The interplay between innate and adaptive immunity is important in multiple sclerosis (MS). The inflammasome complex, which activates caspase-1 to process pro–IL-1β and pro–IL-18, is rapidly emerging as a pivotal regulator of innate immunity, with nucleotide-binding domain, leucine-rich repeat containing protein family, pyrin domain containing 3 (NLRP3) (cryopyrin or NALP3) as a prominent player. Although the role of NLRP3 in host response to pathogen associated molecular patterns and danger associated molecular patterns is well documented, its role in autoimmune diseases is less well studied. To investigate the role of NLRP3 protein in MS, we used a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). Nlrp3 expression was elevated in the spinal cords during EAE, and Nlrp3−/− mice had a dramatically delayed course and reduced severity of disease. This was accompanied by a significant reduction of the inflammatory infiltrate including macrophages, dendritic cells, CD4, and CD8+ T cells in the spinal cords of the Nlrp3−/− mice, whereas microglial accumulation remained the same. Nlrp3−/− mice also displayed improved histology in the spinal cords with reduced destruction of myelin and astrogliosis. Nlrp3−/− mice with EAE produced less IL-18, and the disease course was similar to Il18−/− mice. Furthermore, Nlrp3−/− and Il18−/− mice had similarly reduced IFN-γ and IL-17 production. Thus, NLRP3 plays a critical role in the induction of the EAE, likely through effects on capase-1–dependent cytokines which then influence Th1 and Th17.

The μ-opioid receptor (OPRM1) is the principal receptor target for both endogenous and exogenous opioid analgesics. There are substantial individual differences in human responses to painful stimuli and to opiate drugs that are attributed to genetic variations in OPRM1. In searching for new functional variants, we employed comparative genome analysis and obtained evidence for the existence of an expanded human OPRM1 gene locus with new promoters, alternative exons and regulatory elements. Examination of polymorphisms within the human OPRM1 gene locus identified strong association between single nucleotide polymorphism (SNP) rs563649 and individual variations in pain perception. SNP rs563649 is located within a structurally conserved internal ribosome entry site (IRES) in the 5′-UTR of a novel exon 13-containing OPRM1 isoforms (MOR-1K) and affects both mRNA levels and translation efficiency of these variants. Furthermore, rs563649 exhibits very strong linkage disequilibrium throughout the entire OPRM1 gene locus and thus affects the functional contribution of the corresponding haplotype that includes other functional OPRM1 SNPs. Our results provide evidence for an essential role for MOR-1K isoforms in nociceptive signaling and suggest that genetic variations in alternative OPRM1 isoforms may contribute to individual differences in opiate responses.

Netrin-1 is a secreted protein that directs long-range axon guidance during early stages of neural circuit formation and continues to be expressed in the mammalian forebrain during the postnatal period of peak synapse formation. Here we demonstrate a synaptogenic function of netrin-1 in rat and mouse cortical neurons and investigate the underlying mechanism. We report that netrin-1 and its receptor DCC are widely expressed by neurons in the developing mammalian cortex during synapse formation and are enriched at synapses in vivo. We detect DCC protein distributed along the axons and dendrites of cultured cortical neurons and provide evidence that newly translated netrin-1 is selectively transported to dendrites. Using gain and loss of function manipulations, we demonstrate that netrin-1 increases the number and strength of excitatory synapses made between developing cortical neurons. We show that netrin-1 increases the complexity of axon and dendrite arbors, thereby increasing the probability of contact. At sites of contact, netrin-1 promotes adhesion, while locally enriching and reorganizing the underlying actin cytoskeleton through Src family kinase signaling and m-Tor-dependent protein translation to locally cluster presynaptic and postsynaptic proteins. Finally, we demonstrate using whole-cell patch-clamp electrophysiology that netrin-1 increases the frequency and amplitude of mEPSCs recorded from cortical pyramidal neurons. These findings identify netrin-1 as a synapse-enriched protein that promotes synaptogenesis between mammalian cortical neurons.

BackgroundOpioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to μ-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood.ResultsWe recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o complex, MOR1K couples to the stimulatory Gαs complex.ConclusionThe major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.