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.

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.

Vital role of the calpain-calpastatin system for placental-integrity-dependent embryonic survival.

Although the calpain-calpastatin system has been implicated in a number of pathological conditions, its normal physiological role remains largely unknown. To investigate the functions of this system, we generated conventional and conditional calpain-2 knockout mice. The conventional calpain-2 knockout embryos died around embryonic day 15, preceded by cell death associated with caspase activation and DNA fragmentation in placental trophoblasts. In contrast, conditional knockout mice in which calpain-2 is expressed in the placenta but not in the fetus were spared. These results suggest that calpain-2 contributes to trophoblast survival via suppression of caspase activation. Double-knockout mice also deficient in calpain-1 and calpastatin resulted in accelerated and rescued embryonic lethality, respectively, suggesting that calpain-1 and -2 at least in part share similar in vivo functions under the control of calpastatin. Triple-knockout mice exhibited early embryonic lethality, a finding consistent with the notion that this protease system is vital for embryonic survival.