Challenges and opportunities for modeling aging and cancer.

Age is among the main risk factors for cancer, and any cancer study in adults is faced with an aging tissue and organism. Yet, pre-clinical studies are carried out using young mice and are not able to address the impact of aging and associated comorbidities on disease biology and treatment outcomes. Here, we discuss the limitations of current mouse cancer models and suggest strategies for developing novel models to address these major gaps in knowledge and experimental approaches.

Mapping systemic lupus erythematosus heterogeneity at the single-cell level.

Patients with systemic lupus erythematosus (SLE) display a complex blood transcriptome whose cellular origin is poorly resolved. Using single-cell RNA sequencing, we profiled ~276,000 peripheral blood mononuclear cells from 33 children with SLE with different degrees of disease activity and 11 matched controls. Increased expression of interferon-stimulated genes (ISGs) distinguished cells from children with SLE from healthy control cells. The high ISG expression signature (ISGhi) derived from a small number of transcriptionally defined subpopulations within major cell types, including monocytes, CD4+ and CD8+ T cells, natural killer cells, conventional and plasmacytoid dendritic cells, B cells and especially plasma cells. Expansion of unique subpopulations enriched in ISGs and/or in monogenic lupus-associated genes classified patients with the highest disease activity. Profiling of ~82,000 single peripheral blood mononuclear cells from adults with SLE confirmed the expansion of similar subpopulations in patients with the highest disease activity. This study lays the groundwork for resolving the origin of the SLE transcriptional signatures and the disease heterogeneity towards precision medicine applications.

Isolation of functional presynaptic complexes from CNS neurons: a cell-free preparation for the study of presynaptic compartments In vitro.

The difficulty in developing successful treatments to facilitate nerve regeneration has prompted a number of new in vitro experimental methods. We have recently shown that functional presynaptic boutons can be formed when neuronal cells are cocultured with surface-modified artificial substrates including poly(d-lysine)-coated beads and supported lipid bilayer-coated beads (Lucido(2009) J. Neurosci.29, 12449-12466; Gopalakrishnan(2010) ACS Chem. Neurosci.1, 86-94). We demonstrate here, using confocal microscopy combined with immunocytochemistry, that it is possible to isolate such in vitro presynaptic endings in an exclusive fashion onto glass substrates through a simple "sandwich/lift-off" technique (Perez(2006) Adv. Funct. Mater.16, 306-312). Isolated presynaptic complexes are capable of releasing and recycling neurotransmitter in response to an external chemical trigger. These bead-presynaptic complexes are facile to prepare and are readily dispersible in solution. They are thus compatible with many experimental methods whose focus is the study of the neuronal presynaptic compartment.

Lipid bilayer membrane-triggered presynaptic vesicle assembly.

The formation of functional synapses on artificial substrates is a very important step in the development of engineered in vitro neural networks. Spherical supported bilayer lipid membranes (SS-BLMs) are used here as a novel substrate to demonstrate presynaptic vesicle accumulation at an in vitro synaptic junction. Confocal fluorescence microscopy, cryo-transmission electron microscopy (cryo-TEM), and fluorescence recovery after photobleaching (FRAP) experiments have been used to characterize the SS-BLMs. Conventional immunocytochemistry combined with confocal fluorescence microscopy was used to observe the formation of presynaptic vesicles at the neuron-SS-BLM contacts. These results indicate that lipid phases may play a role in the observed phenomenon, in addition to the chemical and electrostatic interactions between the neurons and SS-BLMs. The biocompatibility of lipid bilayers along with their membrane tunability makes the suggested approach a useful "toolkit" for many neuroengineering applications including artificial synapse formation and synaptogenesis in vivo.

Rapid assembly of functional presynaptic boutons triggered by adhesive contacts.

CNS synapse assembly typically follows after stable contacts between "appropriate" axonal and dendritic membranes are made. We show that presynaptic boutons selectively form de novo following neuronal fiber adhesion to beads coated with poly-d-lysine (PDL), an artificial cationic polypeptide. As demonstrated by atomic force and live confocal microscopy, functional presynaptic boutons self-assemble as rapidly as 1 h after bead contact, and are found to contain a variety of proteins characteristic of presynaptic endings. Interestingly, presynaptic compartment assembly does not depend on the presence of a biological postsynaptic membrane surface. Rather, heparan sulfate proteoglycans, including syndecan-2, as well as others possibly adsorbed onto the bead matrix or expressed on the axon surface, are required for assembly to proceed by a mechanism dependent on the dynamic reorganization of F-actin. Our results indicate that certain (but not all) nonspecific cationic molecules like PDL, with presumably electrostatically mediated adhesive properties, can effectively bypass cognate and natural postsynaptic ligands to trigger presynaptic assembly in the absence of specific target recognition. In contrast, we find that postsynaptic compartment assembly depends on the prior presence of a mature presynaptic ending.

Morphine and pain-related stimuli enhance cell surface availability of somatic delta-opioid receptors in rat dorsal root ganglia.

The present study demonstrates that perikaryaldelta-opioid receptors (deltaORs) in rat dorsal root ganglion (DRG) neurons bind and internalize opioid ligands circulating in the CSF. Using confocal and electron microscopy, we found that prolonged morphine treatment increased the cell surface density of these perikaryal deltaORs and, by way of consequence, receptor-mediated internalization of the fluorescent deltorphin (DLT) analog omega-Bodipy 576/589 deltorphin-I 5-aminopentylamide (Fluo-DLT) in all three types of DRG neurons (small, medium, and large). In contrast, chronic inflammatory pain induced by the injection of complete Freund's adjuvant (CFA) into one hindpaw selectively increased Fluo-DLT internalization in small and medium-sized DRG neurons ipsilateral to the inflammation. Based on our previous studies in the spinal cord of mu-opioid receptor (muOR) knock-out mice, it may be assumed that the enhanced membrane recruitment of deltaORs observed after sustained morphine is attributable to stimulation of muORs. However, the selectivity of the effect induced by inflammatory pain suggests that it involves a different mechanism, namely a modality-specific and pain-related activation of C and Adelta fibers. Indeed, stimulation by capsaicin of transient receptor potential vanilloid 1 receptors, which are selectively expressed by small diameter (< 600 microm2) DRG neurons, increased Fluo-DLT internalization exclusively in this cell population. The present results, therefore, demonstrate that DRG neurons express perikaryal deltaORs accessible to CSF-circulating ligands and that the density and, hence, presumably also the responsiveness, of these receptors may be modulated by both pain-related stimuli and sustained exposure to muOR agonists.

Prolonged morphine treatment selectively increases membrane recruitment of delta-opioid receptors in mouse basal ganglia.

In recent years, we demonstrated that prolonged (48-h) treatment of rats or mice with selective m-opioid receptor ((mu)OR) agonists induced a translocation of delta-opioid receptors ((delta)ORs) from intracellular compartments to neuronal plasma membranes in the dorsal horn of the spinal cord. It remained to be determined whether this phenomenon also occurred in the brain. To resolve this issue, we analyzed by immunogold histochemistry the subcellular distribution of (delta)ORs in the nucleus accumbens, dorsal neostriatum, and frontal cortex in mice treated or not with morphine (48 h). We observed that prolonged treatment with morphine induced a translocation of (delta)ORs from intracellular to subplasmalemmal and membrane compartments in dendrites from both the nucleus accumbens and the dorsal neostriatum but not from the frontal cortex. We propose that this (mu)OR-(delta)OR interaction might prolong and modulate the sensitivity of neurons to opiates in specific target regions.