N6-Furfuryladenine is protective in Huntington’s disease models by signaling huntingtin phosphorylation

© 2018 National Academy of Sciences. All Rights Reserved. The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington’s disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA ad-ducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems

Specificity in PI3K-PKB/AKT-PTEN Signaling: Subcellular Locus-specific Functions of Pathway Targets

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.Ph

ROS-specific Huntingtin Interactions: G418 kill curve in TruHD fibroblasts

Optimization step in the development of an inducible system expressing YFP-tagged huntingtin-specific intrabodies for stable transfection in TruHD fibroblasts

ROS-specific Huntingtin Interactions: Inducible huntingtin-specific chromobody expression by nucleofection

Optimization step in the development of an inducible system expressing YFP-tagged huntingtin-specific intrabodies for stable transfection in TruHD fibroblasts

ROS-specific Huntingtin Interactions: GFP reactivation assay optimization in HEK 293 cells

Optimization step in the lead up to phenotypic analysis of ROS-dependent huntingtin interacting proteins

ROS-specific Huntingtin Interactions: GFP reactivation assay WT vs HD mouse striatal cells

Optimization step in the lead up to phenotypic analysis of ROS-dependent huntingtin interacting proteins

ROS-specific Huntingtin Interactions: Fractionation Optimization

Optimization step in the lead up to mass spec identification of ROS-specific huntingtin protein-protein interactions

ROS-specific Huntingtin Interactions: Comparing transfection methods for inducible expression of huntingtin-specific chromobody

Optimization step in the development of an inducible system expressing YFP-tagged huntingtin-specific intrabodies for stable transfection in TruHD fibroblasts

Legislative Documents

Also, variously referred to as: Senate bills; Senate documents; Senate legislative documents; legislative documents; and General Court documents

ROS-specific Huntingtin Interactions: GFP reactivation assay WT vs HD mouse striatal cells second attempt

<p>Optimization step in the lead up to phenotypic analysis of ROS-dependent huntingtin interacting proteins.</p