The NRF transcription factors NRF1, NRF2, and NRF3, are a subset of Cap‘n’collar transcriptional regulators which modulate the expression of genes harboring antioxidant-response element (ARE) sequences within their genomic loci. Despite the emerging physiological importance of NRF family members, the repertoire of their genetic targets remains incompletely defined. Here we use RNA-sequencing-based transcriptional profiling and quantitative proteomics to delineate the overlapping and differential genetic programs effected by the three NRF transcription factors. We then create consensus target gene sets regulated by NRF1, NRF2, and NRF3 and define the integrity of these gene sets for probing NRF activity in mammalian cell culture and human tissues. Together, our data provide a quantitative assessment of how NRF family members sculpt proteomes and transcriptomes, providing a framework to understand the critical physiological importance of NRF transcription factors and to establish pharmacologic approaches for therapeutically activating these transcriptional programs in disease.

The mitochondrial DNA of trypanosomes, called kinetoplast DNA (kDNA) contains thousands of minicircles that are topologically linked into a single structure that resembles a medieval chainmail. This biological chainmail is characterized by two parameters: the link type between minicircles, and the number of minicircles linked to each minicircle (i.e. the minicircle valence). In previous works, a protocol was proposed to determine the mean value of the minicircle valence. In these experiments, minicircles were excised from the network and the products compared with those obtained from fragmenting idealized structures. These idealized structures assumed a negligible variance in the distribution of valences of the initial network. It is therefore unclear to what extent this theoretical analysis captures the true topology of the kDNA network when kDNA samples are extracted from unsynchronized cells or from cells with silenced kDNA replication genes. Subsequent studies proposed that there is a critical percolation density during network formation. We asked whether this density can be estimated using fragmentation reactions. The goal of this work is to develop a mathematical method that can be used to estimate the mean valence of networks when the variance of the valence is non-negligible. We first show microscopy data on Crithidia fasciculata that, in agreement with the original experimental results, show a distribution of valences with nonzero variance. Second, we use computer simulations of network fragmentation to show that the predicted and actual mean valence are different when the valence distribution has nonzero variance. We propose a more general mathematical formulation and computer simulations of kDNA fragmentation to estimate this value. Last, we show that fragmentation experiments may lead to errors in the estimation of the critical percolation density since the collapsing density depends on the initial density of the network, and on the fragmentation reaction.

Summary: Autophagy-lysosomal impairment is an early and prominent feature of neurodegeneration. Autophagy activation reduces protein aggregates and lipid level abnormalities. We performed a high-content imaging-based screen assessing 940,000 small molecules to identify those that reduce lipid droplet numbers. Of 77 validated, structurally diverse hits, 24 increased autophagy flux reporter activity, consistent with accelerated lipid droplet clearance by lipophagy. Of these, we show that CCT020312 activates autophagy independently of mammalian target of rapamycin (mTOR) inhibition, to avoid immunosuppression. CCT020312 reduced insoluble phosphorylated tau levels and tau-mediated neuronal stress vulnerability, as well as reducing intracellular Aβ levels within directly induced neurons bearing epigenetic marks of aging derived from Alzheimer’s patient fibroblasts. Moreover, CCT020312 cleared mutant prion protein aggregates and normalized trafficking deficiencies in axons of a cellular model of familial prion disease. Autophagy is widely considered a promising strategy to attenuate neurodegeneration, and here we introduce a strategy to discover new pharmacology.