Synergistic Toxicity of Polyglutamine-Expanded TATA-Binding Protein in Glia and Neuronal Cells: Therapeutic Implications for Spinocerebellar Ataxia 17.

Spinocerebellar ataxia 17 (SCA17) is caused by polyglutamine (polyQ) repeat expansion in the TATA-binding protein (TBP) and is among a family of neurodegenerative diseases in which polyQ expansion leads to preferential neuronal loss in the brain. Although previous studies have demonstrated that expression of polyQ-expanded proteins in glial cells can cause neuronal injury via noncell-autonomous mechanisms, these studies investigated animal models that overexpress transgenic mutant proteins. Since glial cells are particularly reactive to overexpressed mutant proteins, it is important to investigate the in vivo role of glial dysfunction in neurodegeneration when mutant polyQ proteins are endogenously expressed. In the current study, we generated two conditional TBP-105Q knock-in mouse models that specifically express mutant TBP at the endogenous level in neurons or in astrocytes. We found that mutant TBP expression in neuronal cells or astrocytes alone only caused mild neurodegeneration, whereas severe neuronal toxicity requires the expression of mutant TBP in both neuronal and glial cells. Coculture of neurons and astrocytes further validated that mutant TBP in astrocytes promoted neuronal injury. We identified activated inflammatory signaling pathways in mutant TBP-expressing astrocytes, and blocking nuclear factor κB (NF-κB) signaling in astrocytes ameliorated neurodegeneration. Our results indicate that the synergistic toxicity of mutant TBP in neuronal and glial cells plays a critical role in SCA17 pathogenesis and that targeting glial inflammation could be a potential therapeutic approach for SCA17 treatment.SIGNIFICANCE STATEMENT Mutant TBP with polyglutamine expansion preferentially affects neuronal viability in SCA17 patients. Whether glia, the cells that support and protect neurons, contribute to neurodegeneration in SCA17 remains mostly unexplored. In this study, we provide both in vivo and in vitro evidence arguing that endogenous expression of mutant TBP in neurons and glia synergistically impacts neuronal survival. Hyperactivated inflammatory signaling pathways, particularly the NF-κB pathway, underlie glia-mediated neurotoxicity. Moreover, blocking NF-κB activity with small chemical inhibitors alleviated such neurotoxicity. Our study establishes glial dysfunction as an important component of SCA17 pathogenesis and suggests targeting glial inflammation as a potential therapeutic approach for SCA17 treatment.

Identification of a Novel Autoimmune Peptide Epitope of Prostein in Prostate Cancer.

There is a demand for novel targets and approaches to diagnose and treat prostate cancer (PCA). In this context, serum and plasma samples from a total of 609 individuals from two independent patient cohorts were screened for IgG reactivity against a sum of 3833 human protein fragments. Starting from planar protein arrays with 3786 protein fragments to screen 80 patients with and without PCA diagnosis, 161 fragments (4%) were chosen for further analysis based on their reactivity profiles. Adding 71 antigens from literature, the selection of antigens was corroborated for their reactivity in a set of 550 samples using suspension bead arrays. The antigens prostein (SLC45A3), TATA-box binding protein (TBP), and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) showed higher reactivity in PCA patients with late disease compared with early disease. Because of its prostate tissue specificity, we focused on prostein and continued with mapping epitopes of the 66-mer protein fragment using patient samples. Using bead-based assays and 15-mer peptides, a minimal peptide epitope was identified and refined by alanine scanning to the KPxAPFP. Further sequence alignment of this motif revealed homology to transmembrane protein 79 (TMEM79) and TGF-beta-induced factor 2 (TGIF2), thus providing a reasoning for cross-reactivity found in females. A comprehensive workflow to discover and validate IgG reactivity against prostein and homologous targets in human serum and plasma was applied. This study provides useful information when searching for novel biomarkers or drug targets that are guided by the reactivity of the immune system against autoantigens.

Plasmodium falciparum: Preinitiation complex occupancy of active and inactive promoters during erythrocytic stage.

Over 80% of Plasmodium falciparum genes are developmentally regulated during the parasite's life cycle with most genes expressed in a "just in time" fashion. However, the molecular mechanisms of gene regulation are still poorly understood. Analysis of P. falciparum genome shows that the parasite appears to encode relatively few transcription factors homologous to those in other eukaryotes. We used Chromatin immunoprecipitation (ChIP) to study interaction of PfTBP and PfTFIIE with stage specific Plasmodium promoters. Our results indicate that PfTBP and PfTFIIE are bound to their cognate sequence in active and inactive erythrocytic-expressed promoters. In addition, TF occupancy appears to extend beyond the promoter regions, since PfTBP interaction with the coding and 3' end regions was also detected. No PfTBP or PfTFIIE interaction was detected on csp and pfs25 genes which are not active during the erythrocytic asexual stage. Furthermore, PfTBP and PfTFIIE binding did not appear to correlate with histone 3 and/or 4 acetylation, suggesting that histone acetylation may not be a prerequisite for PfTBP or PfTFIIE promoter interaction. Based on our observations we concluded that the PfTBP/PfTFIIE-containing preinitiation complex (PIC) would be preassembled on promoters of all erythrocytic-expressed genes in a fashion independent of histone acetylation, providing support for the "poised" model. Contrary to the classical model of eukaryotic gene regulation, PIC interaction with Plasmodium promoters occurred independent of transcriptional activity and to the notion that chromatin acetylation leads to PIC assembly.

Preparation and characterization of a monoclonal antibody specific to Plasmodium falciparum TATA binding protein.

PfTBP is a transcriptional factor required by all three types of RNA polymerases in eukaryotic cells. In order to obtain a specific monoclonal antibody (MAb) against PfTBP, a DNA fragment of 684 base pairs (bp) that contained the complete PfTBP gene was amplified by polymerase chain reaction (PCR) and inserted into the pGEX prokaryotic expression vector. The recombinant protein (GST-PfTBP) was expressed in Escherichia coli, purified, and used as antigen to immunize mice. MAbs against PfTBP were obtained and hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Western blotting and immunofluorescence assays showed that MAb Pf.r1 recognized the PfTBP protein in nuclear extracts from Plasmodium falciparum as well as a native protein in the nuclei of this parasite. This MAb will be a helpful tool for the identification of the TBP associated factors (TAFs), which are apparently highly divergent with other eukaryotes. This information could help to identify new candidate gene products to develop novel drugs or vaccines.

Antigen-binding properties of monoclonal antibodies reactive with human TATA-binding protein and use in immunoaffinity chromatography.

The TATA-binding protein (TBP) plays a central role in the assembly of most eukaryotic transcription initiation complexes. We have characterized 3 monoclonal antibodies (mAbs) that react in the far amino-terminal (N-terminal) domain of the human TBP molecule (residues 1-99). One of these mAbs (designated 1TBP22) is a polyol-responsive monoclonal antibody (PR-mAb) and was adapted to an immunoaffinity chromatography procedure for purifying bacterially expressed, recombinant human TBP. The epitope for mAb 1TBP22 maps to residues 55-99, which includes the polyglutamine region. However, mAb 1TBP22 does not react with poly-l-glutamine. Human TBP, contained on the pET11a plasmid, was expressed in Escherichia coli Rosetta (DE3)pLysS. The cell lysate from 330 ml of induced culture was treated with polyethyleneimine (PEI) at 0.5 M NaCl to precipitate the nucleic acids. After centrifugation, the supernatant fluid was applied to an immunoadsorbent containing mAb 1TBP22. After extensive washing, the TBP was eluted with buffer containing 0.75 M ammonium sulfate and 40% propylene glycol. Human TPB purified by the immunoaffinity chromatography method was found to be active in gel-shift assays and transcription assays. Preliminary data indicate that this mAb might be useful for purifying protein complexes containing TBP from HeLa cell extracts.