Metabolic resistance of Aß3pE-42, a target epitope of the anti-Alzheimer therapeutic antibody, donanemab.

The amyloid ß peptide (Aß), starting with pyroglutamate (pE) at position 3 and ending at position 42 (Aß3pE-42), predominantly accumulates in the brains of Alzheimer's disease. Consistently, donanemab, a therapeutic antibody raised against Aß3pE-42, has been shown to be effective in recent clinical trials. Although the primary Aß produced physiologically is Aß1-40/42, an explanation for how and why this physiological Aß is converted to the pathological form remains elusive. Here, we present experimental evidence that accounts for the aging-associated Aß3pE-42 deposition: Aß3pE-42 was metabolically more stable than other Aßx-42 variants; deficiency of neprilysin, the major Aß-degrading enzyme, induced a relatively selective deposition of Aß3pE-42 in both APP transgenic and App knock-in mouse brains; Aß3pE-42 deposition always colocalized with Pittsburgh compound B-positive cored plaques in APP transgenic mouse brains; and under aberrant conditions, such as a significant reduction in neprilysin activity, aminopeptidases, dipeptidyl peptidases, and glutaminyl-peptide cyclotransferase-like were up-regulated in the progression of aging, and a proportion of Aß1-42 may be processed to Aß3pE-42. Our findings suggest that anti-Aß therapies are more effective if given before Aß3pE-42 deposition.

Proline-rich transmembrane protein 2 regulates the magnitude and frequency of dopamine release by repetitive neuronal stimuli in the striatum of L-dopa-treated mice.

Mutations in proline-rich transmembrane protein 2 (PRRT2) cause paroxysmal kinesigenic dyskinesia (PKD). Recently, we reported that a Prrt2 mutation exacerbated L-dopa-induced motor deficits in mice, suggesting that the basal ganglia might contribute to PKD pathology. Here, we demonstrated that the Prrt2 mutation enhanced depolarization stimuli-induced extracellular dopamine levels in the mouse striatum, which were attenuated by repeated stimulation. L-dopa administration maintained high dopamine levels in Prrt2-KI mice even during repetitive stimuli but did not affect dopamine levels in wild-type mice. Thus, the enhanced and prolonged responsiveness of dopamine release in nigrostriatal dopaminergic neurons to sequential excitation may be partially implicated in Prrt2-related dyskinesia.

The dopaminergic system promotes neprilysin-mediated degradation of amyloid-ß in the brain.

Deposition of amyloid-ß (Aß) in the brain can impair neuronal function and contribute to cognitive decline in Alzheimer's disease (AD). Here, we found that dopamine and the dopamine precursor levodopa (also called l-DOPA) induced Aß degradation in the brain. Chemogenetic approaches in mice revealed that the activation of dopamine release from ventral tegmental area (VTA) neurons increased the abundance and activity of the Aß-degrading enzyme neprilysin and reduced the amount of Aß deposits in the prefrontal cortex in a neprilysin-dependent manner. Aged mice had less dopamine and neprilysin in the anterior cortex, a decrease that was accentuated in AD model mice. Treating AD model mice with levodopa reduced Aß deposition and improved cognitive function. These observations demonstrate that dopamine promotes brain region-specific, neprilysin-dependent degradation of Aß, suggesting that dopamine-associated strategies have the potential to treat this aspect of AD pathology.

Identification and Characterization of Synaptic Vesicle Membrane Protein VAT-1 Homolog as a New Catechin-Binding Protein.

(-)-Epigallocatechin-3-gallate (EGCg), a major constituent of green tea extract, is well-known to exhibit many beneficial actions for human health by interacting with numerous proteins. In this study we identified synaptic vesicle membrane protein VAT-1 homolog (VAT1) as a novel EGCg-binding protein in human neuroglioma cell extracts using a magnetic pull-down assay and LC-tandem mass spectrometry. We prepared recombinant human VAT1 and analyzed its direct binding to EGCg and its alkylated derivatives using surface plasmon resonance. For EGCg and the derivative NUP-15, we measured an association constant of 0.02-0.85 ×103 M-1s-1 and a dissociation constant of nearly 8 × 10-4 s-1. The affinity Km(affinity) of their binding to VAT1 was in the 10-20 µM range and comparable with that of other EGCg-binding proteins reported previously. Based on the common structure of the compounds, VAT1 appeared to recognize a catechol or pyrogallol moiety around the B-, C- and G-rings of EGCg. Next, we examined whether VAT1 mediates the effects of EGCg and NUP-15 on expression of neprilysin (NEP). Treatments of mock cells with these compounds upregulated NEP, as observed previously, whereas no effect was observed in the VAT1-overexpressing cells, indicating that VAT1 prevented the effects of EGCg or NUP-15 by binding to and inactivating them in the cells overexpressing VAT1. Further investigation is required to determine the biological significance of the VAT1-EGCg interaction.

Proline-rich transmembrane protein 2 knock-in mice present dopamine-dependent motor deficits.

Mutations of proline-rich transmembrane protein 2 (PRRT2) lead to dyskinetic disorders such as paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements precipitated by suddenly initiated motion, and some convulsive disorders. Although previous studies have shown that PKD might be caused by cerebellar dysfunction, PRRT2 has not been sufficiently analyzed in some motor-related regions, including the basal ganglia, where dopaminergic neurons are most abundant in the brain. Here, we generated several types of Prrt2 knock-in (KI) mice harboring mutations, such as c.672dupG, that mimics the human pathological mutation c.649dupC and investigated the contribution of Prrt2 to dopaminergic regulation. Regardless of differences in the frameshift sites, all truncating mutations abolished Prrt2 expression within the striatum and cerebral cortex, consistent with previous reports of similar Prrt2 mutant rodents, confirming the loss-of-function nature of these mutations. Importantly, administration of l-dopa, a precursor of dopamine, exacerbated rotarod performance, especially in Prrt2-KI mice. These findings suggest that dopaminergic dysfunction in the brain by the PRRT2 mutation might be implicated in a part of motor symptoms of PKD and related disorders.

Enhancement of Neprilysin Activity by Natural Polyphenolic Compounds and Their Derivatives in Cultured Neuroglioma Cells.

The onset of Alzheimer's disease (AD) is characterized by accumulation of amyloid ß peptide (Aß) in the brain. Neprilysin (NEP) is one of the major Aß-degrading enzymes. Given findings that NEP expression in the brain declines from the early stage of AD before apparent neuronal losses are observed, enhancement of NEP activity and expression may be a preventive and therapeutic strategy relevant to disease onset. We screened for compounds that could enhance the activity and expression of NEP using a polyphenol library previously constructed by our research group and investigated the structure-activity relationships of the identified polyphenols. We found that amentoflavone, apigenin, kaempferol, and chrysin enhanced the activity and expression of NEP, suggesting that chemical structures involving a double bond between positions 2 and 3 in the C ring of flavones are important for NEP enhancement, while catechol or pyrogallol structures, except for the galloyl group of catechins, abolished these effects. Moreover, natural compounds, such as quercetin, were not effective per se, but were changed to effective compounds by adding a lipophilic moiety. Using our study findings, we propose improvements for dietary habits with experimental evidence, and provide a basis for the development of novel small molecules as disease-modifying drugs for AD.

Alterations of ATG4A and LC3B in neurons derived from Alzheimer's disease patients.

We investigated the alterations in autophagy-related molecules in neurons differentiated from induced pluripotent stem cells obtained from patients with Alzheimer's disease (AD). Consistent with our previous microarray data, ATG4A protein was upregulated in the neurons derived from a familial AD patient with an APP-E693Δ mutation who showed accumulation of intracellular amyloid ß peptide (Aß). This upregulation was reversed by inhibiting Aß production, suggesting that the intracellular Aß may be responsible for the upregulation of ATG4A. The LC3B-II/LC3B-I ratio, an index of autophagosome formation, was lower in the neurons derived from the AD patient with APP-E693Δ as well as the neurons derived from other familial and sporadic AD patients. These findings indicate that dysregulation of autophagy-related molecules may accelerate the pathogenesis of AD.

The role of TREM2 N-glycans in trafficking to the cell surface and signal transduction of TREM2.

Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer's disease, as well as other neurodegenerative disorders. TREM2 is glycosylated in vitro and in vivo, but the significance of the modification is unknown. We previously established a sensitive and specific reporter cell model involving cultured Jurkat cells stably expressing a luciferase reporter gene and a gene encoding a TREM2DAP12 fusion protein to monitor TREM2-dependent signalling. In the present study, we prepared modified reporter cells to investigate the role of the N-glycans at N20 and N79. We show that the N-glycans at N79 have a requisite role in translocation of TREM2 to the cell surface, while the N-glycans at both N20 and N79 have a critical role in intracellular signal transduction. Our results indicate that structural changes to the TREM2 N-glycans may cause microglial dysfunction that contributes to the pathogenesis of neurodegenerative disorders and that maintaining the integrity of TREM2 N-glycosylation and the responsible glycosyltransferases may be a novel therapeutic strategy to treat these disorders.

Recent Advances in the Modeling of Alzheimer's Disease.

Since 1995, more than 100 transgenic (Tg) mouse models of Alzheimer's disease (AD) have been generated in which mutant amyloid precursor protein (APP) or APP/presenilin 1 (PS1) cDNA is overexpressed ( 1st generation models ). Although many of these models successfully recapitulate major pathological hallmarks of the disease such as amyloid ß peptide (Aß) deposition and neuroinflammation, they have suffered from artificial phenotypes in the form of overproduced or mislocalized APP/PS1 and their functional fragments, as well as calpastatin deficiency-induced early lethality, calpain activation, neuronal cell death without tau pathology, endoplasmic reticulum stresses, and inflammasome involvement. Such artifacts bring two important uncertainties into play, these being (1) why the artifacts arise, and (2) how they affect the interpretation of experimental results. In addition, destruction of endogenous gene loci in some Tg lines by transgenes has been reported. To overcome these concerns, single App knock-in mouse models harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice) were developed ( 2nd generation models ). While these models are interesting given that they exhibit Aß pathology, neuroinflammation, and cognitive impairment in an age-dependent manner, the model with the Artic mutation, which exhibits an extensive pathology as early as 6 months of age, is not suitable for investigating Aß metabolism and clearance because the Aß in this model is resistant to proteolytic degradation and is therefore prone to aggregation. Moreover, it cannot be used for preclinical immunotherapy studies owing to the discrete affinity it shows for anti-Aß antibodies. The weakness of the latter model (without the Arctic mutation) is that the pathology may require up to 18 months before it becomes sufficiently apparent for experimental investigation. Nevertheless, this model was successfully applied to modulating Aß pathology by genome editing, to revealing the differential roles of neprilysin and insulin-degrading enzyme in Aß metabolism, and to identifying somatostatin receptor subtypes involved in Aß degradation by neprilysin. In addition to discussing these issues, we also provide here a technical guide for the application of App knock-in mice to AD research. Subsequently, a new double knock-in line carrying the AppNL-F and Psen1 P117L/WT mutations was generated, the pathogenic effect of which was found to be synergistic. A characteristic of this 3rd generation model is that it exhibits more cored plaque pathology and neuroinflammation than the AppNL-G-F line, and thus is more suitable for preclinical studies of disease-modifying medications targeting Aß. Furthermore, a derivative AppG-F line devoid of Swedish mutations which can be utilized for preclinical studies of ß-secretase modifier(s) was recently created. In addition, we introduce a new model of cerebral amyloid angiopathy that may be useful for analyzing amyloid-related imaging abnormalities that can be caused by anti-Aß immunotherapy. Use of the App knock-in mice also led to identification of the α-endosulfine-K ATP channel pathway as components of the somatostatin-evoked physiological mechanisms that reduce Aß deposition via the activation of neprilysin. Such advances have provided new insights for the prevention and treatment of preclinical AD. Because tau pathology plays an essential role in AD pathogenesis, knock-in mice with human tau wherein the entire murine Mapt gene has been humanized were generated. Using these mice, the carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) was discovered as a mediator linking tau pathology to neurodegeneration and showed that tau humanization promoted pathological tau propagation. Finally, we describe and discuss the current status of mutant human tau knock-in mice and a non-human primate model of AD that we have successfully created.

Local Action of Neprilysin Exacerbates Pressure Overload Induced Cardiac Remodeling.

[Figure: see text].

Nasal vaccine delivery attenuates brain pathology and cognitive impairment in tauopathy model mice.

Pathological aggregates of tau proteins accumulate in the brains of neurodegenerative tauopathies including Alzheimer's disease and frontotemporal lobar degeneration (FTLD-tau). Although immunotherapies of these disorders against tau are emerging, it is unknown whether nasal delivery, which offers many benefits over traditional approaches to vaccine administration, is effective or not for tauopathy. Here, we developed vaccination against a secreted form of pathological tau linked to FTLD-tau using a Sendai virus (SeV) vector infectious to host nasal mucosa, a key part of the immune system. Tau vaccines given as nasal drops induced tissue tau-immunoreactive antibody production and ameliorated cognitive impairment in FTLD-tau model mice. In vivo imaging and postmortem neuropathological assays demonstrated the suppression of phosphorylated tau accumulation, neurotoxic gliosis, and neuronal loss in the hippocampus of immunized mice. These findings suggest that nasal vaccine delivery may provide a therapeutic opportunity for a broad range of populations with human tauopathy.

Galectin 3-binding protein suppresses amyloid-ß production by modulating ß-cleavage of amyloid precursor protein.

Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of ß-amyloid (Aß) peptides. Aß is produced from amyloid precursor protein (APP) that is sequentially cleaved by ß- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aß production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aß production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aß production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed Aß production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aß production by directly interacting with APP and thereby inhibiting APP processing by ß-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

Activity-dependent cleavage of dyskinesia-related proline-rich transmembrane protein 2 (PRRT2) by calpain in mouse primary cortical neurons.

Mutations of PRRT2 (proline-rich transmembrane protein 2) cause several neurological disorders, represented by paroxysmal kinesigenic dyskinesia (PKD), which is characterized by attacks of involuntary movements triggered by sudden voluntary movements. PRRT2 is reported to suppress neuronal excitation, but it is unclear how the function of PRRT2 is modulated during neuronal excitation. We found that PRRT2 is processed to a 12 kDa carboxy-terminal fragment (12K-CTF) by calpain, a calcium-activated cysteine protease, in a neuronal activity-dependent manner, predominantly via NMDA receptors or voltage-gated calcium channels. Furthermore, we clarified that 12K-CTF is generated by sequential cleavages at Q220 and S244. The amino-terminal fragment (NTF) of PRRT2, which corresponds to PKD-related truncated mutants, is not detected, probably due to rapid cleavage at multiple positions. Given that 12K-CTF lacks most of the proline-rich domain, this cleavage might be involved in the activity-dependent enhancement of neuronal excitation perhaps through transient retraction of PRRT2's function. Therefore, PRRT2 might serve as a buffer for neuronal excitation, and lack of this function in PKD patients might cause neuronal hyperexcitability in their motor circuits.

Aminophospholipids are signal-transducing TREM2 ligands on apoptotic cells.

Variants of triggering receptor expressed on myeloid cells 2 (TREM2) are associated with an increased incidence of Alzheimer's disease, as well as other neurodegenerative disorders. Using a newly developed, highly sensitive reporter cell model, consisting of Jurkat T cells stably overexpressing a reporter gene and a gene encoding TREM2DAP12 fusion protein, we show here that TREM2-dependent signal transduction in response to apoptotic Neuro2a cells is mediated by aminophospholipid ligands, phosphatidylserine and phosphatidylethanolamine, which are not exposed on the intact cell surface, but become exposed upon apoptosis. We also show that signal-transducing TREM2 ligands different from aminophospholipids, which appear to be derived from neurons, might be present in membrane fractions of mouse cerebral cortex. These results may suggest that TREM2 regulates microglial function by transducing intracellular signals from aminophospholipids on apoptotic cells, as well as unidentified ligands in the membranes of the cerebral cortex.

iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid ß Combination for Alzheimer's Disease.

In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer's disease (AD). Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid ß peptide (Aß), a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aß-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aß effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aß cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aß effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development.

A simplified and sensitive method to identify Alzheimer's disease biomarker candidates using patient-derived induced pluripotent stem cells (iPSCs).

We developed a simplified and sensitive method to identify Alzheimer's disease (AD) biomarker candidates by a quantitative and targeted proteomic analysis (combination of liquid chromatography tandem mass spectrometry and multiplexed-multiple reaction monitoring/selected reaction monitoring analysis) of culture media from neurons differentiated from induced pluripotent stem cells (iPSCs) established from AD patients. We found that alpha-1-acid glycoprotein (ORM1) was decreased in the culture media of AD-iPSC-derived neurons, consistent with previous observations for AD patient cerebrospinal fluid, thus validating our new strategy. Moreover, our method is applicable for identifying biomarker candidates for other neurodegenerative disorders using patient-derived iPSCs.

Elucidating Pathogenic Mechanisms of Early-onset Alzheimer's Disease in Down Syndrome Patients.

Down syndrome (DS) patients demonstrate the neuropathology of Alzheimer's disease (AD) characterized by the formation of senile plaques and neurofibrillary tangles by age 40-50 years. It has been considered for a number of years that 1.5-fold expression of the gene for the amyloid precursor protein (APP) located on chromosome 21 leading to overproduction of amyloid-ß peptide (Aß) results in the early onset of AD in adults with DS. However, the mean age of onset of familial AD with the Swedish mutation on APP which has high affinity for ß-secretase associated with a dramatic increase in Aß production is about 55 years. This paradox indicates that there is a poor correlation between average ages of AD onset and the theoretical amount of Aß production and that there are factors exacerbating AD on chromosome 21. We therefore focused on dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), since overexpressing transgenic mice show AD-like brain pathology. The overexpression of DYRK1A caused suppression of the activity of neprilysin (NEP), which is a major Aß-degrading enzyme in the brain, and phosphorylation at the NEP cytoplasmic domain. NEP activity was markedly reduced in fibroblasts derived from DS patients compared with that in fibroblasts derived from healthy controls. This impaired activity of NEP was rescued by DYRK1A inhibition. These results show that DYRK1A overexpression causes suppression of NEP activity through its phosphorylation in DS patients. Our results suggest that DYRK1A inhibitors could be effective against AD not only in adults with DS but also in sporadic AD patients.

Neprilysin Is Suppressed by Dual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1A (DYRK1A) in Down-Syndrome-Derived Fibroblasts.

Amyloid-ß peptide (Aß) accumulation is a triggering event leading to the Alzheimer's disease (AD) pathological cascade. Almost all familial AD-linked gene mutations increase Aß production and accelerate the onset of AD. The Swedish mutation of amyloid precursor protein (APP) affects ß-secretase activity and increases Aß production up to ca. 6-fold in cultured cells; the onset age is around 50. Down syndrome (DS) patients with chromosome 21 trisomy present AD-like pathologies at earlier ages (40s) compared with sporadic AD patients, because APP gene expression is 1.5-fold higher than that in healthy people, thus causing a 1.5-fold increase in Aß production. However, when comparing the causal relationship of Aß accumulation with the onset age between the above two populations, early DS pathogenesis does not appear to be accounted for by the increased Aß production alone. In this study, we found that neprilysin, a major Aß-degrading enzyme, was downregulated in DS patient-derived fibroblasts, compared with healthy people-derived fibroblasts. Treatment with harmine, an inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), which is located in the DS critical region of chromosome 21, and gene knockdown of DYRK1A, upregulated neprilysin in fibroblasts. These results suggest that a decrease in the Aß catabolic rate may be, at least in part, one of the causes for accelerated AD-like pathogenesis in DS patients if a similar event occurs in the brains, and that neprilysin activity may be regulated directly or indirectly by DYRK1A-mediated phosphorylation. DYRK1A inhibition may be a promising disease-modifying therapy for AD via neprilysin upregulation.

Paradigm shift from diagnosing patients based on common symptoms to categorizing patients into subtypes with different pathogenic mechanisms to guide treatment for Alzheimer's disease.

Alzheimer's disease (AD) is a major cause of dementia in the elderly, and the number of AD patients is rapidly growing as life expectancy increases. However, disease-modifying drugs are not yet available. According to the amyloid hypothesis, disease onset is triggered by aggregation and accumulation of amyloid-ß peptide, followed by the formation of neurofibrillary tangles composed of hyperphosphorylated tau, and synaptic loss/neuronal cell death leading to dementia. Based on this hypothesis, various clinical trials for treatment of AD have been conducted, but most were discontinued due to failure to achieve cognitive improvement or appearance of adverse effects. Here we discuss the reasons for the failure of these trials. We suggest that biomarkers of specific, distinct molecular mechanisms of amyloidogenesis should be developed concomitantly with disease-modifying drugs (the so-called companion diagnosis) to aid the proper design of clinical trials, as well as to enable personalized treatment of individual AD patients.