Full-length Amyloid Precursor Protein Research Articles

Eta-secretase-like processing of the amyloid precursor protein (APP) by the rhomboid protease RHBDL4

The amyloid precursor protein (APP) is a key protein in Alzheimer’s disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resemble those generated by the η-secretase or MT5-MMP, which was described to generate so called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, β-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly less Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.

γ-Secretase Modulator BPN15606 Reduced Aβ42 and Aβ40 and Countered Alzheimer-Related Pathologies in a Mouse Model of Down Syndrome.

Due to increased gene dose for the amyloid precursor protein (APP), elderly adults with Down syndrome (DS) are at a markedly increased risk of Alzheimer's disease (AD), known as DS-AD. How the increased APP gene dose acts and which APP products are responsible for DS-AD is not well understood, thus limiting strategies to target pathogenesis. As one approach to address this question, we used a novel class of γ-secretase modulators that promote γ-site cleavages by the γ-secretase complex, resulting in lower levels of the Aβ42 and Aβ40 peptides. Ts65Dn mice, which serve as a model of DS, were treated via oral gavage with 10 mg/kg/weekday of BPN15606 (a potent and novel pyridazine-containing γ-secretase modulators). Treatment started at 3 months-of-age and lasted for 4 months. Demonstrating successful target engagement, treatment with BPN15606 significantly decreased levels of Aβ40 and Aβ42 in the cortex and hippocampus; it had no effect on full-length APP or its C-terminal fragments in either 2 N or Ts65Dn mice. Importantly, the levels of total amyloid-β were not impacted, pointing to BPN15606-mediated enhancement of processivity of γ-secretase. Additionally, BPN15606 rescued hyperactivation of Rab5, a protein responsible for regulating endosome function, and normalized neurotrophin signaling deficits. BPN15606 treatment also normalized the levels of synaptic proteins and tau phosphorylation, while reducing astrocytosis and microgliosis, and countering cognitive deficits. Our findings point to the involvement of increased levels of Aβ42 and/or Aβ40 in contributing to several molecular and cognitive traits associated with DS-AD. They speak to increased dosage of the APP gene acting through heightened levels of Aβ42 and/or Aβ40 as supporting pathogenesis. These findings further the interest in the potential use of γ-secretase modulators for treating and possibly preventing AD in individuals with DS. ANN NEUROL 2024;96:390-404.

STAU1 exhibits a dual function by promoting amyloidogenesis and tau phosphorylation in cultured cells

Staufen-1 (STAU1) is a double-stranded RNA-binding protein (RBP) involved in a variety of pathological conditions. In this study, we investigated the potential role of STAU1 in Alzheimer's disease (AD), in which two hallmarks are well-established as cerebral β-amyloid protein (Aβ) deposition and Tau-centered neurofibrillary tangles. We found that STAU1 protein level was significantly increased in cells that stably express full-length APP and the brain of APP/PS1 mice, an animal model of AD. STAU1 knockdown, as opposed to overexpression, significantly decreased the protein levels of β-amyloid converting enzyme 1 (BACE1) and Aβ. We further found that STAU1 extended the half-life of the BACE1 mRNA through binding to the 3′ untranslated region (3’UTR). Transcriptome analysis revealed that STAU1 enhanced the expression of growth arrest and DNA damage 45 β (GADD45B) upstream of P38 MAPK signaling, which contributed to STAU1-induced regulation of Tau phosphorylation at Ser396 and Thr181. Together, STAU1 promoted amyloidogenesis by inhibiting BACE1 mRNA decay, and augmented Tau phosphorylation through activating GADD45B in relation to P38 MAPK. Targeting STAU1 that acts on both amyloidogenesis and tauopathy may serve as an optimistic approach for AD treatment.

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.

Proteolytic cleavage of amyloid precursor protein by ADAM10 promotes proliferation and migration via activating MAPKs pathway in tongue squamous cell carcinoma in vitro.

APP, well-studied in the development of Alzheimer's disease, has been recently identified as the key gene correlated with TSCC. Here, we investigate the function of APP and its proteolytic cleavage by ADAM10 in the pathogenesis of TSCC. A total of 63 TSCC patients and 30 healthy controls were included and the results of IHC assay showed high expressions of ADAM10 and APP in TSCC tissues compared to paired para-carcinoma tissues. Interestingly, APP expression in TSCC patients was correlated with ADAM10 expression and their combined expression was related to the poor patients' survival. We found that APP was ɑ-cleaved in TSCC cells to form sAPPα, and the serum level of sAPPα but not sAPPβ in TSCC patients was higher than healthy controls. Both overexpression with full-length APP and sAPPα promoted TSCC cell proliferation, migration and invasion. Downregulation of APP or ADAM10 by siRNA decreased the generation of sAPPα and inhibited the activity of ERK1/2 and p38 pathways, thereby reducing TSCC cell proliferation, migration and invasion. Treatment with ERK1/2 or p38 agonist or sAPPα overexpression reversed the effects of APP or ADAM10 knockdown. In conclusion, our data demonstrated the pathogenic roles of APP cleaved by ADAM10 to activate ERK1/2 and p38 pathways in TSCC cells. Both high expressions of ADAM10 and APP were related to poor prognosis. Targeting APP cleaved by ADAM10 might be a potential strategy in TSCC treatment.

Axon-Autonomous Effects of the Amyloid Precursor Protein Intracellular Domain (AICD) on Kinase Signaling and Fast Axonal Transport.

The amyloid precursor protein (APP) is a key molecular component of Alzheimer's disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.

APP family member dimeric complexes are formed predominantly in synaptic compartments

BackgroundThe amyloid precursor protein (APP), a key player in Alzheimer’s disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions.ResultsWe investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls.ConclusionsTogether, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

APP-BACE1 Interaction and Intracellular Localization Regulate Aβ Production in iPSC-Derived Cortical Neurons.

Alzheimer's disease (AD) is characterized pathologically by amyloid β (Aβ)-containing plaques. Generation of Aβ from amyloid precursor protein (APP) by two enzymes, β- and γ-secretase, has therefore been in the AD research spotlight for decades. Despite this, how the physical interaction of APP with the secretases influences APP processing is not fully understood. Herein, we compared two genetically identical human iPSC-derived neuronal cell types: low Aβ-secreting neuroprogenitor cells (NPCs) and high Aβ-secreting mature neurons, as models of low versus high Aβ production. We investigated levels of substrate, enzymes and products of APP amyloidogenic processing and correlated them with the proximity of APP to β- and γ-secretase in endo-lysosomal organelles. In mature neurons, increased colocalization of full-length APP with the β-secretase BACE1 correlated with increased β-cleavage product sAPPβ. Increased flAPP/BACE1 colocalization was mainly found in early endosomes. In the same way, increased colocalization of APP-derived C-terminal fragment (CTF) with presenilin-1 (PSEN1), the catalytic subunit of γ-secretase, was seen in neurons as compared to NPCs. Furthermore, most of the interaction of APP with BACE1 in low Aβ-secreting NPCs seemed to derive from CTF, the remaining APP part after BACE1 cleavage, indicating a possible novel product-enzyme inhibition. In conclusion, our results suggest that interaction of APP and APP cleavage products with their secretases can regulate Aβ production both positively and negatively. β- and γ-Secretases are difficult targets for AD treatment due to their ubiquitous nature and wide range of substrates. Therefore, targeting APP-secretase interactions could be a novel treatment strategy for AD. Colocalization of APP species with BACE1 in a novel model of low- versus high-Aβ secretion-Two genetically identical human iPSC-derived neuronal cell types: low Aβ-secreting neuroprogenitor cells (NPCs) and high Aβ secreting mature neurons, were compared. Increased full-length APP (flAPP)/BACE1 colocalization in early endosomes was seen in neurons, while APP-CTF/BACE1 colocalization was much higher than flAPP/BACE1 colocalization in NPCs, although the cellular location was not determined.

BACE cleavage of APP does not drive the diabetic phenotype of PLB4 mice

BackgroundAlzheimer's Disease (AD) and Type 2 Diabetes Mellitus (T2DM), two prevalent diseases related to ageing, often share common pathologies including increased inflammation, endoplasmic reticulum (ER) stress, and impaired metabolic homeostasis predominantly affecting different organs. Therefore, it was unexpected to find in a previous study that neuronal hBACE1 knock-in (PLB4 mouse) leads to both an AD- and T2DM- like phenotype. The complexity of this co-morbidity phenotype required a deeper systems approach to explore the age-related changes in AD and T2DM-like pathologies of the PLB4 mouse. Therefore, we here analysed key neuronal and metabolic tissues comparing associated pathologies to those of normal ageing. MethodsGlucose tolerance, insulin sensitivity and protein turnover were assessed in 5-h fasted 3- and 8-month-old male PLB4 and wild-type mice. Western Blot and quantitative PCR were performed to determine regulation of homeostatic and metabolic pathways in insulin-stimulated brain, liver and muscle tissue. ResultsNeuronal hBACE1 expression caused early pathological cleavage of APP (increased monomeric Aβ (mAβ) levels at 3 months), in parallel with brain ER stress (increased phosphorylation of the translation regulation factor (p-eIF2α) and the chaperone binding immunoglobulin protein (BIP)). However, APP processing shifted over time (higher full-length APP and secreted APPβ levels, alongside lower mAβ and secreted APPα at 8 months), together with increased ER stress (phosphorylated/total inositol-requiring enzyme 1α (IRE1α)) in brain and liver. Metabolically, systemic glucose intolerance was evident from 3 months, yet metabolic signalling varied greatly between tissues and ages, and was confined to the periphery (increased muscle insulin receptors (IR), dipeptidyl-peptidase-4 (DPP4) levels, and decreased phosphorylated protein Kinase B (p-Akt), alongside increased liver DPP4 and fibroblast growth factor 21 (FGF21)), all of which normalised to wild-type levels at 8 months. ConclusionOur data suggest that the murine nervous system is affected early by APP misprocessing as a result of hBACE1 introduction, which coincided with ER stress, but not IR changes, and was alleviated with age. Peripheral metabolic alterations occurred early and revealed tissue-specific (liver vs. muscle) adaptations in metabolic markers but did not correlate with neuronal APP processing. Compensatory vs. contributory neuronal mechanisms associated with hBACE1 expression at different ages may explain why mice intrinsically do not develop AD pathologies and may offer new insights for future interventions.

Development of a MATLAB-based Toolkit to Quantify Neurodegenerative Histopathology Markers Applied to the Evaluation of a CRISPR-based Therapeutic for Alzheimer’s Disease (P3-6.003)

<h3>Objective:</h3> The objective of this study was to develop a comprehensive, easy-to-use set of MATLAB-based tools to quantify Alzheimer’s disease (AD) associated histopathological markers in immunolabeled brain tissue. We then utilized this MATLAB-based toolkit to assess the efficacy of a novel CRISPR-based, amyloid precursor protein (APP)-editing approach in a mouse model of AD. <h3>Background:</h3> AD is characterized immunohistologically by the presence of extracellular amyloid plaque deposits and associated neuroinflammation throughout the cerebral cortex and hippocampus. Amyloid plaques are aggregates of amyloid beta (Aβ) peptides that are released by successive cleavages of full length APP. Our lab has developed a novel CRISPR-based, APP-editing approach that effectively deletes the YENPTY endocytic domain at the C-terminus of APP, thus attenuating pathologic beta-cleavage and up-regulating neuroprotective alpha-cleavage. In order to assess the efficacy of our approach in vivo, we used CRISPRs to generate germ-line and somatic edits in an APP knock in (APP NL/G/F) mouse model of AD. <h3>Design/Methods:</h3> <h3>Immunohistochemistry (IHC):</h3> Tissues from appropriately aged control and CRISPR-edited APP NL/G/F mice were immunostained for Aβ plaques (6E10 antibodies), astrocytes (anti-GFAP antibodies) and microglia (anti-Iba1 antibodies). <h3>MATLAB analyses:</h3> After images were optimally contrasted, a color-channel filter was applied to isolate cells and plaques from the background. Our MATLAB program was then able to segment individual cells/plaques and measure their area, distance to nearby cells/plaques, and percentage of overlap. Activated microglia were defined as those with an area of 25 μm² or greater. <h3>Results:</h3> MATLAB quantification of IHC-stained sections revealed a robust decrease in Aβ deposition and reactive astrogliosis in CRISPR-treated APP NL/G/F tissues. These results matched supporting qualitative assessments and biochemical analyses. <h3>Conclusions:</h3> Our MATLAB toolkit can reliably quantify common neuropathological IHC markers and may be useful for evaluating the efficacy of neurotherapeutics. <b>Disclosure:</b> Mr. Satyadev has nothing to disclose. Dr. Aulston has nothing to disclose. Miss Branes has nothing to disclose. Miss Checka has nothing to disclose. Dr. Roy has stock in CRISPRAlz. The institution of Dr. Roy has received research support from Beam Therapeutics. Dr. Roy has received intellectual property interests from a discovery or technology relating to health care.

Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca2+ Signaling Pathways.

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling.

Neurexin-β Mediates the Synaptogenic Activity of Amyloid Precursor Protein.

In addition to its role in Alzheimer's disease, amyloid precursor protein (APP) has physiological roles in synapse development and function. APP induces presynaptic differentiation when presented to axons, but the mechanism is unknown. Here we show that APP binds neurexin to mediate this synaptogenic activity. APP specifically binds β not α neurexins modulated by splice site 4. Binding to neurexin heparan sulfate glycan and LNS protein domains is required for high-affinity interaction and for full-length APP to recruit axonal neurexin. The synaptogenic activity of APP is abolished by triple knockdown of neurexins in hippocampal neurons pooled from male and female rats. Based on these and previous results, our model is that a dendritic-axonal trans dimer of full-length APP binds to axonal neurexin-β to promote synaptic differentiation and function. Furthermore, soluble sAPPs also bind neurexin-β and inhibit its interaction with neuroligin-1, raising the possibility that disruption of neurexin function by altered levels of full-length APP and its cleavage products may contribute to early synaptic deficits in Alzheimer's disease.SIGNIFICANCE STATEMENT The prevailing model for the basis of Alzheimer's disease is the amyloid cascade triggered by altered cleavage of amyloid precursor protein (APP). APP also has physiological roles at the synapse, but the molecular basis for its synaptic functions is not well understood. Here, we show that APP binds the presynaptic organizing protein neurexin-β and that neurexin is essential for the synaptogenic activity of APP. Furthermore, soluble APP forms generated by APP cleavage also bind neurexin-β and can block interaction with transmembrane synaptogenic ligands of neurexin. These findings reveal a role for neurexin-APP interaction in synapse development and raise the possibility that disruptions of neurexin function may contribute to synaptic and cognitive deficits in the critical early stage of Alzheimer's disease.

Genetic Mapping of APP and Amyloid-β Biology Modulation by Trisomy 21.

Individuals who have Down syndrome (DS) frequently develop early onset Alzheimer’s disease (AD), a neurodegenerative condition caused by the build-up of aggregated amyloid-β and tau proteins in the brain. Amyloid-β is produced by amyloid precursor protein (APP), a gene located on chromosome 21. People who have Down syndrome have three copies of chromosome 21 and thus also an additional copy of APP; this genetic change drives the early development of Alzheimer’s disease in these individuals. Here we use a combination of next-generation mouse models of Down syndrome (Tc1, Dp3Tyb, Dp(10)2Yey and Dp(17)3Yey) and a knockin mouse model of amyloid-β accumulation (AppNL-F) to determine how chromosome 21 genes, other than APP, modulate APP/amyloid-β in the brain when in three copies. Using both male and female mice, we demonstrate that three copies of other chromosome 21 genes are sufficient to partially ameliorate amyloid-β accumulation in the brain. We go on to identify a subregion of chromosome 21 that contains the gene/genes causing this decrease in amyloid-β accumulation and investigate the role of two lead candidate genes Dyrk1a and Bace2. Thus an additional copy of chromosome 21 genes, other than APP, can modulate APP/amyloid-β in the brain under physiological conditions. This work provides critical mechanistic insight into the development of disease and an explanation for the typically later age of onset of dementia in people who have ADDS, compared to those who have familial AD caused by triplication of APP.

Safety, Tolerability, and Immunogenicity of the ACI-24 Vaccine in Adults With Down Syndrome

Individuals with Down syndrome (DS) are at high risk of developing Alzheimer disease due to an increased dose of the amyloid precursor protein gene, APP, which leads to increased levels of full-length APP and its products, including amyloid-β (Aβ). The liposome-based antiamyloid ACI-24 vaccine is intended to treat neurological disorders caused by misfolded Aβ pathological protein. However, the safety, tolerability, and immunogenicity of the ACI-24 vaccine among adults with DS have not been fully examined. To assess the safety and tolerability of the ACI-24 vaccine among adults with DS as well as its ability to induce immunogenicity measured by anti-Aβ immunoglobulin G titers. This multicenter double-blind placebo-controlled dose-escalation phase 1b randomized clinical trial was conducted at 3 US academic medical centers with affiliated Down syndrome clinics between March 30, 2016, and June 29, 2020. A total of 20 adults with DS were screened; of those, 16 adults were eligible to participate. Eligibility criteria included men or women aged 25 to 45 years with cytogenetic diagnosis of either trisomy 21 or complete unbalanced translocation of chromosome 21. Between April 27, 2016, and July 2, 2018, participants were randomized 3:1 into 2 dose-level cohorts (8 participants per cohort, with 6 participants receiving the ACI-24 vaccine and 2 receiving placebo) in a 96-week study. Participants received 48 weeks of treatment followed by an additional 48 weeks of safety follow-up. Participants were randomized to receive 7 subcutaneous injections of ACI-24, 300 μg or 1000 μg, or placebo. Primary outcomes were measures of safety and tolerability as well as antibody titers. Among 16 enrolled participants, the mean (SD) age was 32.6 (4.4) years; 9 participants were women, and 7 were men. All participants were White, and 1 participant had Hispanic or Latino ethnicity. Treatment adherence was 100%. There were no cases of meningoencephalitis, death, or other serious adverse events (AEs) and no withdrawals as a result of AEs. Most treatment-emergent AEs were of mild intensity (110 of 132 events [83.3%]) and unrelated or unlikely to be related to the ACI-24 vaccine (113 of 132 events [85.6%]). No amyloid-related imaging abnormalities with edema or cerebral microhemorrhage and no evidence of central nervous system inflammation were observed on magnetic resonance imaging scans. Increases in anti-Aβ immunoglobulin G titers were observed in 4 of 12 participants (33.3%) receiving ACI-24 (2 receiving 300 μg and 2 receiving 1000 μg) compared with 0 participants receiving placebo. In addition, a greater increase was observed in plasma Aβ1-40 and Aβ1-42 levels among individuals receiving ACI-24. In this study, the ACI-24 vaccine was safe and well tolerated in adults with DS. Evidence of immunogenicity along with pharmacodynamic and target engagement were observed, and anti-Aβ antibody titers were not associated with any adverse findings. These results support progression to clinical trials using an optimized formulation of the ACI-24 vaccine among individuals with DS. ClinicalTrials.gov Identifier: NCT02738450.

Quantitative proteomic analysis of cerebrospinal fluid reveals CD163, A2M and full-length APP as potential diagnostic biomarkers of paediatric bacterial meningitis.

BackgroundBacterial meningitis (BM) is a life-threatening infectious disease of the central nervous system in infants and children. To date, no diagnostic methods for the early and precise diagnosis of paediatric BM have been developed.MethodsA label-free cerebrospinal fluid (CSF) quantitative proteomic analysis of 8 patients with confirmed or suspected BM, 9 patients with confirmed or suspected viral meningitis (VM) and 6 non-CNS-infected hospital patients was performed via high-resolution LC–MS/MS.ResultsOur CSF proteomic analysis allowed the identification of critical differences between the BM and non-BM groups. Compared to the proteomes of the non-BM groups, the proteome of the paediatric BM group was characterized by upregulation of complement and coagulation cascades, regulation of IGF transport, uptake by IGF-binding proteins and acute inflammatory response, downregulation of developmental growth, and metabolism of carbohydrates. Moreover, the levels of CD163, A2M and full-length APP in CSF showed excellent diagnostic performance for paediatric BM, with AUC values of 0.911 (95% CI: 0.839–0.984), 0.908 (95% CI: 0.816–1.000) and 0.944 (95% CI: 0.86, 1.000), respectively. Among them, A2M and full-length APP are reported here for the first time as potential diagnostic biomarkers of BM. The findings imply that peptidase regulator activity plays an important role in BM and provide potential novel targets for precision medicine in paediatric BM.ConclusionsCD163, A2M and full-length APP are validated as potential diagnostic biomarkers of paediatric BM.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12953-022-00191-5.

Enhanced cleavage of APP by co-expressed Bace1 alters the distribution of APP and its fragments in neuronal and non-neuronal cells.

Alzheimer's disease amyloid-beta peptides (Aβ) are generated via sequential cleavage of the amyloid precursor protein (APP) by β-secretase (Bace1) and γ-secretase. Though the precise subcellular location(s) of Bace1-mediated APP cleavage remains unresolved, current models suggest APP internalization into Bace1-containing endosomes is a critical step. However, direct evidence for this model is lacking, and previous reports that probed the APP/Bace1 interaction (using co-expressed APP and Bace1 differentially labeled with fluorescent protein tags) did not determine if APP fluorescence originated from full-length APP (fl-APP) molecules that had internalized from the cell surface pool. We adapted the bungarotoxin-ligand (BTX) system to label surface APP and track internalized fluorescent APP/BTX puncta in rodent primary neurons co-expressing fluorescently-tagged Bace1. Subsequently, we employed imaging and biochemical-based approaches to measure N- and C-terminal APP epitope levels in primary neurons, N2a neuroblastoma, and HeLa cell lines. We hypothesized that surface-labeled APP/BTX puncta would, upon internalization, colocalize with fluorescently-tagged Bace1. Unexpectedly, we observed a dramatic loss of internalized APP in co-transfected neurons and ~ 80-90% loss of surface-resident fl-APP, which we also observed in HeLa and N2a cells. Loss of surface fl-APP could be reversed by a Bace1 inhibitor, suggesting that enhanced Bace1-mediated APP cleavage was responsible for the altered processing and mis-sorting. Importantly, in a C-terminally-tagged APP construct, the majority of C-terminal fluorescence was preserved in HeLa cells despite the loss of N-terminal APP signal. This phenomenon was not only recapitulated in cultured neurons, but also showed a progressive disappearance of the APP N-terminal tag, reflecting continual cleavage of fl-APP by Bace1 away from the cell body. Our results strongly suggested that in APP/Bace1 co-expression approaches, there was significant early and aberrant Bace1-mediated APP cleavage that perturbed fl-APP trafficking from the secretory pathway onwards, resulting in a substantial loss of surface fl-APP, which in turn led to a marked reduction in APP internalization. In C-terminally-tagged APP constructs, a large fraction of the APP fluorescence signal therefore likely arose from fluorescently-tagged β-C-terminal-fragment (β-CTF) or downstream proteolytic derivatives instead of fl-APP. Thus, care is needed in interpreting results where APP is detected only with a C-terminal tag in the presence of Bace1 co-expression, and previous findings may need to be reinterpreted if it is unclear whether fl-APP is present in normal physiological levels.

Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β-amyloid load in Alzheimer's disease model.

Inducing gamma oscillations with non‐invasive light flicker has been reported to impact Alzheimer's disease‐related pathology. However, it is unclear which signaling pathways are involved in reducing amyloid load. Here, we found that gamma frequency light flicker increased anchoring of amyloid precursor protein (APP) to the plasma membrane for non‐amyloidogenic processing, and then physically interacted with KCC2, a neuron‐specific K+‐Cl− cotransporter, suggesting that it is essential to maintain surface GABAA receptor α1 levels and reduce β‐amyloid (Aβ) production. Stimulation with such light flicker limited KCC2 internalization and subsequent degradation via both tyrosine phosphorylation and ubiquitination, leading to an increase in surface‐KCC2 levels. Specifically, PKC‐dependent phosphorylation of APP on a serine residue was induced by gamma frequency light flicker, which was responsible for maintaining plasma membrane levels of full‐length APP, leading to its reduced trafficking to endosomes and inhibiting the β‐secretase cleavage pathway. The activated PKC from the gamma frequency light flicker subsequently phosphorylated serine of KCC2 and stabilized it onto the cell surface, which contributed to the upregulation of surface GABAA receptor α1 levels. Together, these data indicate that enhancement of APP trafficking to the plasma membrane via light flicker plays a critical modulatory role in reduction of Aβ load in Alzheimer's disease.

Studying Alzheimer’s Disease Using &amp;lt;i&amp;gt;Drosophila melanogaster&amp;lt;/i&amp;gt; as a Powerful Tool

Background: Alzheimer’s disease (AD) is an increasingly prevalent neurodegenerative disease characterized by protein aggregation in the form of amyloid plaques containing beta-amyloid peptides and neurofibrillary tangles containing hyperphosphorylated tau protein. The central molecular events underlying AD pathogenesis remain controversial and poorly defined. Drosophila melanogaster has emerged as an important genetic resource for studying the pathology of AD. Many AD models have been created using Drosophila, taking advantage of its short generation times, sophisticated genetic tools, and abundance of homologs to human genes. Purpose: This review summarizes different models for studying AD in Drosophila melanogaster, including the full-length APP, C99, Aβ42 and Tau models, explaining how the models were built and what we have learned from them. Conclusion: Four main AD Drosophila models are introduced in this review, which can serve as a future method to investigate genes and drugs that can modify symptoms.

An App knock-in rat model for Alzheimer\u2019s disease exhibiting A\u03b2 and tau pathologies, neuronal death and cognitive impairments

A major obstacle in Alzheimer’s disease (AD) research is the lack of predictive and translatable animal models that reflect disease progression and drug efficacy. Transgenic mice overexpressing amyloid precursor protein (App) gene manifest non-physiological and ectopic expression of APP and its fragments in the brain, which is not observed in AD patients. The App knock-in mice circumvented some of these problems, but they do not exhibit tau pathology and neuronal death. We have generated a rat model, with three familiar App mutations and humanized Aβ sequence knocked into the rat App gene. Without altering the levels of full-length APP and other APP fragments, this model exhibits pathologies and disease progression resembling those in human patients: deposit of Aβ plaques in relevant brain regions, microglia activation and gliosis, progressive synaptic degeneration and AD-relevant cognitive deficits. Interestingly, we have observed tau pathology, neuronal apoptosis and necroptosis and brain atrophy, phenotypes rarely seen in other APP models. This App knock-in rat model may serve as a useful tool for AD research, identifying new drug targets and biomarkers, and testing therapeutics.

MicroRNA-455-3p improves synaptic, cognitive functions and extends lifespan: Relevance to Alzheimer's disease

BackgroundMicroRNA-455-3p is one of the highly conserved miRNAs involved in multiple cellular functions in humans and we explored its relevance to learning and memory functions in Alzheimer's disease (AD). Our recent in vitro studies exhibited the protective role of miR-455-3p against AD toxicities in reducing full-length APP and amyloid-β (Aβ) levels, and also in reducing defective mitochondrial biogenesis, impaired mitochondrial dynamics and synaptic deficiencies. In the current study, we sought to determine the function of miR-455-3p in mouse models. MethodsFor the first time we generated both transgenic (TG) and knockout (KO) mouse models of miR-455-3p. We determined the lifespan extension, cognitive function, mitochondrial biogenesis, mitochondrial dynamics, mitochondrial morphology, dendritic spine density, synapse numbers and synaptic activity in miR-455-3p TG and KO mice. ResultsMiR-455-3p TG mice lived 5 months longer than wild-type (WT) counterparts, whereas KO mice lived 4 months shorter than WT mice. Morris water maze test showed improved cognitive behavior, spatial learning and memory in miR-455-3p TG mice relative to age-matched WT mice and miR-455-3p KO mice. Further, mitochondrial biogenesis, dynamics and synaptic activities were enhanced in miR-455-3p TG mice, while these were reduced in KO mice. Overall, overexpressed miR-455-3p in mice displayed protective effects, whereas depleted miR-455-3p in mice exhibited deleterious effects in relation to lifespan, cognitive behavior, and mitochondrial and synaptic activities. ConclusionBoth mouse models could be ideal research tools to understand the molecular basis of aging and its relevance to AD and other age-related diseases.