Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.

Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder typified by tremor, rigidity, akinesia and postural instability due in part to the loss of dopamine within the nigrostriatal system. The pathologic features of this disorder include the loss of substantia nigra dopamine neurons and attendant striatal terminals, the presence of large protein-rich neuronal inclusions containing fibrillar α-synuclein and increased numbers of activated microglia. Evidence suggests that both misfolded α-synuclein and oxidative stress play an important role in the pathogenesis of sporadic PD. Here we review evidence that α-synuclein activates glia inducing inflammation and that Nrf2-directed phase-II antioxidant enzymes play an important role in PD. We also provide new evidence that the expression of antioxidant enzymes regulated in part by Nrf2 is increased in a mouse model of α-synuclein overexpression. We show that misfolded α-synuclein directly activates microglia inducing the production and release of the proinflammatory cytokine, TNF-α, and increasing antioxidant enzyme expression. Importantly, we demonstrate that the precise structure of α-synuclein is important for induction of this proinflammatory pathway. This complex α-synuclein-directed glial response highlights the importance of protein misfolding, oxidative stress and inflammation in PD and represents a potential locus for the development of novel therapeutics focused on induction of the Nrf2-directed antioxidant pathway and inhibition of protein misfolding.

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.H3K9me3 | peptide inhibitor | prostate cancer | transcription factor S TAT3, a member of the STAT family, is a key signaling protein that transduces extracellular signals to the nucleus and regulates transcription of genes (1). Following ligand stimulation, STAT3 is phosphorylated on Y705 tyrosine residue, dimerizes, and translocates to the nucleus to bind its cognate DNA-response elements, activating gene transcription (1). Constitutively activated STAT3 mediates deregulated growth, survival, and angiogenesis (2, 3). STAT3 is widely recognized as a potential drug target for cancer therapy, and various approaches, including targeting of upstream tyrosine kinases and direct inhibitors of STAT3 dimerization, have been advanced to inhibit STAT3 signaling in cancers (4). However, unphosphorylated STAT3 (U-STAT3) has also been shown to influence gene transcription, both in response to cytokines and in cancer cells, albeit by mechanisms that are distinct from those activated by phosphorylated STAT3 (5). We have developed a highly selective inhibitor of STAT3 ND, ST3-Hel2A-2 (ST3-H2A2), that binds to the N-terminal domain (ND) and inhibits STAT3 signaling (6). STAT3 ND is involved in the interactions of two STAT dimers on neighboring sites to form a more stable tetramer and the interactions with histone-modifier proteins to induce changes in chromatin structure (reviewed in ref. 7). These complex interactions may greatly affect STAT3-dependent transcriptional activity, suggesting that the STAT3 ND mediates important regulatory functions of STAT3 in normal cells (8) and in cancer (9). ST3-H2A2 induces death in breast cancer cells MDA-MB-231 an...

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD ؉ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4 -5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP ؊/؊ cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.Poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30) is a chromatin-associated enzyme that catalyzes the transfer of successive units of the ADP-ribose moiety from NAD ϩ to itself and other nuclear acceptor proteins (1). PARP is a zinc fingercontaining protein, which allows enzyme binding to either double or single strand DNA breaks without any apparent sequence preference (2, 3). The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification (4,5). Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with breaking and rejoining DNA strands, and it plays a pivotal role in DNA damage repair (2, 6 -8).Recent studies have implicated PARP in transcription of eukaryotic genes (9 -16). PARP-dependent gene regulation involves poly(ADP-ribosyl)ation of transcription factors, which, in turn, prevents their binding to specific promoter sequences (10). The basal transcription factors TFIIF and TEF-1 as well as transcription factors TATA box-binding protein, YY1, SP-1, cAMP-response element-binding protein, p53, and NFB are all highly specific substrates for poly(ADP-ribosyl)ation (10,11,14,16). PARP may also interact directly with gene promoters. For instance, recombinant full-length PARP bound the DNA sequences within the MCAT1 regulatory element (11) and to the DF4 protein binding site of the Pax-6 gene neuroretinaspecific enhancer (17). Furthermore, PARP involvement in the active...

Background: Unphosphorylated STAT3 (U-STAT3) regulates gene expression, but the mechanisms of its DNA binding are not fully understood.Results: U-STAT3 binds to the same γ-activated sequence (GAS) DNA-binding site as phosphorylated STAT3. It also binds to AT-rich DNA structures.Conclusion: U-STAT3 regulates gene expression by binding to GAS and influencing chromatin organization.Significance: Our data provide an explanation of mechanisms of U-STAT3 binding to DNA.