Reverting the mode of action of the mitochondrial FOF1-ATPase by Legionella pneumophila preserves its replication niche

Legionella pneumophila, the causative agent of Legionnaires'; disease, a severe pneumonia, injects via a type-IV-secretion-system (T4SS) more than 300 proteins into macrophages, its main host cell in humans. Certain of these proteins are implicated in reprogramming the metabolism of infected cells by reducing mitochondrial oxidative phosphorylation (OXPHOS) early after infection. Here we show that despite reduced OXPHOS, the mitochondrial membrane potential (Δψm) is maintained during infection of primary human monocyte-derived macrophages (hMDMs). We reveal that L. pneumophila reverses the ATP-synthase activity of the mitochondrial FOF1-ATPase to ATP-hydrolase activity in a T4SS-dependent manner, which leads to a conservation of the Δψm, preserves mitochondrial polarization and prevents macrophage cell death. Analyses of T4SS effectors known to target mitochondrial functions revealed that LpSpl is partially involved in conserving the Δψm, but not LncP and MitF. The inhibition of the L. pneumophila-induced 'reverse mode' of the FOF1-ATPase collapsed the Δψm and caused cell death in infected cells. Single-cell analyses suggested that bacterial replication occurs preferentially in hMDMs that conserved the Δψm and showed delayed cell death. This direct manipulation of the mode of activity of the FOF1-ATPase is a newly identified feature of L. pneumophila allowing to delay host cell death and thereby to preserve the bacterial replication niche during infection.

Establishing Irreversible Electroporation Electric Field Potential Threshold in A Suspension In Vitro Model for Cardiac and Neuronal Cells

Aims: Irreversible electroporation is an ablation technique being adapted for the treatment of atrial fibrillation. Currently, there are many differences reported in the in vitro and pre-clinical literature for the effective voltage threshold for ablation. The aim of this study is a direct comparison of different cell types within the cardiovascular system and identification of optimal voltage thresholds for selective cell ablation. Methods: Monophasic voltage pulses were delivered in a cuvette suspension model. Cell viability and live–dead measurements of three different neuronal lines, cardiomyocytes, and cardiac fibroblasts were assessed under different voltage conditions. The immediate effects of voltage and the evolution of cell death was measured at three different time points post ablation. Results: All neuronal and atrial cardiomyocyte lines showed cell viability of less than 20% at an electric field of 1000 V/cm when at least 30 pulses were applied with no significant difference amongst them. In contrast, cardiac fibroblasts showed an optimal threshold at 1250 V/cm with a minimum of 50 pulses. Cell death overtime showed an immediate or delayed cell death with a proportion of cell membranes re-sealing after three hours but no significant difference was observed between treatments after 24 h. Conclusions: The present data suggest that understanding the optimal threshold of irreversible electroporation is vital for achieving a safe ablation modality without any side-effect in nearby cells. Moreover, the evolution of cell death post electroporation is key to obtaining a full understanding of the effects of IRE and selection of an optimal ablation threshold.

Neonatal Mesenchymal Stem Cell Treatment Improves Myelination Impaired by Global Perinatal Asphyxia in Rats

The effect of perinatal asphyxia (PA) on oligodendrocyte (OL), neuroinflammation, and cell viability was evaluated in telencephalon of rats at postnatal day (P)1, 7, and 14, a period characterized by a spur of neuronal networking, evaluating the effect of mesenchymal stem cell (MSCs)-treatment. The issue was investigated with a rat model of global PA, mimicking a clinical risk occurring under labor. PA was induced by immersing fetus-containing uterine horns into a water bath for 21 min (AS), using sibling-caesarean-delivered fetuses (CS) as controls. Two hours after delivery, AS and CS neonates were injected with either 5 μL of vehicle (10% plasma) or 5 × 104 MSCs into the lateral ventricle. Samples were assayed for myelin-basic protein (MBP) levels; Olig-1/Olig-2 transcriptional factors; Gglial phenotype; neuroinflammation, and delayed cell death. The main effects were observed at P7, including: (i) A decrease of MBP-immunoreactivity in external capsule, corpus callosum, cingulum, but not in fimbriae of hippocampus; (ii) an increase of Olig-1-mRNA levels; (iii) an increase of IL-6-mRNA, but not in protein levels; (iv) an increase in cell death, including OLs; and (v) MSCs treatment prevented the effect of PA on myelination, OLs number, and cell death. The present findings show that PA induces regional- and developmental-dependent changes on myelination and OLs maturation. Neonatal MSCs treatment improves survival of mature OLs and myelination in telencephalic white matter.

Stroke promotes the development of brain atrophy and delayed cell death in hypertensive rats

Post-stroke cognitive impairment (PSCI) is a major source of disability, affecting up to two thirds of stroke survivors with no available therapeutic options. The condition remains understudied in preclinical models due to its delayed presentation. Although hypertension is a leading risk factor for dementia, how ischemic stroke contributes to this neurodegenerative condition is unknown. In this study, we used a model of hypertension to study the development of PSCI and its mechanisms. Spontaneously hypertensive rats (SHR) were compared to normotensive rats and were subjected to 1-h middle cerebral artery occlusion or sham surgery. Novel object recognition, passive avoidance test and Morris water maze were used to assess cognition. In addition, brain magnetic resonance images were obtained 12-weeks post-stroke and tissue was collected for immunohistochemistry and protein quantification. Stroked animals developed impairment in long-term memory at 4-weeks post-stroke despite recovery from motor deficits, with hypertensive animals showing some symptoms of anhedonia. Stroked SHRs displayed grey matter atrophy and had a two-fold increase in apoptosis in the ischemic borderzone and increased markers of inflammatory cell death and DNA damage at 12 weeks post-stroke. This indicates that preexisting hypertension exacerbates the development of secondary neurodegeneration after stroke beyond its acute effects on neurovascular injury.

The inner membrane protein YhdP modulates the rate of anterograde phospholipid flow in Escherichia coli

The outer membrane (OM) of Gram-negative bacteria is a selective permeability barrier that allows uptake of nutrients while simultaneously protecting the cell from harmful compounds. The basic pathways and molecular machinery responsible for transporting lipopolysaccharides (LPS), lipoproteins, and β-barrel proteins to the OM have been identified, but very little is known about phospholipid (PL) transport. To identify genes capable of affecting PL transport, we screened for genetic interactions with mlaA*, a mutant in which anterograde PL transport causes the inner membrane (IM) to shrink and eventually rupture; characterization of mlaA*-mediated lysis suggested that PL transport can occur via a high-flux, diffusive flow mechanism. We found that YhdP, an IM protein involved in maintaining the OM permeability barrier, modulates the rate of PL transport during mlaA*-mediated lysis. Deletion of yhdP from mlaA* reduced the rate of IM transport to the OM by 50%, slowing shrinkage of the IM and delaying lysis. As a result, the weakened OM of ΔydhP cells was further compromised and ruptured before the IM during mlaA*-mediated death. These findings demonstrate the existence of a high-flux, diffusive pathway for PL flow in Escherichia coli that is modulated by YhdP.Significance StatementThe outer membrane (OM) of Gram-negative bacteria serves as a barrier that protects cells from harmful chemical compounds, including many antibiotics. Understanding how bacteria build this barrier is an important step in engineering strategies to circumvent it. A long-standing mystery in the field is how phospholipids (PLs) are transported from the inner membrane (IM) to the OM. We previously discovered that a mutation in the gene mlaA causes rapid flow of PLs to the OM, eventually resulting in IM rupture. Here, we found that deletion of the gene yhdP delayed cell death in the mlaA mutant by slowing flow of PLs to the OM. These findings reveal a high-flux, diffusive pathway for PL transport in Gram-negative bacteria modulated by YhdP.

Dentate gyrus neurons that are born at the peak of development, but not before or after, die in adulthood

In the dentate gyrus of the rodent hippocampus, neurogenesis begins prenatally and continues to the end of life. Adult-born neurons often die in the first few weeks after mitosis, but then survive indefinitely. In contrast, neurons born at the peak of development are initially stable but can die later in adulthood. Physiological and pathological changes in hippocampal structure may therefore result from both the addition of new neurons and the loss of older neurons. However, it is unknown whether neurons born at other stages of development also undergo delayed cell death. Here, we used BrdU to label dentate granule cells that were born in rats on embryonic day 19 (E19; before the developmental peak), postnatal day 6 (P6; peak) and P21 (after the peak). We quantified BrdU+ neurons in separate groups of rats at 2 and 6 months post-BrdU injection. Consistent with previous work, there was a 15% loss of P6-born neurons between 2 and 6 months of age. In contrast, E19- or P21-born neurons were stable throughout young adulthood. Delayed death of P6-born neurons suggests they may play a unique role in hippocampal plasticity and pathology in adulthood.

New astroglial injury-defined biomarkers for neurotrauma assessment

Traumatic brain injury (TBI) is an expanding public health epidemic with pathophysiology that is difficult to diagnose and thus treat. TBI biomarkers should assess patients across severities and reveal pathophysiology, but currently, their kinetics and specificity are unclear. No single ideal TBI biomarker exists. We identified new candidates from a TBI CSF proteome by selecting trauma-released, astrocyte-enriched proteins including aldolase C (ALDOC), its 38kD breakdown product (BDP), brain lipid binding protein (BLBP), astrocytic phosphoprotein (PEA15), glutamine synthetase (GS) and new 18-25kD-GFAP-BDPs. Their levels increased over four orders of magnitude in severe TBI CSF. First post-injury week, ALDOC levels were markedly high and stable. Short-lived BLBP and PEA15 related to injury progression. ALDOC, BLBP and PEA15 appeared hyper-acutely and were similarly robust in severe and mild TBI blood; 25kD-GFAP-BDP appeared overnight after TBI and was rarely present after mild TBI. Using a human culture trauma model, we investigated biomarker kinetics. Wounded (mechanoporated) astrocytes released ALDOC, BLBP and PEA15 acutely. Delayed cell death corresponded with GFAP release and proteolysis into small GFAP-BDPs. Associating biomarkers with cellular injury stages produced astroglial injury-defined (AID) biomarkers that facilitate TBI assessment, as neurological deficits are rooted not only in death of CNS cells, but also in their functional compromise.