Mesenchymal inflammation drives genotoxic stress in hematopoietic stem cells and predicts disease evolution in human pre-leukemia

Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system but the molecular mechanisms and their relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the preleukemic disorder Shwachman-Diamond syndrome induces mitochondrial dysfunction, oxidative stress and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the mouse model and a range of human preleukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome, the principal leukemia predisposition syndrome. Collectively, our findings reveal a concept of mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as an actionable determinant of disease outcome in human preleukemia

Pathological structural conversion of α-synuclein at the mitochondria induces neuronal toxicity

Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity

Stress-induced changes in synaptic morphology in rat CA1 hippocampus [poster presentation]

[Poster]: Ultrastructural studies in our laboratory have demonstrated that restraint stress significantly reduces axospinous synaptic density in the CA3 subfield of the rat hippocampus (Sandi et al. Euro. J. Neurosci. 17: 2446-2476). Because the CA1 subfield receives direct innervation from CA3 and is the major output area of the hippocampus, we examined the effects of restraint stress upon synaptic parameters in this strategic region. An unbiased stereological approach was used to compare numerical asymmetrical synaptic densities (Nv) and post-synaptic membrane lengths, between control and stress (restraint stress; 6h/day for 21 days started at c. 13wks of age) groups in CA1 hippocampus of rats (n=3, per experimental group). Measurements were taken throughout the stratum radiatum (StRad) and lacunosum moleculare (LacMol). Our data show that no statistically significant changes occur in Nv or post synaptic membrane length within the StRad of CA1. However, further statistical analysis (Student’s two-tailed t-test) reveal a significant increase in the lengths of individual post-synaptic membranes in the LacMol of CA1 (control group mean = 252.4nm; stress group mean = 355.2nm; p=0.027). These results indicate a stress-induced remodelling of synaptic connectivity in LacMol of CA1 that may underlie neurobiological mechanisms related to stress

Stress increases 'Axo-spinous' post-synaptic density size in CA1 stratum lacunosum-moleculare: a three-dimensional ultrastructural study [poster presentation]

Stress alters the synaptic connectivity of hippocampal neurones (Sandi et al., Euro. J. Neurosci. 17: 2446-2476). Stereological studies in our laboratory indicate that chronic restraint stress (CRS; 6h/day, 21days) significantly increases the size of the post-synaptic density (PSD) membranes of asymmetric axo-spinous synapses in dorsal anterior CA1 stratum lacunosum-moleculare. No change occured in the absolute number of these synapses between groups. To further characterise these stress-associated synaptic alterations, 3-D reconstructions of asymmetric axo-spinous PSD membranes were performed using electron-microscope images obtained from serial ultrathin sections. Thirty synapses per animal were reconstructed (4 animals per group). The associated dendritic spines of the identified synapses were also reconstructed in 3-D. Our preliminary findings indicate a significant increase in PSD membrane surface area of asymmetric axo-spinous synapses following CRS (+36%; p=0.026) and a highly significant increase in PSD membrane volume (+77%; p=0.003). No significant alterations were detected in the surface area or volume of reconstructed dendritic spines. These results verify our earlier stereological findings that CRS significantly increases the size of asymmetric axo-spinous synaptic junctions in dorsal-anterior CA1 stratum lacunosum-moleculare. This reinforces our previous conclusion that a structural remodelling of asymmetric ‘excitatory’ axo-spinous synapses occurs in this region of CA1 hippocampus following CRS

Origins of Enterovirus Replication Organelles Established by Whole-Cell Electron Microscopy

ABSTRACT Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles. IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes

Volume electron microscopy.

Life exists in three dimensions, but until the turn of the century most electron microscopy methods provided only 2D image data. Recently, electron microscopy techniques capable of delving deep into the structure of cells and tissues have emerged, collectively called volume electron microscopy (vEM). Developments in vEM have been dubbed a quiet revolution as the field evolved from established transmission and scanning electron microscopy techniques, so early publications largely focused on the bioscience applications rather than the underlying technological breakthroughs. However, with an explosion in the uptake of vEM across the biosciences and fast-paced advances in volume, resolution, throughput and ease of use, it is timely to introduce the field to new audiences. In this Primer, we introduce the different vEM imaging modalities, the specialized sample processing and image analysis pipelines that accompany each modality and the types of information revealed in the data. We showcase key applications in the biosciences where vEM has helped make breakthrough discoveries and consider limitations and future directions. We aim to show new users how vEM can support discovery science in their own research fields and inspire broader uptake of the technology, finally allowing its full adoption into mainstream biological imaging

Assessment of indicators and collection methodology to estimate nutrient digestibility in buffaloes

Dry fecal matter production was estimated from neutral detergent indicators on indigestible fiber, indigestible acid detergent fiber, indigestible dry matter, incubated for 144 hours and 288 hours, as well as chromium oxide (Cr2O3) and enriched and purified isolated lignin (LIPE®) in two sampling schemes (3 and 5 days) on buffaloes. Sample consisted of five castrated animals with average weight of 300 ± 0.6 kg fed on elephant grass cv Cameroon (Pennisetum purpureum). Experimental design consisted of randomized blocks in subdivided plots. Production of dry fecal matter was overestimated when using Cr2O3, indigestible acid detergent fiber 144 hours, indigestible neutral detergent fiber 144 hours, indigestible neutral detergent fiber 288 hours and indigestible dry matter 144 hours, while indigestible acid detergent fiber 288 hours, indigestible dry matter 288 hours and LIPE® did not differ from total collection. The same result was observed for apparent digestibility of nutrients. There was no difference in dry fecal matter production and digestibility between both collection periods of 3 and 5 days, demonstrating that a collection period of three days can be used to estimate dry fecal matter production in buffaloes. A three-day period of sample collection, in order to estimate dry fecal matter production and apparent digestibility coefficients, is therefore recommended. The use of LIPE®, fibers in indigestible acid detergent and indigestible dry matter as indicators, both latter incubated for 288 hours, result in accurate estimates of dry fecal matter production in confined buffaloes, fed on a forage based diet