Targeting Kremen1 Downregulation with RVG-9R/siRNA Complexes in the Triple-Transgenic Mouse Model of Alzheimer's Disease

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)

Kelly E. Baker (Creator)

Institution

East Carolina University (ECU )

Web Site: http://www.ecu.edu/lib/

Abstract: Alzheimer's disease (AD) is a progressive disease characterized by cognitive decline and memory loss. Memory loss observed in AD results from the loss of neurons and synapses which may be caused by the disruption of the canonical Wnt signaling pathway by Dickkopf-1 (Dkk-1). Under normal conditions , the canonical Wnt signaling pathway is responsible for normal neuronal development , synaptic plasticity , and overall normal brain function. Amyloid-[beta] (A[beta]) plaques and neurofibrillary tangles are two characteristic morphological changes observed in AD. An increased level of A[beta] has been associated with increased expression of Dkk-1 , which may be linked to synaptic loss seen in AD. Kremen1 (Krm1) is a receptor for Dkk-1. Published and unpublished observations from our laboratory showed that silencing Krm1 with miR-431 can promote regenerative axon growth and prevent synaptic loss in a cell culture model. This study focused on downregulating Krm1 the triple-transgenic mouse model of AD (3xTg-AD). It was hypothesized that application of siRNA-431 in vivo would downregulate Krm1 thereby preventing synaptic loss and memory deficits in the 3xTg-AD mouse model of AD. Tail vein injections of RVG-9R/siRNA complexes and control injections were administered to 3xTg-AD mice and wild-type (WT) mice at 4 , 6 , or 12 months of age. Within each age cohort there were three different groups: 3xTg-AD mice injected with RVG/siRNA , 3xTg-AD mice injected with control peptide/siRNA , and WT mice injected with saline. Each group of mice was approximately half male and half female. Following the injections , the Barnes Maze was administered to each mouse in order to assess memory function. Data gathered from the Barnes Maze shows 3xTg-AD mice have a longer primary latency in the probe phase compared to WT mice. Of mice tested , fewer 3xTg-AD mice have been successful in finding the target hole during probe phase compared to WT mice. After completion of the Barnes Maze , mice were sacrificed and brains were collected for analysis. The brains were analyzed for Krm1 downregulation at the protein and mRNA levels via Western blot and qPCR , respectively. In 4 month old mice , WT mice showed the lowest levels of Krm1 protein and mRNA expression levels and 3xTg-AD CPep/siRNA treated mice showed the highest. The 4 month old 3xTg-AD RVG-9R/siRNA treated mice had Krm1 protein and mRNA expression levels that fell between the other two groups. Immunofluorescence was performed on coronal brain sections to analyze number of synapses. Six month old 3xTg-AD CPep/siRNA treated mice had significantly fewer synapses than both the WT and 3xTg-AD RVG-9R/siRNA treated groups. In conclusion , IF , qPCR , and Western blot data reveal the potential for RVG-9R/siRNA treatment to target and downregulate Kremen1 in vivo and provide protection from synaptic loss. However , further studies are need to confirm the ability of RVG-9R/siRNA treatment to downregulate Kremen1.

Additional Information

Publication

Thesis

Language: English

Date: 2017

Keywords

Kremen1, Wnt Signaling, 3xTg-AD Mouse Model

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