Integration of protein-protein interaction networks and human genome-wide RNAi screens produces mechanistic insight into Wnt/β-catenin signaling.

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC₅₀ ≥ 1 μM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC₅₀ 30 nM), selective, metabolically stable, and brain penetrable (AUC_brain/AUC_plasma = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1β-induced astrocyte-derived EV release (51 ± 13%; p < 0.001). This inhibition led to a reduction of cytokine upregulation in liver and attenuation of the infiltration of immune cells into the brain (80 ± 23%; p < 0.01). A structurally similar but inactive analog had no effect in vitro or in vivo.

The discovery of the causative gene for Huntington’s disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two Drosophila models of Huntington’s disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington’s disease, and potentially for other neurodegenerative disorders.

The COVID-19 pandemic has highlighted the need for human respiratory tract-based assay platforms for efficient discovery and development of antivirals and disease-modulating therapeutics. Physiologically relevant tissue models of the lower respiratory tract (LRT), including the respiratory bronchioles and the alveolar sacs, are of high interest because they are the primary site of severe SARS-CoV-2 infection and are most affected during the terminal stage of COVID-19. Current epithelial lung models used to study respiratory viral infections include lung epithelial cells at the air–liquid interface (ALI) with fibroblasts and endothelial cells, but such models do not have a perfusable microvascular network to investigate both viral infectivity and viral infection-induced thrombotic events. Using a high throughput, 64-chip microfluidic plate-based platform, we have developed two novel vascularized, LRT multi-chip models for the alveoli and the small airway. Both models include a perfusable microvascular network consisting of human primary microvascular endothelial cells, fibroblasts and pericytes. The established biofabrication protocols also enable the formation of differentiated lung epithelial layers at the ALI on top of the vascularized tissue bed. We validated the physiologically relevant cellular composition, architecture and perfusion of the vascularized lung tissue models using fluorescence microscopy, flow cytometry, and electrical resistance measurements. These vascularized, perfusable microfluidic lung tissue on high throughput assay platforms will enable the development of respiratory viral infection and disease models for research investigation and drug discovery.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient–derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.