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  Glial and vascular contributions to neurodegenerative diseases
    PP30
Mechanistic investigation of immune regulatory roles of Kv1.3 channels in microglia Christine Bowen1, Young Lin1, Hai Nguyen2, Tia Dowling1, Pritha Bagchi Bagchi1, Duc Duong1, Prateek Kumar1, Hailian
Xiao1, Heike Wulff1, Nicholas Seyfried1, Srikant Rangaraju1
1Emory University, Atlanta, USA, 2University of California Davis, Davis, USA
 Background: A subset of pro-inflammatory microglia in Alzheimer’s Disease (AD) upregulate Kv1.3 potassium channels[1]. Pharmacological blockade of Kv1.3 reduces Aβ pathology and dampens pro-inflammatory function of microglia[2]. To identify Kv1.3-regulated neuroinflammatory mechanisms and pathways in microglia, we applied proximity-labeling to examine the protein-protein interactome of Kv1.3 channels in vitro, and conditional microglia-specific Kv1.3 deletion in vivo.
Methods: TurboID, a biotin ligase that biotinylates proteins within 10nm proximity, was fused to N-term or C-term of Kv1.3 and stably-transduced in BV2 microglia. Kv1.3 expression and function were confirmed via qPCR, electrophysiology, western blot and flow cytometry. Mass spectrometry (MS) of biotinylated proteins from BV2-Kv1.3-TurboID lines, under resting and lipopolysaccharide(LPS)-treated conditions, was performed to identify N and C-term interactomes. Kcna3(Kv1.3)- floxed mice were generated, validated using CMV-Cre mice, and crossed to Tmem119-Cre-Ert2 mice for microglia-specific Kv1.3 deletion. Results: Proximity-based proteomics of BV2-Kv1.3-TurboID microglia identified distinct N-term (n=818) and C-term Kv1.3 (n=706) interactors. Kv1.3 N-term interacts with translation (Rpl10, Eef1a1), plasma membrane proteins (e.g. Calr1, Psma1) and mitochondrial trafficking proteins (Timm23), while the C-term interacts with immune response proteins (Cd68, Tlr2, Csf1), some of which are modified by LPS- stimulation (C3) (n=15), and dependent on a PDZ-binding C-term motif (n=62). We confirmed loss of Kv1.3 mRNA and channels in brain and spleen after crossing Kcna3-floxed mice with CMV-Cre mice. Tamoxifen treatment of Tmem119-cre/Kcna3-floxed mice reduced microglial Kv1.3 expression by 50% without impacting peripheral tissues.
Discussion and Conclusions: We identified novel N and C-term domain- specific as well as context-dependent interactors of Kv1.3 channels in microglia. While the N-terminus regulates protein processing, transport and localization, the C-terminus regulates immune signaling which is influenced by LPS-stimulation. Ongoing studies will verify the functional relevance of these candidate interactors. We also generated a novel conditional Kv1.3 deletion model to investigate the roles of microglial Kv1.3 channels in neuroinflammatory and neurodegenerative diseases.
Background: Alzheimer’s disease (AD) often coexists with other aging- associated diseases including diabetes and cardiovascular diseases. The precondition for these diseases is metabolic syndrome (MetS). An important cause of MetS is the deficiency of SIRT3, a mitochondrial NAD+ dependent deacetylase that regulates metabolism. We have demonstrated that Sirt3 gene deletion leads to decreased mitochondrial function and inflammasome formation leading to neuroinflammation [1]. Later, we reported exacerbation of β amyloid plaque deposition, and microglial activation in APP/PS1/Sirt3-/- mice, a comorbid AD model [2]. When cocultures of Sirt3-silenced mouse brain microvascular and microglial cell lines were exposed to palmitic acid, significant cytotoxicity was observed [3]. Therefore, the objective of this study is to determine how amyloid pathology and MetS interact.
Materials and Methods: RNA-seq analysis of the brain samples of APP/PS1/Sirt3-/- mice were performed. Cultured BV2 cells, a mouse microglial cell line, were exposed to SIRT3 activators. Immunoblotting and ELISA were used to analyze proteins.
Results: RNA-seq analysis revealed upregulation of inflammatory genes. A key finding was the decreased expression of insulin degrading enzyme (IDE), an Aβ peptide degrading enzyme, following Sirt3 gene deletion. Activation of Sirt3 by nicotinamide riboside in vivo and in vitro resulted in IDE upregulation. Honokiol is another activator of SIRT3. We tested its novel derivatives, honokiol DCA (DCA) and honokiol hexa (Hexa) in BV2 cells. SIRT3 and IDE levels increased in BV2 cells exposed to DCA and Hexa. Hexa showed maximum decrease in lysine acetylation of mitochondrial proteins, a marker for their activation. Hexa also increased the levels of TFAM and mitofusin2, key mitochondrial proteins. Honokiol derivatives improved mitochondrial respiration, especially in state IV.
Discussion and Conclusion: Decreased IDE expression provides a molecular link between MetS and AD. SIRT3 deficiency in MetS can exacerbate AD pathogenesis through microglial dysregulation. SIRT3 is a potential therapeutic target to treat AD.
Background: Microglia are an important regulator of inflammation and neurodegeneration. Microglia take up aggregated proteins, including tau, though this ability is reduced in advanced stages of neurodegenerative disease. Recent work indicates microglial inflammation correlates with tau spread in human patients with Alzheimer’s Disease. Thus, it is important to understand the impact of microglial inflammatory states on their ability to take up and spread tau. Methods: We are investigating how inflammatory state of microglia alters their ability to take up and spread tau to neurons and astrocytes using human induced pluripotent stem cell (hiPSC) models. We are also examining whether tau spread can be blocked using novel bifunctional intrabody technology. These intrabodies specifically bind and degrade tau inside of cells and are delivered via lentiviral or AAV vectors. The intrabodies have a recombinant antibody domain that specifically binds tau and contain a PEST degron, which targets the antibody and its bound cargo for degradation via the proteasome. Excitingly, intrabody treatment of neurons effectively lowers tau and improves proteasome function. Our approach is particularly relevant to microglia that express little MAPT RNA.
Results: Preliminary results show our hiPSC-microglia take up tau, which changes their inflammatory profile distinguishable from LPS treatment. In the future we will: 1) examine whether microglia can transfer tau to neurons or astrocytes, 2) assess whether intrabodies can degrade microglial tau, and whether this restores their ability to clear aggregated proteins and 3) compare tau uptake in inflammation- primed microglia to evaluate if this alters their ability to accumulate or release tau.
Conclusions: Our studies will reveal mechanisms of tau spread via microglia, as well as if degradation of accumulated tau can restore hiPSC microglia function. This will provide novel insights on the mechanisms of disease progression and determine the potential for intrabody- mediated degradation of tau as a therapeutic target.
  PP32
Examination of Microglial Tau Spread and Intrabody Treatment using Human Induced- Pluripotent Stem Cells
Elizabeth Fisher1, Lindsay Tomaszeck1, Lianna D'Brant1,
Natasha Rugenstein1, David Butler1, Sally Temple1
1Neural Stem Cell Institute, Rensselaer, USA
   PP31
SIRT3 activators meliorate microglial dysregulation in neurodegeneration Alpna Tyagi1,2, Jack Arbiser3, Subbiah Pugazhenthi1,2
1University of Colorado-Anschutz Medical Campus, Aurora, USA, 2 Rocky Mountain Regional VA Medical Center, Aurora, USA, 3Emory University School of Medicine, Atlanta, USA
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