Small Molecules Efficiently Reprogram Human Astroglial Cells into Functional Neurons.

We have recently demonstrated that reactive glial cells can be directly reprogrammed into functional neurons by a single neural transcription factor, NeuroD1. Here we report that a combination of small molecules can also reprogram human astrocytes in culture into fully functional neurons. We demonstrate that sequential exposure of human astrocytes to a cocktail of nine small molecules that inhibit glial but activate neuronal signaling pathways can successfully reprogram astrocytes into neurons in 8-10 days. This chemical reprogramming is mediated through epigenetic regulation and involves transcriptional activation of NEUROD1 and NEUROGENIN2. The human astrocyte-converted neurons can survive for >5 months in culture and form functional synaptic networks with synchronous burst activities. The chemically reprogrammed human neurons can also survive for >1 month in the mouse brain in vivo and integrate into local circuits. Our study opens a new avenue using chemical compounds to reprogram reactive glial cells into functional neurons.

ATF4 Gene Network Mediates Cellular Response to the Anticancer PAD Inhibitor YW3-56 in Triple-Negative Breast Cancer Cells.

We previously reported that a pan-PAD inhibitor, YW3-56, activates p53 target genes to inhibit cancer growth. However, the p53-independent anticancer activity and molecular mechanisms of YW3-56 remain largely elusive. Here, gene expression analyses found that ATF4 target genes involved in endoplasmic reticulum (ER) stress response were activated by YW3-56. Depletion of ATF4 greatly attenuated YW3-56-mediated activation of the mTORC1 regulatory genes SESN2 and DDIT4. Using the ChIP-exo method, high-resolution genomic binding sites of ATF4 and CEBPB responsive to YW3-56 treatment were generated. In human breast cancer cells, YW3-56-mediated cell death features mitochondria depletion and autophagy perturbation. Moreover, YW3-56 treatment effectively inhibits the growth of triple-negative breast cancer xenograft tumors in nude mice. Taken together, we unveiled the anticancer mechanisms and therapeutic potentials of the pan-PAD inhibitor YW3-56.

A comprehensive and high-resolution genome-wide response of p53 to stress.

Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.

Folded Conformation, Cyclic Pentamer, Nano-Structure and PAD4 Binding Mode of YW3-56.

The physical and chemical mechanisms of small molecules with pharmacological activity forming nano-structures are developing into a new field of nano-medicine. By using ROESY 2D NMR spectroscopy, trandem mass spectroscopy, transmission electron microscopy and computer-assisted molecular modeling, this paper demonstrated the contribution of the folded conformation, the intra- and intermolecular π-π stacking, the intra- and intermolecular hydrogen bonds, and the receptor binding free energy of 6-dimethylaminonaph-2-yl-{N-S-[1-benzylcarba-moyl-4-(2-chloroacetamidobutyl)]-carboxamide (YW3-56) to the rapid formation of nano-rings and the slow formation of nano-capsules. Thus we have developed a strategy that makes it possible to elucidate the physical and chemical mechanisms of bioactive small molecules forming nano-structures.

PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures.

NETosis, the process wherein neutrophils release highly decondensed chromatin called neutrophil extracellular traps (NETs), has gained much attention as an alternative means of killing bacteria. In vivo, NETs are induced by bacteria and pro-inflammatory cytokines. We have reported that peptidylarginine deiminase 4 (PAD4), an enzyme that converts Arg or monomethyl-Arg to citrulline in histones, is essential for NET formation. The areas of extensive chromatin decondensation along the NETs were rich in histone citrullination. Here, upon investigating the effect of global citrullination in cultured cells, we discovered that PAD4 overexpression in osteosarcoma U2OS cells induces extensive chromatin decondensation independent of apoptosis. The highly decondensed chromatin is released to the extracellular space and stained strongly by a histone citrulline-specific antibody. The structure of the decondensed chromatin is reminiscent of NETs but is unique in that it occurs without stimulation of cells with pro-inflammatory cytokines and bacteria. Furthermore, histone citrullination during chromatin decondensation can dissociate heterochromatin protein 1 beta (HP1ß) thereby offering a new molecular mechanism for understanding how citrullination regulates chromatin function. Taken together, our study suggests that PAD4 mediated citrullination induces chromatin decondensation, implicating its essential role in NET formation under physiological conditions in neutrophils.

Anticancer peptidylarginine deiminase (PAD) inhibitors regulate the autophagy flux and the mammalian target of rapamycin complex 1 activity.

Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.

Cofilin aggregation blocks intracellular trafficking and induces synaptic loss in hippocampal neurons.

Cofilin is an actin-binding protein and a major actin depolymerization factor in the central nervous system (CNS). Cofilin-actin aggregates are associated with neurodegenerative disorders, but how cofilin-actin aggregation induces pathological effects in the CNS remains unclear. Here, we demonstrated that cofilin rods disrupted dendritic microtubule integrity in rat hippocampal cultures. Long term time-lapse imaging revealed that cofilin rods block intracellular trafficking of both mitochondria and early endosomes. Importantly, cofilin rod formation induced a significant loss of SV2 and PSD-95 puncta as well as dendritic spines. Cofilin rods also impaired local glutamate receptor responses. We discovered an inverse relationship between the number of synaptic events and the accumulation of cofilin rods in dendrites. We also detected cofilin rods in aging rat brains in vivo. These results suggest that cofilin aggregation may contribute to neurodegeneration and brain aging by blocking intracellular trafficking and inducing synaptic loss.