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.

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.

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Cerebrospinal fluid (CSF) biomarkers play an important role in diagnosing Alzheimer’s disease (AD) which is characterized by amyloid-β (Aβ) amyloidosis. Here, we used two App knock-in mouse models, AppNL-F/NL-F and AppNL-G-F/NL-G-F, exhibiting AD-like Aβ 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-Aβ42 but normal cognition. Notably, in this group of subjects, CSF-decorin levels positively correlated with CSF-Aβ42 levels indicating that the change in CSF-decorin is associated with early Aβ 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 Aβ plaque load and with decorin levels in choroid plexus. In addition, a low concentration of human Aβ42 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 Aβ amyloidosis in the brain may reflect pathological changes in choroid plexus, present in a subtype of AD subjects.

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 ( App NL-G-F and App NL-F mice). We have now generated App knock-in mice devoid of the Swedish mutations ( App G-F mice) and evaluated its characteristics. Amyloid β peptide (Aβ) pathology was exhibited by App G-F mice from 6 to 8 months of age and was accompanied by neuroinflammation. Aβ-secretase inhibitor, verubecestat, attenuated Aβ production in App G-F mice, but not in App NL-G-F mice, indicating that the App G-F mice are more suitable for preclinical studies of β-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 β (CTF-β) and humanization of Aβ 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.

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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). These models showed the development of amyloid We previously developed single App knock-in mouse models of 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 some additional mutants to address the following two questions: [1] Do the Swedish mutations influence the mode of beta-secretase inhibitor action in vivo? [2] Does the quantity of C-terminal fragment of amyloid precursor protein (APP) generated by beta-secretase (CTF-beta) affect endosomal properties as previously reported as well as other pathological events? Abeta pathology was exhibited by AppG-F mice from 6 to 8 months of age, and was accompanied by microglial and astrocyte activation. We found that a beta-secretase inhibitor, verubecestat, inhibited Abeta 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 beta-secretase inhibition given that most AD patients do not carry Swedish mutations. We also found that the quantity of CTF-beta generated by various App knock-in mutants failed to correlate with endosomal alterations or enlargement, implying that CTF-beta, endosomal abnormalities, or both are unlikely to play a major role in AD pathogenesis. This is the first AD mouse model ever described that recapitulates amyloid pathology in the brain without the presence of Swedish mutations and without relying on the overexpression paradigm. Thus, experimental comparisons between different App knock-in mouse lines will potentially provide new insights into our understanding of the etiology of AD.

The American Society for Microbiology and Molecular and Cellular Biology (MCB) are issuing this Expression of Concern to alert readers to concerns with the data presented in this article. Problems that have been noted are as follows: (i) duplication of Fig. 7 as Fig. 5, (ii) presentation of Fig. 5 as Fig. 4, (iii) omission of Fig. 4, and (iv) missing labels in Fig. 3 and 5. The files uploaded during the final submission of this paper to MCB by the authors represent the source of these figure problems. These problems were brought to the authors' attention; however, a correction to resolve them has not yet been submitted. An update will be published when this matter has been resolved.