Stress granules plug and stabilize damaged endolysosomal membranes.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

Peroxisomal ROS control cytosolic Mycobacterium tuberculosis replication in human macrophages.

Peroxisomes are organelles involved in many metabolic processes including lipid metabolism, reactive oxygen species (ROS) turnover, and antimicrobial immune responses. However, the cellular mechanisms by which peroxisomes contribute to bacterial elimination in macrophages remain elusive. Here, we investigated peroxisome function in iPSC-derived human macrophages (iPSDM) during infection with Mycobacterium tuberculosis (Mtb). We discovered that Mtb-triggered peroxisome biogenesis requires the ESX-1 type 7 secretion system, critical for cytosolic access. iPSDM lacking peroxisomes were permissive to Mtb wild-type (WT) replication but were able to restrict an Mtb mutant missing functional ESX-1, suggesting a role for peroxisomes in the control of cytosolic but not phagosomal Mtb. Using genetically encoded localization-dependent ROS probes, we found peroxisomes increased ROS levels during Mtb WT infection. Thus, human macrophages respond to the infection by increasing peroxisomes that generate ROS primarily to restrict cytosolic Mtb. Our data uncover a peroxisome-controlled, ROS-mediated mechanism that contributes to the restriction of cytosolic bacteria.

Microcephaly-associated protein WDR62 shuttles from the Golgi apparatus to the spindle poles in human neural progenitors.

WDR62 is a spindle pole-associated scaffold protein with pleiotropic functions. Recessive mutations in WDR62 cause structural brain abnormalities and account for the second most common cause of autosomal recessive primary microcephaly (MCPH), indicating WDR62 as a critical hub for human brain development. Here, we investigated WDR62 function in corticogenesis through the analysis of a C-terminal truncating mutation (D955AfsX112). Using induced Pluripotent Stem Cells (iPSCs) obtained from a patient and his unaffected parent, as well as isogenic corrected lines, we generated 2D and 3D models of human neurodevelopment, including neuroepithelial stem cells, cerebro-cortical progenitors, terminally differentiated neurons, and cerebral organoids. We report that WDR62 localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue, and translocates to the mitotic spindle poles in a microtubule-dependent manner. Moreover, we demonstrate that WDR62 dysfunction impairs mitotic progression and results in alterations of the neurogenic trajectories of iPSC neuroderivatives. In summary, impairment of WDR62 localization and function results in severe neurodevelopmental abnormalities, thus delineating new mechanisms in the etiology of MCPH.

ATG7 and ATG14 restrict cytosolic and phagosomal Mycobacterium tuberculosis replication in human macrophages.

Autophagy is a cellular innate-immune defence mechanism against intracellular microorganisms, including Mycobacterium tuberculosis (Mtb). How canonical and non-canonical autophagy function to control Mtb infection in phagosomes and the cytosol remains unresolved. Macrophages are the main host cell in humans for Mtb. Here we studied the contributions of canonical and non-canonical autophagy in the genetically tractable human induced pluripotent stem cell-derived macrophages (iPSDM), using a set of Mtb mutants generated in the same genetic background of the common lab strain H37Rv. We monitored replication of Mtb mutants that are either unable to trigger canonical autophagy (Mtb ΔesxBA) or reportedly unable to block non-canonical autophagy (Mtb ΔcpsA) in iPSDM lacking either ATG7 or ATG14 using single-cell high-content imaging. We report that deletion of ATG7 by CRISPR-Cas9 in iPSDM resulted in increased replication of wild-type Mtb but not of Mtb ΔesxBA or Mtb ΔcpsA. We show that deletion of ATG14 resulted in increased replication of both Mtb wild type and the mutant Mtb ΔesxBA. Using Mtb reporters and quantitative imaging, we identified a role for ATG14 in regulating fusion of phagosomes containing Mtb with lysosomes, thereby enabling intracellular bacteria restriction. We conclude that ATG7 and ATG14 are both required for restricting Mtb replication in human macrophages.

Lysosomal damage drives mitochondrial proteome remodelling and reprograms macrophage immunometabolism.

Transient lysosomal damage after infection with cytosolic pathogens or silica crystals uptake results in protease leakage. Whether limited leakage of lysosomal contents into the cytosol affects the function of cytoplasmic organelles is unknown. Here, we show that sterile and non-sterile lysosomal damage triggers a cell death independent proteolytic remodelling of the mitochondrial proteome in macrophages. Mitochondrial metabolic reprogramming required leakage of lysosomal cathepsins and was independent of mitophagy, mitoproteases and proteasome degradation. In an in vivo mouse model of endomembrane damage, live lung macrophages that internalised crystals displayed impaired mitochondrial function. Single-cell RNA-sequencing revealed that lysosomal damage skewed metabolic and immune responses in alveolar macrophages subsets with increased lysosomal content. Functionally, drug modulation of macrophage metabolism impacted host responses to Mycobacterium tuberculosis infection in an endomembrane damage dependent way. This work uncovers an inter-organelle communication pathway, providing a general mechanism by which macrophages undergo mitochondrial metabolic reprograming after endomembrane damage.

Corrigendum: Adenosine receptor stimulation improves glucocorticoid-induced osteoporosis in a rat model.

[This corrects the article DOI: 10.3389/fphar.2017.00558.].

Visualizing Pyrazinamide Action by Live Single-Cell Imaging of Phagosome Acidification and Mycobacterium tuberculosis pH Homeostasis.

Mycobacterium tuberculosis segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and M. tuberculosis intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that M. tuberculosis can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH), or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by disrupting M. tuberculosis intrabacterial pH homeostasis in cellulo. By using M. tuberculosis mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens. IMPORTANCE We still do not completely understand why tuberculosis (TB) treatment requires the combination of several antibiotics for up to 6 months. M. tuberculosis is a facultative intracellular pathogen, and it is still unknown whether heterogenous and dynamic intracellular populations of bacteria in different cellular environments affect antibiotic efficacy. By developing a dual live imaging approach to monitor mycobacterial pH homeostasis, host cell environment, and antibiotic action, we show here that intracellular localization of M. tuberculosis affects the efficacy of one first-line anti-TB drug. Our observations can be applicable to the treatment of other intracellular pathogens and help to inform the development of more effective combined therapies for tuberculosis that target heterogenous bacterial populations within the host.

Genome editing in stem cells for genetic neurodisorders.

The recent advent of genome editing techniques and their rapid improvement paved the way in establishing innovative human neurological disease models and in developing new therapeutic opportunities. Human pluripotent (both induced or naive) stem cells and neural stem cells represent versatile tools to be applied to multiple research needs and, together with genomic snip and fix tools, have recently made possible the creation of unique platforms to directly investigate several human neural affections. In this chapter, we will discuss genome engineering tools, and their recent improvements, applied to the stem cell field, focusing on how these two technologies may be pivotal instruments to deeply unravel molecular mechanisms underlying development and function, as well as disorders, of the human brain. We will review how these frontier technologies may be exploited to investigate or treat severe neurodevelopmental disorders, such as microcephaly, autism spectrum disorder, schizophrenia, as well as neurodegenerative conditions, including Parkinson's disease, Huntington's disease, Alzheimer's disease, and spinal muscular atrophy.

Human stem cell-based models for studying host-pathogen interactions.

The use of human cell lines and primary cells as in vitro models represents a valuable approach to study cellular responses to infection. However, with the advent of new molecular technologies and tools available, there is a growing need to develop more physiologically relevant systems to overcome cell line model limitations and better mimic human disease. Since the discovery of human stem cells, its use has revolutionised the development of in vitro models. This is because after differentiation, these cells have the potential to reflect in vivo cell phenotypes and allow for probing questions in numerous fields of the biological sciences. Moreover, the possibility to combine the advantages of stem cell-derived cell types with genome editing technologies and engineered 3D microenvironments, provides enormous potential for producing in vitro systems to investigate cellular responses to infection that are both relevant and predictive. Here, we discuss recent advances in the use of human stem cells to model host-pathogen interactions, highlighting emerging technologies in the field of stem cell biology that can be exploited to investigate the fundamental biology of infection. TAKE AWAYS: hPSC overcome current limitations to study host-pathogen interactions in vitro. Genome editing can be used in hPSC to study cellular responses to infection. hPSC, 3D models and genome editing can recreate physiological in vitro systems.

Activity of the SNARE Protein SNAP29 at the Endoplasmic Reticulum and Golgi Apparatus.

Snap29 is a conserved regulator of membrane fusion essential to complete autophagy and to support other cellular processes, including cell division. In humans, inactivating SNAP29 mutations causes CEDNIK syndrome, a rare multi-systemic disorder characterized by congenital neuro-cutaneous alterations. The fibroblasts of CEDNIK patients show alterations of the Golgi apparatus (GA). However, whether and how Snap29 acts at the GA is unclear. Here we investigate SNAP29 function at the GA and endoplasmic reticulum (ER). As part of the elongated structures in proximity to these membrane compartments, a pool of SNAP29 forms a complex with Syntaxin18, or with Syntaxin5, which we find is required to engage SEC22B-loaded vesicles. Consistent with this, in HeLa cells, in neuroepithelial stem cells, and in vivo, decreased SNAP29 activity alters GA architecture and reduces ER to GA trafficking. Our data reveal a new regulatory function of Snap29 in promoting secretory trafficking.

Variation rs2235503 C > A Within the Promoter of MSLN Affects Transcriptional Rate of Mesothelin and Plasmatic Levels of the Soluble Mesothelin-Related Peptide.

Soluble mesothelin-related peptide (SMRP) is a promising biomarker for malignant pleural mesothelioma (MPM), but several confounding factors can reduce SMRP-based test's accuracy. The identification of these confounders could improve the diagnostic performance of SMRP. In this study, we evaluated the sequence of 1,000 base pairs encompassing the minimal promoter region of the MSLN gene to identify expression quantitative trait loci (eQTL) that can affect SMRP. We assessed the association between four MSLN promoter variants and SMRP levels in a cohort of 72 MPM and 677 non-MPM subjects, and we carried out in vitro assays to investigate their functional role. Our results show that rs2235503 is an eQTL for MSLN associated with increased levels of SMRP in non-MPM subjects. Furthermore, we show that this polymorphic site affects the accuracy of SMRP, highlighting the importance of evaluating the individual's genetic background and giving novel insights to refine SMRP specificity as a diagnostic biomarker.

Improving homology-directed repair efficiency in human stem cells.

The generation of induced pluripotent stem cell models of human disease requires efficient modification of one or both alleles depending on dominant or recessive inheritance of the disease. To faithfully recapitulate many disease variants, the introduction of a single base change is required. The introduction of additional silent mutations designed to prevent re-cutting of the modified allele by Cas9 is not an optimal strategy, particularly for non-coding variants. Here, we developed an improved protocol for efficient engineering of single nucleotide variants in human iPS cells. Using a fluorescent BFP->GFP assay to monitor the incorporation of a single base pair change, we optimized the protocol to achieve HDR in 70% of unselected human iPS cells. The additive effects of cold shock, a small molecule enhancer of HDR and chemically modified ssODN dramatically shift the bias of repair in favor of HDR, resulting in a seven-fold higher ratio of HDR to NHEJ from 0.5 to 3.7.

Effects of the antagomiRs 15b and 200b on the altered healing pattern of diabetic mice.

BACKGROUND AND PURPOSE: Diabetic patients with non-healing ulcers have a reduced expression of VEGF. Genetically diabetic mice have an altered expression pattern of VEGF and its receptor, VEGF receptor 2 (VEGFR-2). In diabetic wounds, the microRNAs, miR15b and miR200b, which respectively inhibit VEGF and VEGF-R2 mRNAs, are up-regulated, further affecting the impaired angiogenesis. We investigated whether anti-miRs directed toward miR15b and miR200b could improve wound repair in genetically diabetic mice. EXPERIMENTAL APPROACH: Skin wounds were produced on the backs of female diabetic mice. The anti-miRs (antimiR15b, antimiR200b or antimiR15b/200b) at 10 mg·kg-1 , or vehicle were applied to the wound edge. Mice were killed on days 7, 14 and at time of complete wound closure. Levels of mRNA and protein of angiogenic mediators and their receptors were measured with RT-qPCR and Western blotting. Wounds were examined by histological and immunochemical methods. KEY RESULTS: mRNA expression of VEGF, VEGFR-2, angiopoietin-1 and its receptor TEK were evaluated after 7 and 14 days. Protein levels of VEGF and transglutaminase II were measured at day 7, while VEGFR-2 and Angiopoietin-1 were measured at day 14. Histological features and the time to achieve a complete wound closure were also examined. Treatment with the anti-miRs improved the analysed parameters and the co-treatment resulted the most effective. CONCLUSION AND IMPLICATIONS: The results suggest that the inhibition of miR15b and miR200b may have a potential application in diabetes-related wound disorders.

Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model.

Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.