Increased CSF-decorin predicts brain pathological changes driven by Alzheimer's Aß amyloidosis.

Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer's disease (AD) which is characterized by amyloid-ß (Aß) amyloidosis. Here, we used two App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aß pathology to analyze how the brain pathologies translate to CSF proteomes by label-free mass spectrometry (MS). This identified several extracellular matrix (ECM) proteins as significantly altered in App knock-in mice. Next, we compared mouse CSF proteomes with previously reported human CSF MS results acquired from patients across the AD spectrum. Intriguingly, the ECM protein decorin was similarly and significantly increased in both AppNL-F/NL-F and AppNL-G-F/NL-G-F mice, strikingly already at three months of age in the AppNL-F/NL-F mice and preclinical AD subjects having abnormal CSF-Aß42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aß42 levels indicating that the change in CSF-decorin is associated with early Aß amyloidosis. Importantly, receiver operating characteristic analysis revealed that CSF-decorin can predict a specific AD subtype having innate immune activation and potential choroid plexus dysfunction in the brain. Consistently, in AppNL-F/NL-F mice, increased CSF-decorin correlated with both Aß plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aß42 induces decorin secretion from mouse primary neurons. Interestingly, we finally identify decorin to activate neuronal autophagy through enhancing lysosomal function. Altogether, the increased CSF-decorin levels occurring at an early stage of Aß amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.

An isogenic panel of App knock-in mouse models: Profiling ß-secretase inhibition and endosomal abnormalities.

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) that harbor the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations (AppG-F mice) and evaluated its characteristics. Amyloid ß peptide (Aß) pathology was exhibited by AppG-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aß-secretase inhibitor, verubecestat, attenuated Aß production in AppG-F mice, but not in AppNL-G-F mice, indicating that the AppG-F mice are more suitable for preclinical studies of ß-secretase inhibition given that most patients with AD do not carry the Swedish mutations. Comparison of isogenic App knock-in lines revealed that multiple factors, including elevated C-terminal fragment ß (CTF-ß) and humanization of Aß might influence endosomal alterations in vivo. Thus, experimental comparisons between different isogenic App, knock-in mouse lines will provide previously unidentified insights into our understanding of the etiology of AD.

A third-generation mouse model of Alzheimer's disease shows early and increased cored plaque pathology composed of wild-type human amyloid ß peptide.

We previously developed single App knock-in mouse models of Alzheimer's disease (AD) harboring the Swedish and Beyreuther/Iberian mutations with or without the Arctic mutation (AppNL-G-F and AppNL-F mice, respectively). These models showed Aß pathology, neuroinflammation, and cognitive impairment in an age-dependent manner. The former model exhibits extensive pathology as early as 6 months, but is unsuitable for investigating Aß metabolism and clearance because the Arctic mutation renders Aß resistant to proteolytic degradation and prone to aggregation. In particular, it is inapplicable to preclinical immunotherapy studies due to its discrete affinity for anti-Aß antibodies. The latter model may take as long as 18 months for the pathology to become prominent, which leaves an unfulfilled need for an Alzheimer's disease animal model that is both swift to show pathology and useful for antibody therapy. We thus utilized mutant Psen1 knock-in mice into which a pathogenic mutation (P117L) had been introduced to generate a new model that exhibits early deposition of wild-type human Aß by crossbreeding the AppNL-F line with the Psen1P117L/WT line. We show that the effects of the pathogenic mutations in the App and Psen1 genes are additive or synergistic. This new third-generation mouse model showed more cored plaque pathology and neuroinflammation than AppNL-G-F mice and will help accelerate the development of disease-modifying therapies to treat preclinical AD.

Proteomics Time-Course Study of App Knock-In Mice Reveals Novel Presymptomatic Aß42-Induced Pathways to Alzheimer's Disease Pathology.

BACKGROUND: The 42 amino acids long amyloid-ß peptide, Aß42, may initiate a cascade of events leading to the severe neurodegeneration observed in Alzheimer's disease (AD) brain. However, the underlying molecular mechanisms remain to be established. OBJECTIVE: To find early Aß42-induced AD related mechanisms, we performed a brain proteomics time-course study on a novel App knock-in AD mouse model, AppNL-F, expressing high levels of Aß42 without AßPP overexpression artifacts. METHODS: Hippocampus and cortex were analyzed separately by using 18O-labelling mass spectrometry to reveal alterations in protein levels. Pathway analysis of proteomics data was used to identify altered biological functions. Immunohistochemistry was used to further investigate a significant key regulatory protein. RESULTS: Around 100 proteins were differently expressed in AppNL-F mice at each time point (3, 6, 9, and 18 months of age) as compared to wild type mice. Strikingly, already at 3 months of age-long before Aß plaque development and memory impairment-several pathways, including long-term potentiation and synaptic plasticity, were downregulated, and neuritogenesis was increased. Huntingtin (HTT) was identified as an upstream regulator, i.e., a key protein affecting the levels of several proteins. Increased levels of HTT in hippocampus of AppNL-F mice was supported by immunofluorescence microscopy. CONCLUSION: Notably, the proteome was significantly altered already at 3 months of age, 6 months before the development of plaques. Differentially expressed proteins varied over time, indicating that increased Aß42 levels initiate a cascade of events that eventually manifests in amyloid depositions, inflammation, and decline in memory.

Separation of Synephrine Enantiomers in Citrus Fruits by a Reversed Phase HPLC after Chiral Precolumn Derivatization.

Racemic synephrine, which was transformed into diastereomers by derivatization with 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranosil isothiocyanate, was resolved by a reversed phase HPLC with UV detection at 254 nm. The total contents of synephrine enantiomers in citrus fruit samples were exocarp > mesocarp > endocarp > sarcocarp, suggesting that synephrine content of outer side of citrus fruits was higher than that of the inner side. (R)-Synephrine was detected in exocarp of eleven fresh citrus fruits, except for lemon, lime, and grapefruit samples. (S)-Synephrine was determined in the exocarp of four citrus fruits (mikan, orange, bitter orange, and ponkan samples) and the ratio of (S)-synephrine to total synephrine was 0.5 - 0.9%. The racemization of (R)-synephrine in aqueous solution during heating at 100°C was also examined. An increase in the heating time brought about an increase in the (S)-synephrine content in a linear fashion. The racemization was found to be significantly reduced by the addition of D-fructose, D-maltose, D-glucose, D-mannose or D-galactose, but not D-sucrose or D-mannitol. It is suggested that the reducibility of sugars may result in the inhibition of racemization.

Introduction of pathogenic mutations into the mouse Psen1 gene by Base Editor and Target-AID.

Base Editor (BE) and Target-AID (activation-induced cytidine deaminase) are engineered genome-editing proteins composed of Cas9 and cytidine deaminases. These base-editing tools convert C:G base pairs to T:A at target sites. Here, we inject either BE or Target-AID mRNA together with identical single-guide RNAs (sgRNAs) into mouse zygotes, and compare the base-editing efficiencies of the two distinct tools in vivo. BE consistently show higher base-editing efficiency (10.0-62.8%) compared to that of Target-AID (3.4-29.8%). However, unexpected base substitutions and insertion/deletion formations are also more frequently observed in BE-injected mice or zygotes. We are able to generate multiple mouse lines harboring point mutations in the mouse presenilin 1 (Psen1) gene by injection of BE or Target-AID. These results demonstrate that BE and Target-AID are highly useful tools to generate mice harboring pathogenic point mutations and to analyze the functional consequences of the mutations in vivo.

Loss of neprilysin alters protein expression in the brain of Alzheimer's disease model mice.

Alzheimer's disease (AD) is a neurodegenerative disease displaying extracellular plaques formed by the neurotoxic amyloid ß-peptide (Aß), and intracellular neurofibrillary tangles consisting of protein tau. However, how these pathologies relate to the massive neuronal death that occurs in AD brains remain elusive. Neprilysin is the major Aß-degrading enzyme and a lack thereof increases Aß levels in the brain twofold. To identify altered protein expression levels induced by increased Aß levels, we performed a proteomic analysis of the brain of the AD mouse model APPsw and compared it to that of APPsw mice lacking neprilysin. To this end we established an LC-MS/MS method to analyze brain homogenate, using an (18) O-labeled internal standard to accurately quantify the protein levels. To distinguish between alterations in protein levels caused by increased Aß levels and those induced by neprilysin deficiency independently of Aß, the brain proteome of neprilysin deficient APPsw mice was also compared to that of neprilysin deficient mice. By this approach we identified approximately 600 proteins and the levels of 300 of these were quantified. Pathway analysis showed that many of the proteins with altered expression were involved in neurological disorders, and that tau, presenilin and APP were key regulators in the identified networks. The data have been deposited to the ProteomeXchange Consortium with identifiers PXD000968 and PXD001786 (http://proteomecentral.proteomexchange.org/dataset/PXD000968 and (http://proteomecentral.proteomexchange.org/dataset/PXD001786). Interestingly, the levels of several proteins, including some not previously reported to be linked to AD, were associated with increased Aß levels.

Autophagy-related protein 7 deficiency in amyloid ß (Aß) precursor protein transgenic mice decreases Aß in the multivesicular bodies and induces Aß accumulation in the Golgi.

Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid ß (Aß) peptide, which aggregates into extracellular Aß plaques in AD brains. Before plaque formation, Aß accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in Aß metabolism. In addition to a degradative role of autophagy in Aß metabolism we recently showed that Aß secretion is inhibited in mice lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of Aß secretion leads to intracellular accumulation of Aß. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular localization of the intracellular Aß accumulation which accumulates in Aß precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62(+) aggregates, but these aggregates do not contain Aß. However, knockdown of Atg7 induced Aß accumulation in the Golgi and a concomitant reduction of Aß in the multivesicular bodies. This indicates that Atg7 influences the transport of Aß possibly derived from Golgi to multivesicular bodies.

Aß secretion and plaque formation depend on autophagy.

Alzheimer's disease (AD) is a neurodegenerative disease biochemically characterized by aberrant protein aggregation, including amyloid beta (Aß) peptide accumulation. Protein aggregates in the cell are cleared by autophagy, a mechanism impaired in AD. To investigate the role of autophagy in Aß pathology in vivo, we crossed amyloid precursor protein (APP) transgenic mice with mice lacking autophagy in excitatory forebrain neurons obtained by conditional knockout of autophagy-related protein 7. Remarkably, autophagy deficiency drastically reduced extracellular Aß plaque burden. This reduction of Aß plaque load was due to inhibition of Aß secretion, which led to aberrant intraneuronal Aß accumulation in the perinuclear region. Moreover, autophagy-deficiency-induced neurodegeneration was exacerbated by amyloidosis, which together severely impaired memory. Our results establish a function for autophagy in Aß metabolism: autophagy influences secretion of Aß to the extracellular space and thereby directly affects Aß plaque formation, a pathological hallmark of AD.

Global brain delivery of neprilysin gene by intravascular administration of AAV vector in mice.

Accumulation of amyloid-ß peptide (Aß) in the brain is closely associated with cognitive decline in Alzheimer's disease (AD). Stereotaxic infusion of neprilysin-encoding viral vectors into the hippocampus has been shown to decrease Aß in AD-model mice, but more efficient and global delivery is necessary to treat the broadly distributed burden in AD. Here we developed an adeno-associated virus (AAV) vector capable of providing neuronal gene expression throughout the brains after peripheral administration. A single intracardiac administration of the vector carrying neprilysin gene in AD-model mice elevated neprilysin activity broadly in the brain, and reduced Aß oligomers, with concurrent alleviation of abnormal learning and memory function and improvement of amyloid burden. The exogenous neprilysin was localized mainly in endosomes, thereby effectively excluding Aß oligomers from the brain. AAV vector-mediated gene transfer may provide a therapeutic strategy for neurodegenerative diseases, where global transduction of a therapeutic gene into the brain is necessary.