• Mon. Nov 28th, 2022

Furthermore, Nicoll et al


Jun 20, 2022

Furthermore, Nicoll et al. percentages in the hippocampus are shown as a bar graph (means S.E.M.). NIHMS436659-supplement-Supp_Fig_2.pdf (265K) GUID:?C0723EF9-9DFB-4096-ADF5-BA76BB8F19B2 Supp Fig 3: Supplementary Physique 3. Reactive astrocytes visualized by GFAP antibody in the hippocampus (aCd) Brain sections from mice injected with PBS (a), rAAV5-CAscFv-Gag (b), and rAAV5-CAscFv59 (c) were stained with anti-GFAP antibody and quantified by morphometric analysis (d). Scale bars are 200 m (aCc). (d) GFAP-immunoreactive area percentages in the hippocampus are shown as a bar graph (means S.E.M.). NIHMS436659-supplement-Supp_Fig_3.pdf (3.3M) GUID:?B1E8D396-3949-4F2C-8253-0102FFA785A1 Abstract Accumulation of amyloid -protein (A) in the brain is thought to be a causal event in Alzheimers disease (AD). Immunotherapy targeting A holds great promise for reducing A in the brain. Here, we evaluated the efficacy and safety of anti-A single-chain antibody (scFv59) delivery via recombinant adeno-associated virus (rAAV) on reducing A deposits in an AD mouse model (TgAPPswe/PS1dE9). First, delivery of scFv59 to the brain was optimized by injecting rAAV serotypes 1, 2, and 5 into the right lateral ventricle. Symmetrical high expression of scFv59 was found throughout the hippocampus and partly in the neocortex in both hemispheres via rAAV1 or rAAV5 while scFv59 expression via rAAV2 was mostly limited to one hemisphere. rAAV1, however, induced apoptosis and microglial activation but rAAV5 did not. Therefore, rAAV5 was selected for therapeutic scFv59 delivery in TgAPPswe/PS1dE9 mice. Radequinil rAAV5 was similarly injected into the ventricle of 10-month-old TgAPPswe/PS1dE9 mice and 5 months later its efficacy and safety were evaluated. Immunoreactive A deposits reduced in the hippocampus. A42 levels in cerebrospinal fluid (CSF) tended to increase and the A40:42 ratio decreased in CSF, suggesting that A42 was relocated from the parenchyma to CSF. Hemorrhages associated with a focal increase in blood vessel amyloid were found in the brain. While immunotherapy has great potential for clearing cerebral A, caution for cerebrovascular effects should be exercised when rAAV-mediated anti-A immunotherapy is usually applied. gene and one of the serotype specific genes [15]. Produced viral particles were released from the cells by rapid freeze and thaw and purified by iodixanol gradient centrifugation. The iodixanol gradient fraction was further purified by HPLC using a 5-ml HiTrap Q column (GE Healthcare, Piscataway, NJ) as described before [16]. A control rAAV5 Radequinil encoding scFv-Gag was similarly prepared. The titers of rAAV virions that contained the vector genomes were Rabbit polyclonal to CDH2.Cadherins comprise a family of Ca2+-dependent adhesion molecules that function to mediatecell-cell binding critical to the maintenance of tissue structure and morphogenesis. The classicalcadherins, E-, N- and P-cadherin, consist of large extracellular domains characterized by a series offive homologous NH2 terminal repeats. The most distal of these cadherins is thought to beresponsible for binding specificity, transmembrane domains and carboxy-terminal intracellulardomains. The relatively short intracellular domains interact with a variety of cytoplasmic proteins,such as b-catenin, to regulate cadherin function. Members of this family of adhesion proteinsinclude rat cadherin K (and its human homolog, cadherin-6), R-cadherin, B-cadherin, E/P cadherinand cadherin-5 determined by the quantitative dot-blot assay as described previously [14]. Experimental animals and Radequinil stereotaxic injection of rAAV-scFvs C57BL/6 mice (6C8 weeks old) purchased from Jackson Laboratory (Bar Harbor, ME) were used to optimize intracranial delivery of scFv59 by testing 3 differently pseudotyped rAAVs. Mice were randomly assigned to 4 treatment groups in such a manner as there was no significant intergroup difference in body weight: PBS, rAAV1-CAscFv59, rAAV2-CAscFv59 and rAAV5-CAscFv59 group (n = 10 for Radequinil each group). Mice were anesthetized by pentobarbital and placed on a stereotaxic instrument with a motorized stereotaxic injector (Stoelting, Wood Dale, IL). A midline incision was made to expose the bregma. A hole in the skull was made by a drill 0.5 mm posterior to the bregma and 1.0 mm right to the midline. rAAV [2.5 1010 vector genomes (vg) in 10 l PBS/mouse] was injected unilaterally into the Radequinil right ventricle at the depth of 2 mm at a rate of 1 1 l/min. After allowing the needle to remain in place for 5 min, the needle was slowly raised at a rate of 0.1 cm/min. Control mice received the same amount of PBS. Three months after the rAAV injection, the experimental mice were terminated by a lethal injection of sodium pentobarbital to determine expression levels of scFv59 in the brain. An AD mouse model, B6.Cg-Tg(APPswe, PSEN1dE9) 85Dbo/J mice (TgAPPswe/PS1dE9 mice) [17] purchased from Jackson Laboratory, was used to study the therapeutic effects of intracerebroventricular injection of the optimized rAAV-CAscFv59. Ten-month-old TgAPPswe/PS1dE9 mice (n = 10, 8.