they have been useful for discovering new molecular mechanisms, such as identifying proteins that bridge APP processing with tau phosphorylation and for identifying molecular pathways that bridge APP processing dysfunction with impaired cholesterol biosynthesis. Perhaps the greatest use of AD-iPSCs has been in discovering compounds via drug screening, that reduce amyloid beta (Aβ) in neurons, such as the anti-inflammatory compound, cromolyn, and antiparasitic drugs, avermectins. In addition, high content screening using AD-iPSCs has led to the identification of statins that can reduce levels of phosphorylated tau (p-Tau) in neurons. Some of these compounds have made it through to testing in human clinical trials. Improvements in omic technologies including single cell RNA sequencing and proteomics as well as advances in production of iPSC-cerebral organoids and tri-cultures is likely to result in the further discovery of new drugs and treatments for AD. Some caveats remain in the field, including, long experimental conditions to create mature neurons, high costs of media that limit research capabilities, and a lack of reproducibility using current iPSC-cerebral organoid protocols. Despite these current limitations, AD-iPSCs remain an excellent cellular model for studying AD mechanisms and for drug discovery.

Apicidin attenuates memory deficits by reducing the Aβ load in APP/PS1 mice.

Amyloid beta (Aβ) is an important pathological feature of Alzheimer's disease (AD). A disintegrin and metalloproteinase 10 (ADAM10) can reduce the production of toxic Aβ by activating the nonamyloidogenic pathway of amyloid precursor protein (APP). We previously found that apicidin, which is a histone deacetylase (HDAC) inhibitor, can promote the expression of ADAM10 and reduce the production of Aβ in vitro. This study was designed to determine the potential of apicidin treatment to reverse learning and memory impairments in an AD mouse model and the possible correlation of these effects with ADAM10. Nine-month-old APP/PS1 mice and C57 mice received intraperitoneal injections of apicidin or vehicle for 2months. At 11 months of age, we evaluated the memory performance of mice with Morris water maze (MWM) and context fear conditioning tests. The Aβ levels were assessed in mouse brain using the immunohistochemical method and ELISA. The expression of corresponding protein involved in proteolytic processing of APP and the phosphorylation of tau were assessed by Western blotting. Apicidin reversed the deficits of spatial reference memory and contextual fear memory, attenuated the formation of Aβ-enriched plaques, and decreased the levels of soluble and insoluble Aβ40/42 in APP/PS1 mice. Moreover, apicidin significantly increased the expression of ADAM10, improved the level of sAPPα, and reduced the production of sAPPβ, but did not affect the levels of phosphorylated tau in APP/PS1 mice. Apicidin significantly improves the AD symptoms of APP/PS1 mice by regulating the expression of ADAM10, which may contribute to decreasing the levels of Aβ rather than decreasing the phosphorylation of tau.