Inhibition of IL-34 Unveils Tissue-Selectivity and Is Sufficient to Reduce Microglial Proliferation in a Model of Chronic Neurodegeneration.

The proliferation and activation of microglia, the resident macrophages in the brain, is a hallmark of many neurodegenerative diseases such as Alzheimer's disease (AD) and prion disease. Colony stimulating factor 1 receptor (CSF1R) is critically involved in regulating microglial proliferation, and CSF1R blocking strategies have been recently used to modulate microglia in neurodegenerative diseases. However, CSF1R is broadly expressed by many cell types and the impact of its inhibition on the innate immune system is still unclear. CSF1R can be activated by two independent ligands, CSF-1 and interleukin 34 (IL-34). Recently, it has been reported that microglia development and maintenance depend on IL-34 signaling. In this study, we evaluate the inhibition of IL-34 as a novel strategy to reduce microglial proliferation in the ME7 model of prion disease. Selective inhibition of IL-34 showed no effects on peripheral macrophage populations in healthy mice, avoiding the side effects observed after CSF1R inhibition on the systemic compartment. However, we observed a reduction in microglial proliferation after IL-34 inhibition in prion-diseased mice, indicating that microglia could be more specifically targeted by reducing IL-34. Overall, our results highlight the challenges of targeting the CSF1R/IL34 axis in the systemic and central compartments, important for framing any therapeutic effort to tackle microglia/macrophage numbers during brain disease.

Development of a characterised tool kit for the interrogation of NLRP3 inflammasome-dependent responses.

Inflammation is an established contributor to disease and the NLRP3 inflammasome is emerging as a potential therapeutic target. A number of small molecule inhibitors of the NLRP3 pathway have been described. Here we analysed the most promising of these inhibitor classes side by side to assess relative potency and selectivity for their respective putative targets. Assessed using ASC inflammasome-speck formation, and release of IL-1ß, in both human monocyte/macrophage THP1 cells and in primary mouse microglia, we compared the relative potency and selectivity of P2X7 inhibitors, inflammasome inhibitors (diarylsulfonylurea vs. the NBC series), and caspase-1 inhibitors. In doing so we are now able to provide a well characterised small molecule tool kit for interrogating and validating inflammasome-dependent responses with a range of nanomolar potency inhibitors against established points in the inflammasome pathway.

Triazoloamides as potent γ-secretase modulators with reduced hERG liability.

Synthesis and SAR studies of novel aryl triazoles as gamma secretase modulators (GSMs) are presented in this communication. Starting from our aryl triazole leads, optimization studies were continued and the series progressed towards novel amides and lactams. Triazole 57 was identified as the most potent analog in this series, displaying single-digit nanomolar Aß42 IC(50) in cell-based assays and reduced affinity for the hERG channel.

Role for Runx1 in the proliferation and neuronal differentiation of selected progenitor cells in the mammalian nervous system.

Neurogenesis requires factors that regulate the decision of dividing progenitors to leave the cell cycle and activate the neuronal differentiation program. It is shown here that the murine runt-related gene Runx1 is expressed in proliferating cells on the basal side of the olfactory epithelium. These include both Mash1+ olfactory receptor neuron (ORN) progenitors and NeuroD+ ORN precursors. Disruption of Runx1 function in vivo does not cause a change in Mash1 expression but leads to a decrease in the number of NeuroD+ neuronal precursors and an increase in differentiated ORNs. These effects result in premature and ectopic ORN differentiation. It is shown further that exogenous Runx1 expression in cultured olfactory neural progenitors causes an expansion of the mitotic cell population. In agreement with these findings, exogenous Runx1 expression also promotes cortical neural progenitor cell proliferation without inhibiting neuronal differentiation. These effects are phenocopied by a chimeric protein containing ETO, the eight twenty one transcriptional repressor, fused to the Runx1 DNA-binding domain, which suggests the involvement of transcription repression mechanisms. Consistent with this possibility, Runx1 represses transcription driven by the promoter of the cell cycle inhibitor p21Cip 1 in cortical progenitors. Together, these findings suggest a previously unrecognized role for Runx1 in coordinating the proliferation and neuronal differentiation of selected populations of neural progenitors.

Identification of two novel elements involved in human MUCI gene expression in vivo.

BACKGROUND: MUC1, a membrane-tethered glycoprotein that is expressed on a number of epithelial cell types in vivo, is over-expressed in adenocarcinomas and thought to play a significant role in tumour progression and metastasis. Hence, elucidation of the mechanisms of regulation of MUC1 gene expression is of considerable biological importance. Our aim was to evaluate regulation of MUC1 expression in vivo. MATERIALS AND METHODS: DNase I hypersensitive sites (DHS) were mapped in chromatin from human cell lines and human MUC1 transgenic mice. MUC1 expression was evaluated by RT-PCR and Northern blots. RESULTS: We identified two novel DHS in the MUC1 promoter at -750 bp and -250 bp from the transcriptional start site. These DHS were detected in human cell lines and in a human MUC1 transgene in mice. The -750 DHS was apparent in many cell types irrespective of the level of MUC1 expression but the -250 DHS was only evident in cells that express MUC1 and its intensity correlated with the abundance of MUC1 transcripts. The -250 DHS became undetectable in cell lines representing a transition from colon adenoma to carcinoma, commensurate with a significant reduction in MUC1 expression. CONCLUSIONS: The -750 and -250 regions are conserved between the human MUC1 and mouse Muc1 genes and may be associated with functionally important genetic elements. The DHS at -250 is in the vicinity of previously defined purine/pyrimidine mirror repeat elements that may form intramolecular H-DNA structures, which can alter the accessibility of chromatin to regulatory proteins.

Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene shows a complex pattern of expression, with temporal and spatial regulation that is not accounted for by elements in the promoter. One approach to identifying the regulatory elements for CFTR is the mapping of DNase I hypersensitive sites (DHS) within the locus. We previously identified at least 12 clusters of DHS across the CFTR gene and here further evaluate DHS in introns 2, 3, 10, 16, 17a, 18, 20 and 21 to assess their functional importance in regulation of CFTR gene expression. Transient transfections of enhan- cer/reporter constructs containing the DHS regions showed that those in introns 20 and 21 augmented the activity of the CFTR promoter. Structural analysis of the DNA sequence at the DHS suggested that only the one intron 21 might be caused by inherent DNA structures. Cell specificity of the DHS suggested a role for the DHS in introns 2 and 18 in CFTR expression in some pancreatic duct cells. Finally, regulatory elements at the DHS in introns 10 and 18 may contribute to upregulation of CFTR gene transcription by forskolin and mitomycin C, respectively. These data support a model of regulation of expression of the CFTR gene in which multiple elements contribute to tightly co-ordinated expression in vivo.

Role for Hes1-induced phosphorylation in Groucho-mediated transcriptional repression.

Transcriptional corepressors of the Groucho/transducin-like Enhancer of split (Gro/TLE) family regulate a number of developmental pathways in both invertebrates and vertebrates. They form transcription repression complexes with members of several DNA-binding protein families and participate in the regulation of the expression of numerous genes. Despite their pleiotropic roles, little is known about the mechanisms that regulate the functions of Gro/TLE proteins. It is shown here that Gro/TLEs become hyperphosphorylated in response to neural cell differentiation and interaction with the DNA-binding cofactor Hairy/Enhancer of split 1 (Hes1). Hyperphosphorylation of Gro/TLEs is correlated with a tight association with the nuclear compartment through interaction with chromatin, suggesting that hyperphosphorylated Gro/TLEs may mediate transcriptional repression via chromatin remodeling mechanisms. Pharmacological inhibition of protein kinase CK2 reduces the Hes1-induced hyperphosphorylation of Gro/TLEs and causes a decrease in the chromatin association of the latter. Moreover, the transcription repression activity of Gro/TLEs is reduced by protein kinase CK2 inhibition. Consistent with these observations, Gro/TLEs are phosphorylated in vitro by purified protein kinase CK2. Taken together, these results implicate protein kinase CK2 in Gro/TLE functions. They suggest further that this kinase is involved in a hyperphosphorylation mechanism activated by Hes1 that promotes the transcription repression functions of Hes1-Gro/TLE protein complexes.