Q134R: Small Chemical Compound with NFAT Inhibitory Properties Improves Behavioral Performance and Synapse Function in Mouse Models of Amyloid Pathology

Inhibition of the protein phosphatase calcineurin (CN) ameliorates pathophysiologic and cognitive changes in aging rodents and mice with aging-related Alzheimer\u27s disease (AD)-like pathology. However, concerns over adverse effects have slowed the transition of common CN-inhibiting drugs to the clinic for the treatment of AD and AD-related disorders. Targeting substrates of CN, like the nuclear factor of activated T cells (NFATs), has been suggested as an alternative, safer approach to CN inhibitors. However, small chemical inhibitors of NFATs have only rarely been described. Here, we investigate a newly developed neuroprotective hydroxyquinoline derivative (Q134R) that suppresses NFAT signaling, without inhibiting CN activity. Q134R partially inhibited NFAT activity in primary rat astrocytes, but did not prevent CN-mediated dephosphorylation of a non-NFAT target, either in vivo, or in vitro. Acute (≤1 week) oral delivery of Q134R to APP/PS1 (12 months old) or wild-type mice (3–4 months old) infused with oligomeric Aβ peptides led to improved Y maze performance. Chronic (≥3 months) oral delivery of Q134R appeared to be safe, and, in fact, promoted survival in wild-type (WT) mice when given for many months beyond middle age. Finally, chronic delivery of Q134R to APP/PS1 mice during the early stages of amyloid pathology (i.e., between 6 and 9 months) tended to reduce signs of glial reactivity, prevented the upregulation of astrocytic NFAT4, and ameliorated deficits in synaptic strength and plasticity, without noticeably altering parenchymal Aβ plaque pathology. The results suggest that Q134R is a promising drug for treating AD and aging-related disorders

Cell-Specific DNA Methylation Patterns of Retina-Specific Genes

Many studies have demonstrated that epigenetic mechanisms are important in the regulation of gene expression during embryogenesis, gametogenesis, and other forms of tissue-specific gene regulation. We sought to explore the possible role of epigenetics, specifically DNA methylation, in the establishment and maintenance of cell type-restricted gene expression in the retina. To assess the relationship between DNA methylation status and expression level of retinal genes, bisulfite sequence analysis of the 1000 bp region around the transcription start sites (TSS) of representative rod and cone photoreceptor-specific genes and gene expression analysis were performed in the WERI and Y79 human retinoblastoma cell lines. Next, the homologous genes in mouse were bisulfite sequenced in the retina and in non-expressing tissues. Finally, bisulfite sequencing was performed on isolated photoreceptor and non-photoreceptor retinal cells isolated by laser capture microdissection. Differential methylation of rhodopsin (RHO), retinal binding protein 3 (RBP3, IRBP) cone opsin, short-wave-sensitive (OPN1SW), cone opsin, middle-wave-sensitive (OPN1MW), and cone opsin, long-wave-sensitive (OPN1LW) was found in the retinoblastoma cell lines that inversely correlated with gene expression levels. Similarly, we found tissue-specific hypomethylation of the promoter region of Rho and Rbp3 in mouse retina as compared to non-expressing tissues, and also observed hypomethylation of retinal-expressed microRNAs. The Rho and Rbp3 promoter regions were unmethylated in expressing photoreceptor cells and methylated in non-expressing, non-photoreceptor cells from the inner nuclear layer. A third regional hypomethylation pattern of photoreceptor-specific genes was seen in a subpopulation of non-expressing photoreceptors (Rho in cones from the Nrl −/− mouse and Opn1sw in rods). These results demonstrate that a number of photoreceptor-specific genes have cell-specific differential DNA methylation that correlates inversely with their expression level. Furthermore, these cell-specific patterns suggest that DNA methylation may play an important role in modulating photoreceptor gene expression in the developing mammalian retina

Dynamics of Regulatory Networks in the Developing Mouse Retina

<div><p>Understanding gene regulation is crucial to dissect the molecular basis of human development and disease. Previous studies on transcription regulatory networks often focused on their static properties. Here we used retinal development as a model system to investigate the dynamics of regulatory networks that are comprised of transcription factors, microRNAs and other protein-coding genes. We found that the active sub-networks are topologically different at early and late stages of retinal development. At early stages, the active sub-networks tend to be highly connected, while at late stages, the active sub-networks are more organized in modular structures. Interestingly, network motif usage at early and late stages is also distinct. For example, network motifs containing reciprocal feedback regulatory relationships between two regulators are overrepresented in early developmental stages. Additionally, our analysis of regulatory network dynamics revealed a natural turning point at which the regulatory network undergoes drastic topological changes. Taken together, this work demonstrates that adding a dynamic dimension to network analysis can provide new insights into retinal development, and we suggest the same approach would likely be useful for the analysis of other developing tissues.</p> </div

Topological measures of the static and active networks of six time points.

<p>Out-degree is the number of regulated genes by a TF or miRNA. In-degree is the number of regulating TFs or miRNAs of a target gene. Clustering coefficient measures the inter-connectivity around a node. Average path length is the average length of all shortest paths among all node pairs. Betweenness is the average number of shortest paths between all node pairs passing through a node. Reachability is the fraction of nodes that can be reached from a node in the network. The mean and standard deviation (mean±SD) of 300 random networks for each time point are presented in Random Networks row. Examples of early and late time point active sub-networks are illustrated in the last row.</p

Enrichment analysis on six Gene Ontology terms (biological processes).

<p>Enrichment analysis for the genes specifically expressed in six development time points. X-axis is the time points and Y axis is the e-value of the terms at each time point.</p

Expression correlations between miRNAs and their predicted targets.

<p>The distributions for the predicted targets in TargetScan database (solid) and for the random targets (dot) are shown. The column plot represents the Z-values of each interval against 1000 miRNA target randomization on the right Y-axis.</p

Network motif dynamics.

<p>Dynamics of two distinct patterns of network motif classes (A and B). Five network motifs are shown for each cluster. The network motifs are illustrated in the corresponding boxes. The expression of protein-coding genes (C), or miRNAs (D) cannot separate the early and late stages, while the Z-values for network motifs (E) show clear separation between early and late developmental stages.</p

Hypomethylation of mouse photoreceptor-specific genes in expressing photoreceptors and regional hypomethylation in non-expressing photoreceptors.

<p>Bisulfite sequencing of photoreceptor-specific genes from non-expressing tissues (testes, kidney, and brain) and non-expressing cells from the INL, as well as expressing cells from the ONL (primarily rods from wild type mice and cones from Nrl −/− mice) was performed. Thirty sequences (10 clones for each of 3 biological replicates) were batched together in a true-to-scale map, which represents the % methylation for each CpG site by a color gradient (white, unmethylated; dark blue, 100% methylated). <i>Opn1mw</i> has only 2 CpG sites within the 1000 bp upstream of the TSS and none in the first exon. Both of these CpG sites (−762 and −510), were methylated in all tissues and cell types and the true-to-scale maps are not shown.</p

DNA methylation of photoreceptor genes in human cell lines is inversely correlated with expression levels.

<p><i>A,</i> QPCR of <i>OPN1LW</i>, <i>OPN1MW</i>, <i>OPN1SW</i>, <i>RHO</i> and <i>RBP3</i> in Y79, WERI, and HEK293 cells, normalized to expression of human retinal cDNA. <i>B–F,</i> % DNA methylation of photoreceptor genes in 3 cell lines plotted versus the CpG site position (noted by hash marks) relative to the TSS (position “0”). Expression level and % methylation are the average of biological triplicates ± SEM.</p

Bisulfite sequencing of mouse photoreceptor-specific genes and miRNAs shows increased DNA methylation in non-expressing tissues.

<p><i>A–F,</i> Genomic DNA was isolated from 4 mouse tissues and bisulfite sequenced. % DNA methylation of photoreceptor genes was plotted versus the CpG site position (noted by hash marks) relative to the TSS (position “0”) for genes or the 5′ end of the miRNA stem-loop sequence (position “0”). % methylation is the average of 3 animals ± SEM. Reliable sequence was not obtained for <i>Rho</i> sites −270, −166, and −129 due to lack of sequence complexity that preceded these sites.</p