Spinophilin participates in information transfer at immunological synapses

The adaptive immune response is initiated by the presentation of peptides bound to major histocompatibility complex molecules on dendritic cells (DCs) to antigen-specific T lymphocytes at a junction termed the immunological synapse. Although much attention has been paid to cytoplasmic events on the T cell side of the synapse, little is known concerning events on the DC side. We have sought signal transduction components of the neuronal synapse that were also expressed by DCs. One such protein is spinophilin, a scaffolding protein of neuronal dendritic spines that regulates synaptic transmission. In inactive, immature DCs, spinophilin is located throughout the cytoplasm but redistributes to the plasma membrane upon stimulus-induced maturation. In DCs interacting with T cells, spinophilin is polarized dynamically to contact sites in an antigen-dependent manner. It is also required for optimal T cell activation because DCs derived from mice lacking spinophilin exhibit defects in antigen presentation both in vitro and in vivo. Thus, spinophilin may play analogous roles in information transfer at both neuronal and immunological synapses

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity

Macrophage Migration Inhibitory Factor (MIF) Makes Complex Contributions to Pain-Related Hyperactivity of Nociceptors after Spinal Cord Injury

Neuropathic pain is a major, inadequately treated challenge for people with spinal cord injury (SCI). While SCI pain mechanisms are often assumed to be in the CNS, rodent studies have revealed mechanistic contributions from primary nociceptors. These neurons become chronically hyperexcitable after SCI, generating ongoing electrical activity that promotes ongoing pain. A major question is whether extrinsic chemical signals help to drive ongoing electrical activity after SCI. People living with SCI exhibit acute and chronic elevation of circulating levels of macrophage migration inhibitory factor (MIF), a cytokine implicated in preclinical pain models. Probable nociceptors isolated from male rats and exposed to an MIF concentration reported in human plasma (1 ng/ml) showed hyperactivity similar to that induced by SCI, although, surprisingly, a 10-fold higher concentration failed to increase excitability. Conditioned behavioral aversion to a chamber associated with peripheral MIF injection suggested that MIF stimulates affective pain. A MIF inhibitor, Iso-1, reversed SCI-induced hyperexcitability. Unlike chronic SCI-induced hyperexcitability, acute MIF-induced hyperexcitability was only partially abrogated by inhibiting ERK signaling. Unexpectedly, MIF concentrations that induced hyperactivity in nociceptors from naive animals, after SCI induced a long-lasting conversion from a highly excitable nonaccommodating type to a rapidly accommodating, hypoexcitable type, possibly as a homeostatic response to prolonged depolarization. Treatment with conditioned medium from cultures of DRG cells obtained after SCI was sufficient to induce MIF-dependent hyperactivity in neurons from naive rats. Thus, changes in systemic and DRG levels of MIF may help to maintain SCI-induced nociceptor hyperactivity that persistently promotes pain.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major challenge for people with spinal cord injury (SCI). Pain can drastically impair quality of life, and produces substantial economic and social burdens. Available treatments, including opioids, remain inadequate. This study shows that the cytokine macrophage migration inhibitory factor (MIF) can induce pain-like behavior and plays an important role in driving persistent ongoing electrical activity in injury-detecting sensory neurons (nociceptors) in a rat SCI model. The results indicate that SCI produces an increase in MIF release within sensory ganglia. Low MIF levels potently excite nociceptors, but higher levels trigger a long-lasting hypoexcitable state. These findings suggest that therapeutic targeting of MIF in neuropathic pain states may reduce pain and sensory dysfunction by curbing nociceptor hyperactivity

Highly conserved molecular pathways, including Wnt signaling, promote functional recovery from spinal cord injury in lampreys

© The Author(s), 2018. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Scientific Reports 8 (2018): 742, doi:10.1038/s41598-017-18757-1.In mammals, spinal cord injury (SCI) leads to dramatic losses in neurons and synaptic connections, and consequently function. Unlike mammals, lampreys are vertebrates that undergo spontaneous regeneration and achieve functional recovery after SCI. Therefore our goal was to determine the complete transcriptional responses that occur after SCI in lampreys and to identify deeply conserved pathways that promote regeneration. We performed RNA-Seq on lamprey spinal cord and brain throughout the course of functional recovery. We describe complex transcriptional responses in the injured spinal cord, and somewhat surprisingly, also in the brain. Transcriptional responses to SCI in lampreys included transcription factor networks that promote peripheral nerve regeneration in mammals such as Atf3 and Jun. Furthermore, a number of highly conserved axon guidance, extracellular matrix, and proliferation genes were also differentially expressed after SCI in lampreys. Strikingly, ~3% of differentially expressed transcripts belonged to the Wnt pathways. These included members of the Wnt and Frizzled gene families, and genes involved in downstream signaling. Pharmacological inhibition of Wnt signaling inhibited functional recovery, confirming a critical role for this pathway. These data indicate that molecular signals present in mammals are also involved in regeneration in lampreys, supporting translational relevance of the model.We gratefully acknowledge support from the National Institutes of Health (R03NS078519 to OB; R01GM104123 to JJS; R01NS078165 to JRM), The Feinstein Institute for Medical Research and The Marine Biological Laboratory, including the Charles Evans Foundation Research Award, the Albert and Ellen Grass Foundation Faculty Research Award, and The Eugene and Millicent Bell Fellowship Fund in Tissue Engineering

Sequencing of the Sea Lamprey (Petromyzon marinus) Genome Provides Insights into Vertebrate Evolution

Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ∼500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms

Non-mammalian model systems for studying neuro-immune interactions after spinal cord injury

Mammals exhibit poor recovery after injury to the spinal cord, where the loss of neurons and neuronal connections can be functionally devastating. In contrast, it has long been appreciated that many non-mammalian vertebrate species exhibit significant spontaneous functional recovery after spinal cord injury (SCI). Identifying the biological responses that support an organism\u27s inability or ability to recover function after SCI is an important scientific and medical question. While recent advances have been made in understanding the responses to SCI in mammals, we remain without an effective clinical therapy for SCI. A comparative biological approach to understanding responses to SCI in non-mammalian vertebrates will yield important insights into mechanisms that promote recovery after SCI. Presently, mechanistic studies aimed at elucidating responses, both intrinsic and extrinsic to neurons, that result in different regenerative capacities after SCI across vertebrates are just in their early stages. There are several inhibitory mechanisms proposed to impede recovery from SCI in mammals, including reactive gliosis and scarring, myelin associated proteins, and a suboptimal immune response. One hypothesis to explain the robust regenerative capacity of several non-mammalian vertebrates is a lack of some or all of these inhibitory signals. This review presents the current knowledge of immune responses to SCI in several non-mammalian species that achieve anatomical and functional recovery after SCI. This subject is of growing interest, as studies increasingly show both beneficial and detrimental roles of the immune response following SCI in mammals. A long-term goal of biomedical research in all experimental models of SCI is to understand how to promote functional recovery after SCI in humans. Therefore, understanding immune responses to SCI in non-mammalian vertebrates that achieve functional recovery spontaneously may identify novel strategies to modulate immune responses in less regenerative species and promote recovery after SCI

Exploratory study for identifying systemic biomarkers that correlate with pain response in patients with intervertebral disc disorders

Molecular events that drive disc damage and low back pain (LBP) may precede clinical manifestation of disease onset and can cause detrimental long-term effects such as disability. Biomarkers serve as objective molecular indicators of pathological processes. The goal of this study is to identify systemic biochemical factors as predictors of response to treatment of LBP with epidural steroid injection (ESI). Since inflammation plays a pivotal role in LBP, this pilot study investigates the effect of ESI on systemic levels of 48 inflammatory biochemical factors (cytokines, chemokines, and growth factors) and examines the relationship between biochemical factor levels and pain or disability in patients with disc herniation (DH), or other diagnoses (Other Dx) leading to low back pain, which included spinal stenosis (SS) and degenerative disc disease (DDD). Study participants (n = 16) were recruited from a back pain management practice. Pain numerical rating score (NRS), Oswestry Disability Index (ODI), and blood samples were collected pre- and at 7 to 10 days post-treatment. Blood samples were assayed for inflammatory mediators using commercial multiplex assays. Mediator levels were compared pre- and post-treatment to investigate the potential correlations between clinical and biochemical outcomes. Our results indicate that a single ESI significantly decreased systemic levels of SCGF-β and IL-2. Improvement in pain in all subjects was correlated with changes in chemokines (MCP-1, MIG), hematopoietic progenitor factors (SCGF-β), and factors that participate in angiogenesis/fibrosis (HGF), nociception (SCF, IFN-α2), and inflammation (IL-6, IL-10, IL-18, TRAIL). Levels of biochemical mediators varied based on diagnosis of LBP, and changes in pain responses and systemic mediators from pre- to post-treatment were dependent on the diagnosis cohort. In the DH cohort, levels of IL-17 and VEGF significantly decreased post-treatment. In the Other Dx cohort, levels of IL-2Rα, IL-3, and SCGF-β significantly decreased post-treatment. In order to determine whether mediator changes were related to pain, correlations between change in pain scores and change in mediator levels were performed. Subjects with DH demonstrated a profile signature that implicated hematopoiesis factors (SCGF-β, GM-CSF) in pain response, while subjects with Other Dx demonstrated a biomarker profile that implicated chemokines (MCP-1, MIG) and angiogenic factors (HGF, VEGF) in pain response. Our findings provide evidence that systemic biochemical factors in patients with LBP vary by diagnosis, and pain response to treatment is associated with a unique profile of biochemical responses in each diagnosis group. Future hypothesis-based studies with larger subject cohorts are warranted to confirm the findings of this pilot exploratory study

Molecular Anatomy of a Living Nerve Terminal: Actin and Synapsin in the Synaptic Vesicle Cycle

Neurotransmission is based on a series of events termed the synaptic vesicle cycle. Several molecular components contributing to the synaptic vesicle cycle are known, including the cytoskeletal protein actin and the synaptic vesicle-associated phosphoprotein synapsin. The studies presented in this thesis were designed to investigate the role of the interaction between synapsin and actin in an intact model of synaptic transmission, the lamprey reticulospinal synapse. To establish the role of actin in the presynaptic terminal, several actin-directed compounds were injected into reticulospinal axons. Axons were maintained at rest or stimulated under various conditions, fixed, and the ultrastructure of the terminal was studied by electron microscopy. Unexpectedly, an actin-based network was revealed that appears to participate in the translocation of synaptic vesicles from endocytic regions of the plasma membrane back to the vesicle cluster. The prevailing hypothesis of synapsin function states that it interacts with actin to maintain organization of the vesicle cluster. In the present experiments, actin-modifying compounds did not visibly affect the vesicle cluster organization. Therefore, to investigate the potential relevance of the interaction between synapsin and actin to the novel actin-based translocation of vesicles, the localization of synapsin was determined in synapses fixed after several conditions of activity, using post-embedding techniques. In active synapses, synapsin was found on synaptic vesicles throughout the vesicle cluster, supporting a role for synapsin in mediating the dynamics of synaptic vesicle distal and proximal to the active zone. Synapsin was also present on several elements of the actin-based translocation network, including: the plasma membrane area where filaments are localized, the actin matrix itself and vesicles enmeshed in the matrix. Consistent with all data obtained, a novel hypothesis is presented, namely that the interaction of synapsin and actin plays an important role in the translocation of locally recycled synaptic vesicles in the presynaptic nerve terminal, facilitating the continuous supply of vesicles necessary to sustain neurotransmitter release. This hypothesis provides that synapsin serves as a direct functional link between the endocytic and exocytic pathways, as suggested by its binding partners that are known to participate in signal transduction, the endocytic pathway and the regulation of actin dynamics