Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.

Plants are obliged to defend themselves against a wide range of biotic and abiotic stresses. Complex regulatory signaling networks mount an appropriate defense response depending on the type of stress that is perceived. In response to abiotic stresses such as drought, cold, and salinity, the function of abscisic acid (ABA) is well documented: elevation of plant ABA levels and activation of ABA-responsive signaling result in regulation of stomatal aperture and expression of stress-responsive genes. In response to pathogens, the role of ABA is more obscure and is a research topic that has long been overlooked. This article aims to evaluate and review the reported modes of ABA action on pathogen defense and highlight recent advances in deciphering the complex role of ABA in plant-pathogen interactions. The proposed mechanisms responsible for positive or negative effects of ABA on pathogen defense are discussed, as well as the regulation of ABA signaling and in planta ABA concentrations by beneficial and pathogenic microorganisms. In addition, the fast-growing number of reports that characterize antagonistic and synergistic interactions between abiotic and biotic stress responses point to ABA as an essential component in integrating and fine-tuning abiotic and biotic stress-response signaling networks.

Plant defense mechanisms against necrotrophic pathogens, such as Botrytis cinerea, are considered to be complex and to differ from those that are effective against biotrophs. In the abscisic acid-deficient sitiens tomato (Solanum lycopersicum) mutant, which is highly resistant to B. cinerea, accumulation of hydrogen peroxide (H 2 O 2 ) was earlier and stronger than in the susceptible wild type at the site of infection. In sitiens, H 2 O 2 accumulation was observed from 4 h postinoculation (hpi), specifically in the leaf epidermal cell walls, where it caused modification by protein cross-linking and incorporation of phenolic compounds. In wildtype tomato plants, H 2 O 2 started to accumulate 24 hpi in the mesophyll layer and was associated with spreading cell death. Transcript-profiling analysis using TOM1 microarrays revealed that defense-related transcript accumulation prior to infection was higher in sitiens than in wild type. Moreover, further elevation of sitiens defense gene expression was stronger than in wild type 8 hpi both in number of genes and in their expression levels and confirmed a role for cell wall modification in the resistant reaction. Although, in general, plant defense-related reactive oxygen species formation facilitates necrotrophic colonization, these data indicate that timely hyperinduction of H 2 O 2 -dependent defenses in the epidermal cell wall can effectively block early development of B. cinerea.

While central nervous system (CNS) microglia have been studied extensively, surprisingly little is known about macrophages populating the peripheral nervous system (PNS Macs).We performed ontogenic, transcriptomic and spatial characterization of sciatic nerve Macs (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and get replaced by bone marrow-derived Macs over time. Using single-cell profiling, we identified a tissue-specific core signature of snMacs and found two spatially-separated snMacs: Relmα + Mgl1 + snMacs in the epineurium and Relmα -Mgl1-snMacs in the endoneurium. Globally, snMacs lack most core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. Single-cell transcriptomics revealed that in response to injury both snMacs respond differently and that the PNS, in contrast to the CNS, is permissive to prolonged engraftment of monocytederived Macs recruited upon injury.

The epileptic encephalopathies are a clinically and aetiologically heterogeneous subgroup of epilepsy syndromes. Most epileptic encephalopathies have a genetic cause and patients are often found to carry a heterozygous de novo mutation in one of the genes associated with the disease entity. Occasionally recessive mutations are identified: a recent publication described a distinct neonatal epileptic encephalopathy (MIM 615905) caused by autosomal recessive mutations in the SLC13A5 gene. Here, we report eight additional patients belonging to four different families with autosomal recessive mutations in SLC13A5. SLC13A5 encodes a high affinity sodium-dependent citrate transporter, which is expressed in the brain. Neurons are considered incapable of de novo synthesis of tricarboxylic acid cycle intermediates; therefore they rely on the uptake of intermediates, such as citrate, to maintain their energy status and neurotransmitter production. The effect of all seven identified mutations (two premature stops and five amino acid substitutions) was studied in vitro, using immunocytochemistry, selective western blot and mass spectrometry. We hereby demonstrate that cells expressing mutant sodium-dependent citrate transporter have a complete loss of citrate uptake due to various cellular loss-of-function mechanisms. In addition, we provide independent proof of the involvement of autosomal recessive SLC13A5 mutations in the development of neonatal epileptic encephalopathies, and highlight teeth hypoplasia as a possible indicator for SLC13A5 screening. All three patients who tried the ketogenic diet responded well to this treatment, and future studies will allow us to ascertain whether this is a recurrent feature in this severe disorder.

There is a growing appreciation that membrane-bound organelles in eukaryotic cells communicate directly with one another through direct membrane contact sites. Mitochondria-associated membranes are specialized subdomains of the endoplasmic reticulum that function as membrane contact sites between the endoplasmic reticulum and mitochondria. These sites have emerged as major players in lipid metabolism and calcium signaling. More recently also autophagy and mitochondrial dynamics have been found to be regulated at ER-mitochondria contact sites. Neurons critically depend on mitochondria-associated membranes as a means to exchange metabolites and signaling molecules between these organelles. This is underscored by the fact that genes affecting mitochondrial and endoplasmic reticulum homeostasis are clearly overrepresented in several hereditary neurodegenerative disorders. Conversely, the processes affected by the contact sites between the endoplasmic reticulum and mitochondria are widely implicated in neurodegeneration. This review will focus on the most recent data addressing the structural composition and function of the mitochondria-associated membranes. In addition, the 3D morphology of the contact sites as observed using volume electron microscopy is discussed. Finally, it will highlight the role of several key proteins associated with these contact sites that are involved not only in dementias, amyotrophic lateral sclerosis and Parkinson’s disease, but also in axonopathies such as hereditary spastic paraplegia and Charcot–Marie–Tooth disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1528-7) contains supplementary material, which is available to authorized users.

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.

The most common form of spinal muscular atrophy (SMA) is a recessive disorder caused by deleterious SMN1 mutations in 5q13, whereas the genetic etiologies of non-5q SMA are very heterogeneous and largely remain to be elucidated. In a Bulgarian family affected by autosomal-dominant proximal SMA, we performed genome-wide linkage analysis and whole-exome sequencing and found a heterozygous de novo c.320C>T (p.Ser107Leu) mutation in bicaudal D homolog 2 (Drosophila) (BICD2). Further analysis of BICD2 in a cohort of 119 individuals with non-5q SMA identified a second de novo BICD2 mutation, c.2321A>G (p.Glu774Gly), in a simplex case. Detailed clinical and electrophysiological investigations revealed that both families are affected by a very similar disease course, characterized by early childhood onset, predominant involvement of lower extremities, and very slow disease progression. The amino acid substitutions are located in two interaction domains of BICD2, an adaptor protein linking the dynein molecular motor with its cargo. Our immunoprecipitation and localization experiments in HeLa and SH-SY5Y cells and affected individuals' lymphoblasts demonstrated that p.Ser107Leu causes increased dynein binding and thus leads to accumulation of BICD2 at the microtubule-organizing complex and Golgi fragmentation. In addition, the altered protein had a reduced colocalization with RAB6A, a regulator of vesicle trafficking between the Golgi and the endoplasmic reticulum. The interaction between p.Glu744Gly altered BICD2 and RAB6A was impaired, which also led to their reduced colocalization. Our study identifies BICD2 mutations as a cause of non-5q linked SMA and highlights the importance of dynein-mediated motility in motor neuron function in humans.