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Myeloperoxidase (MPO) is a potent enzyme expressed mostly by neutrophils, that catalyzes the formation of reactive oxygen species (ROS), mainly hypochlorous acid. MPO is a primary mediator of neutrophils oxidative stress response and was shown to be essential for the formation of neutrophils extracellular traps (NETs). Upon secretion, MPO was also shown to attach to blood endothelial cells and promote their degradation, making it a therapeutic target in multiple clinical incidences, including neurodegenerative disorders. Here, we aimed to assess the role of neutrophil-derived MPO in 5XFAD model of AD. For that purpose, we subjected 5XFAD mice to myeloablative irradiation and bone marrow-repopulation from MPO Knock-out (KO) mice to form 'MPO KO-5XFAD' mice. MPO KO-5XFAD mice exhibited improvement in their risk assessment behavior, as measured by the 'Elevated plus maze/ test and the 'Open field' test, when compared to 5XFAD mice transplanted with WT cells (termed 'WT-5XFAD'). Remarkably, MPO KO-5XFAD mice also showed improved spatial recognition and learning, as was evident in the 'Y-maze' test, the 'Morris water maze' test and by 'Contextual fear conditioning' test. Hippocampal RNA expression analysis by QPCR indicated decreased expression of proinflammatory cytokines in MPO KO-5XFAD mice, along with decreased blood brain barrier damage and reduced expression of murine APOE. Together, these results indicate a substantial involvement of blood-derived oxidative damage in mouse model of AD, and assign MPO as a potential therapeutic target in AD. [ABSTRACT FROM AUTHOR]

The apolipoprotein E4 (ApoE4) is the most prevalent genetic risk factor for sporadic Alzheimer's disease (AD). More than 50% of AD patients express the ApoE4 protein; which increases the risk of prevalence for AD since it lowers the age of onset by as much as 10-20 years. Therefore, eliminating the expression of ApoE4 presents an attractive therapeutic strategy. Methods:The two alleles of ApoE (E3 and E4) differ in one nucleotide. Based on this single-nucleotide polymorphism (SNP), we designed a novel "clustered regularly interspaced short palindromic repeats" (CRISPR) based system that specifically recognizes, binds and targets the ApoE4 allele for knockout by using the VRER CRISPR associated protein 9 (Cas9) variant. Application of this approach in mice astrocytes expressing the human ApoE3 or ApoE4 resulted in a significant decrease of ApoE4 protein levels (-70%) without any significant changes in ApoE3 levels. The cell-culture experiments provide a proof of concept that indeed our CRISPR based system can be used to specifically knockout ApoE4 without affecting ApoE3. These experiments are currently being extended to the in-vivo level. We hope that this study will lead to development of novel gene-therapy applications for AD patients, carriers of the ApoE4 allele in general and ApoE3/4 heterozygous patients in particular. [ABSTRACT FROM AUTHOR]

The presence of the ApoE4 allele has been associated to a greater risk of Alzheimer's disease (AD). Previous studies revealed that Aβ uptake in mouse astrocytes expressing the human ApoE4 is reduced and this effect may be driven by impaired endocytosis/autophagy. One of the factors that was shown to be associated with endocytosis/autophagy is calpain, a Ca2+-dependent cysteine protease which was also shown to be overactivated in the Alzheimer's disease (AD) human brain. The aim of this study was to test the association between the ApoE genotype, calpain expression and autophagy in astrocytes. We used the CRISPR-Cas9 system to knock down the small subunit of the calpain protein in mice astrocytes expressing either the human ApoE3 or ApoE4 alleles. We then investigated, the resulting effects of the Calpain knock down on the expression of autophagy-related proteins and on the uptake of fluorescent Aβ. We found that the levels of mitofusin1, a protein responsible for the fusion and integrity of the mitochondria and p62 a protein that undergoes degradation during autophagy, are higher in the ApoE4 compared to ApoE3. However, in astrocytes treated with CRISPR-Cas9, which reduced the levels calpain by 75%, the levels of both mitofusin1 and p62 were significantly reduced in the ApoE4 astrocytes and were similar to those of ApoE3 astrocytes. No change in these protein levels was found when ApoE3 astrocytes were treated with the CRISPR-Cas9. These results suggest higher autophagy and mitochondrial fusion in the CRISPR-Cas9 treated ApoE4 cells. Moreover, the Calpain directed CRISPR-Cas9 treatment increased the levels of Aβ uptake in both, ApoE4 and ApoE3 astrocytes. Improving the astrocyte capacity to clear the accumulated Aβ by knocking down Calpain, might be an interesting and new approach for a possible treatment of some aspects of the AD pathology. [ABSTRACT FROM AUTHOR]