Dharma Rao Polisetti

Induction of Heme Oxygenase I (HMOX1) by HPP-4382: A Novel Modulator of Bach1 Activity

Oxidative stress is generated by reactive oxygen species (ROS) produced in response to metabolic activity and environmental factors. Increased oxidative stress is associated with the pathophysiology of a broad spectrum of inflammatory diseases. Cellular response to excess ROS involves the induction of antioxidant response element (ARE) genes under control of the transcriptional activator Nrf2 and the transcriptional repressor Bach1. The development of synthetic small molecules that activate the protective anti-oxidant response network is of major therapeutic interest. Traditional small molecules targeting ARE-regulated gene activation (e.g., bardoxolone, dimethyl fumarate) function by alkylating numerous proteins including Keap1, the controlling protein of Nrf2. An alternative is to target the repressor Bach1. Bach1 has an endogenous ligand, heme, that inhibits Bach1 binding to ARE, thus allowing Nrf2-mediated gene expression including that of heme-oxygenase-1 (HMOX1), a well described target of Bach1 repression. In this report, normal human lung fibroblasts were used to screen a collection of synthetic small molecules for their ability to induce HMOX1. A class of HMOX1-inducing compounds, represented by HPP-4382, was discovered. These compounds are not reactive electrophiles, are not suppressed by N-acetyl cysteine, and do not perturb either ROS or cellular glutathione. Using RNAi, we further demonstrate that HPP-4382 induces HMOX1 in an Nrf2-dependent manner. Chromatin immunoprecipitation verified that HPP-4382 treatment of NHLF cells reciprocally coordinated a decrease in binding of Bach1 and an increase of Nrf2 binding to the HMOX1 E2 enhancer. Finally we show that HPP-4382 can inhibit Bach1 activity in a reporter assay that measures transcription driven by the human HMOX1 E2 enhancer. Our results suggest that HPP-4382 is a novel activator of the antioxidant response through the modulation of Bach1 binding to the ARE binding site of target genes.

EFFICACY OF RAGE ANTAGONISTS IN MURINE MODEL OF ALZHEIMER'S DISEASE

Background: The autophagic ubiquitin adaptor p62 mediates clearance of ubiquitinated cellular components through chaperone-assisted selective autophagy and package of ubiquitinated proteins into inclusion bodies as it self-assembles.As hyperphosphorylated tau in the brains of Alzheimer’s disease and related tauopathies is ubiquitinated, p62 is presumed to be involved in the degradation and sequestration of this pathological form of tau. Here, weprovide the first evidence that p62 acts protectively against neuronal death provoked by abnormal tau accumulation.Methods: In the P301Smutant human tau transgenic mouse (PS19), abundant accumulation of fibrillar tau inclusions resembling neurofibrillary tangles (NFTs) in human tauopathies are concurrent with gradual loss of neurons in the brainstem, while massive neuronal death prior to the formation of NFTs rapidly occurs in the hippocampus. We crossbred p62-knockout mice with PS19 mice to investigate roles of p62 in processing of abnormal tau.Results:Deficiency in p62 alone did not induce overt neuropathologies, but accelerated neuronal loss in the hippocampus of P19 mice. Putatively neurotoxic tau oligomers were markedly increased in p62-deficient PS19 mice as assayed by immunoblotting and immunohistochemistry. Formation of granular inclusions packaging p62 and organelles observed in the PS19 hippocampus was nearly completely abolished by p62 deficiency. Sequestration of phosphorylated tau into neuronal somas was characteristic of the PS19 brainstem, and was in sharp contrast with diffuse somatodendritic and synaptic localization of phospho-tau in the hippocampus. Notably, this compartmentalization of phospho-tau in the brainstem was disrupted in p62-deficient PS19 mice, leading to neurodegeneration accompanied by inflammatory gliosis. Formation of NFT-like inclusions in the PS19 brainstem was partially suppressed by p62 deficiency. Finally, biochemical data indicated that insufficient reserve of authophagic capacity in the PS19 hippocampus may account for profound tau-induced neuronal loss in the hippocampus versus slow progression of neurodegeneration in the brainstem. Conclusions: Our findings have demonstrated that p62 protects neurons against tau-induced neurotoxicities by 1) eliminating phosphorylated tau oligomers primarily through selective autophagy; 2) sequestering abnormal tau species into neuronal somas; and 3) packaging damaged organelles tagged with ubiquitins.