Large-scale pathway specific polygenic risk and transcriptomic community network analysis identifies novel functional pathways in Parkinson disease.

Polygenic inheritance plays a central role in Parkinson disease (PD). A priority in elucidating PD etiology lies in defining the biological basis of genetic risk. Unraveling how risk leads to disruption will yield disease-modifying therapeutic targets that may be effective. Here, we utilized a high-throughput and hypothesis-free approach to determine biological processes underlying PD using the largest currently available cohorts of genetic and gene expression data from International Parkinson's Disease Genetics Consortium (IPDGC) and the Accelerating Medicines Partnership-Parkinson's disease initiative (AMP-PD), among other sources. We applied large-scale gene-set specific polygenic risk score (PRS) analyses to assess the role of common variation on PD risk focusing on publicly annotated gene sets representative of curated pathways. We nominated specific molecular sub-processes underlying protein misfolding and aggregation, post-translational protein modification, immune response, membrane and intracellular trafficking, lipid and vitamin metabolism, synaptic transmission, endosomal-lysosomal dysfunction, chromatin remodeling and apoptosis mediated by caspases among the main contributors to PD etiology. We assessed the impact of rare variation on PD risk in an independent cohort of whole-genome sequencing data and found evidence for a burden of rare damaging alleles in a range of processes, including neuronal transmission-related pathways and immune response. We explored enrichment linked to expression cell specificity patterns using single-cell gene expression data and demonstrated a significant risk pattern for dopaminergic neurons, serotonergic neurons, hypothalamic GABAergic neurons, and neural progenitors. Subsequently, we created a novel way of building de novo pathways by constructing a network expression community map using transcriptomic data derived from the blood of PD patients, which revealed functional enrichment in inflammatory signaling pathways, cell death machinery related processes, and dysregulation of mitochondrial homeostasis. Our analyses highlight several specific promising pathways and genes for functional prioritization and provide a cellular context in which such work should be done.

Gene therapy-emulating small molecule treatments in cystic fibrosis airway epithelial cells and patients.

BACKGROUND: Several small molecule corrector and potentiator drugs have recently been licensed for Cystic Fibrosis (CF) therapy. However, other aspects of the disease, especially inflammation, are less effectively treated by these drugs. We hypothesized that small molecule drugs could function either alone or as an adjuvant to licensed therapies to treat these aspects of the disease, perhaps emulating the effects of gene therapy in CF cells. The cardiac glycoside digitoxin, which has been shown to inhibit TNFα/NFκB signaling in CF lung epithelial cells, may serve as such a therapy. METHODS: IB3-1 CF lung epithelial cells were treated with different Vertex (VX) drugs, digitoxin, and various drug mixtures, and ELISA assays were used to assess suppression of baseline and TNFα-activated secretion of cytokines and chemokines. Transcriptional responses to these drugs were assessed by RNA-seq and compared with gene expression in AAV-[wildtype]CFTR-treated IB3-1 (S9) cells. We also compared in vitro gene expression signatures with in vivo data from biopsied nasal epithelial cells from digitoxin-treated CF patients. RESULTS: CF cells exposed to digitoxin exhibited significant suppression of both TNFα/NFκB signaling and downstream secretion of IL-8, IL-6 and GM-CSF, with or without co-treatment with VX drugs. No evidence of drug-drug interference was observed. RNA-seq analysis showed that gene therapy-treated CF lung cells induced changes in 3134 genes. Among these, 32.6% were altered by digitoxin treatment in the same direction. Shared functional gene ontology themes for genes suppressed by both digitoxin and gene therapy included inflammation (84 gene signature), and cell-cell interactions and fibrosis (49 gene signature), while genes elevated by both were enriched for epithelial differentiation (82 gene signature). A new analysis of mRNA data from digitoxin-treated CF patients showed consistent trends in expression for genes in these signatures. CONCLUSIONS: Adjuvant gene therapy-emulating activities of digitoxin may contribute to enhancing the efficacy of currently licensed correctors and potentiators in CF patients.

Altered gene expression in the emerging cerebellar primordium of Neurog1-/- mice.

Expression of the basic helix-loop-helix (bHLH) transcription factor Neurogenin1 (Neurog1) coincides with the emergence of the cerebellum and Neurog1-expressing progenitors are fated to become Purkinje cells and later interneurons. However, the gene regulatory functions of Neurog1 in cerebellar development have not been characterized. We performed a genome-wide analysis of gene expression in the cerebellar primordium of E11.5 Neurog1 null (Neurog1-/-) mice to identify the Neurog1 transcriptome in the emerging cerebellum. This screen identified 117 genes differentially enriched in Neurog1-/- versus control sample sets with a high presence of gene sets enriched for functions in nervous system development. Hierarchical clustering revealed complete stratification of differentially expressed genes based on Neurog1 gene deletion status. In silico analysis of promoter regions identifies high probability Neurog1 regulatory (E-box) binding sites in 94 of the 117 differentially expressed genes and Pax6 binding motifs in 25 of these 94 promoters. Our data provide a framework for investigating Neurog1 transcriptional programs in early cerebellar development and suggest functional Neurog1-Pax6 cross-talk in the activation of downstream targets.

Valproic acid inhibits neurosphere formation by adult subventricular cells by a lithium-sensitive mechanism.

The mood stabilizer valproic acid (VPA) decreases neural progenitor proliferation and promotes neurogenesis in the adult hippocampus. However, the effects of VPA on progenitor cells in the adult subventricular zone (SVZ) are not as well characterized. Here we report VPA blocks neurosphere formation and inhibits DNA synthesis in cultured NSCs from the SVZ of adult mice. Inhibition of DNA synthesis is associated with the up-regulation of the differentiation transcription factors Egr1 and Neurod1 and down-regulation of transcription factors associated with "stemness". Co-treatment of VPA with the mood stabilizer lithium antagonizes the anti-proliferative effects of VPA on adult NSCs and abolishes VPA activation of Egr1. Co-treatment of VPA with the MEK1/2 inhibitor PD980589 similarly abolishes Egr1 activation consistent with VPA activation and lithium antagonism of MEK-ERK signaling in adult NSCs. However, Western blot reveals VPA significantly suppresses ERK2 phosphorylation in adult NSCs grown in proliferating culture conditions and that lithium co-treatment does not attenuate this effect. Combined the data indicate VPA inhibition of adult NSC proliferation and activation of Egr1 by VPA, along with the antagonism of these effects by lithium, are the effects of cumulative changes in multiple signaling pathways and are not attributable to a common kinase target.

Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs.

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.