Azetidine-2-Carboxylic Acid-Induced Oligodendrogliopathy: Relevance to the Pathogenesis of Multiple Sclerosis.

The naturally occurring imino acid azetidine-2-carboxylic acid (Aze) is consumed by humans and can be misincorporated in place of proline in myelin basic protein (MBP) in vitro. To determine Aze effects on the mammalian CNS in vivo, adult CD1 mice were given Aze orally or intraperitoneally. Clinical signs reminiscent of MBP-mutant mice occurred with 600 mg/kg Aze exposure. Aze induced oligodendrocyte (OL) nucleomegaly and nucleoplasm clearing, dilated endoplasmic reticulum, cytoplasmic vacuolation, abnormal mitochondria, and Aze dose-dependent apoptosis. Immunohistochemistry demonstrated myelin blistering and nuclear translocation of unfolded protein response (UPR)/proinflammatory molecules (ATF3, ATF4, ATF6, eIF2α, GADD153, NFκB, PERK, XBP1), MHC I expression, and MBP cytoplasmic aggregation in OL. There were scattered microglial nodules in CNS white matter (WM); other CNS cells appeared unaffected. Mice given Aze in utero and postnatally showed more marked effects than their dams. These OL, myelin, and microglial alterations are found in normal-appearing WM (NAWM) in multiple sclerosis (MS) patients. Thus, Aze induces a distinct oligodendrogliopathy in mice that recapitulates MS NAWM pathology without leukocyte infiltration. Because myelin proteins are relatively stable throughout life, we hypothesize that Aze misincorporation in myelin proteins during myelinogenesis in humans results in a progressive UPR that may be a primary process in MS pathogenesis.

Dinitrogen Insertion and Cleavage by a Metal-Metal Bonded Tricobalt(I) Cluster.

Reduction of a tricobalt(II) tri(bromide) cluster supported by a tris(ß-diketiminate) cyclophane results in halide loss, ligand compression, and metal-metal bond formation to yield a 48-electron CoI3 cluster, Co3LEt/Me (2). Upon reaction of 2 with dinitrogen, all metal-metal bonds are broken, steric conflicts are relaxed, and dinitrogen is incorporated within the internal cavity to yield a formally (µ3-η1:η2:η1-dinitrogen)tricobalt(I) complex, 3. Broken symmetry DFT calculations (PBE0/def2-tzvp/D3) support an N-N bond order of 2.1 in the bound N2 with the calculated N-N stretching frequency (1743 cm-1) comparable to the experimental value (1752 cm-1). Reduction of 3 under Ar in the presence of Me3SiBr results in N2 scission with tris(trimethylsilyl)amine afforded in good yield.

Evaluating Metal Ion Identity on Catalytic Silylation of Dinitrogen Using a Series of Trimetallic Complexes.

We report catalytic silylation of dinitrogen to tris(trimethylsilyl)amine by a series of trinuclear first row transition metal complexes (M = Cr, Mn, Fe, Co, Ni) housed in our tris(ß-diketiminate) cyclophane (L 3- ). Yields are expectedly dependent on metal ion type ranging from 14 to 199 equiv NH4 +/complex after protonolysis for the Mn to Co congeners, respectively. For the series of complexes, the number of turnovers trend observed is Co > Fe > Cr > Ni > Mn, consistent with prior reports of greater efficacy of Co over Fe in other ligand systems for this reaction.

Synthetic Factors Governing Access to Tris(ß-diketimine) Cyclophanes versus Tripodal Tri-ß-aminoenones.

Tris(ß-diketimine) cyclophanes are an important ligand class for investigating cooperative multimetallic interactions of bioinorganic clusters. Discussed herein are the synthetic factors governing access to tris(ß-diketimine) cyclophanes versus tripodal tri-ß-aminoenones. Cyclophanes bearing Me, Et, and MeO cap substituents and ß-Me, Et, or Ph arm substituents are obtained, and a modified condensation method produced α-Me ß-Me cyclophane. These operationally simple procedures produce the ligands in gram quantities and in 22-94% yields.

Anti-Myelin Proteolipid Protein Peptide Monoclonal Antibodies Recognize Cell Surface Proteins on Developing Neurons and Inhibit Their Differentiation.

Using a panel of monoclonal antibodies (mAbs) to myelin proteolipid protein (PLP) peptides, we found that in addition to CNS myelin, mAbs to external face but not cytoplasmic face epitopes immunostained neurons in immature human CNS tissues and in adult hippocampal dentate gyrus and olfactory bulbs, that is neural stem cell niches (NSCN). To explore the pathobiological significance of these observations, we assessed the mAb effects on neurodifferentiation in vitro. The mAbs to PLP 50-69 (IgG1κ and IgG2aκ), and 178-191 and 200-219 (both IgG1κ) immunostained live cell surfaces and inhibited neurite outgrowth of E18 rat hippocampal precursor cells and of PC12 cells, which do not express PLP. Proteins immunoprecipitated from PC12 cell extracts and captured by mAb-coated magnetic beads were identified by GeLC-MS/MS. Each neurite outgrowth-inhibiting mAb captured a distinct set of neurodifferentiation molecules including sequence-similar M6 proteins and other unrelated membrane and extracellular matrix proteins, for example integrins, Eph receptors, NCAM-1, and protocadherins. These molecules are expressed in adult human NSCN and are implicated in the pathogenesis of many chronic CNS disease processes. Thus, diverse anti-PLP epitope autoantibodies may inhibit neuronal precursor cell differentiation via multispecific recognition of cell surface molecules thereby potentially impeding endogenous neuroregeneration in NSCN and in vivo differentiation of exogenous neural stem cells.

Review of the history and current status of cell-transplant approaches for the management of neuropathic pain.

Treatment of sensory neuropathies, whether inherited or caused by trauma, the progress of diabetes, or other disease states, are among the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord would be the logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the transplant of cells or a cell line to treat human disease. The history of the research and development of useful cell-transplant-based approaches offers an understanding of the advantages and problems associated with these technologies, but as an adjuvant or replacement for current pharmacological treatments, cell therapy is a likely near future clinical tool for improved health care.

Potential for Cell-Transplant Therapy with Human Neuronal Precursors to Treat Neuropathic Pain in Models of PNS and CNS Injury: Comparison of hNT2.17 and hNT2.19 Cell Lines.

Effective treatment of sensory neuropathies in peripheral neuropathies and spinal cord injury (SCI) is one of the most difficult problems in modern clinical practice. Cell therapy to release antinociceptive agents near the injured spinal cord is a logical next step in the development of treatment modalities. But few clinical trials, especially for chronic pain, have tested the potential of transplant of cells to treat chronic pain. Cell lines derived from the human neuronal NT2 cell line parentage, the hNT2.17 and hNT2.19 lines, which synthesize and release the neurotransmitters gamma-aminobutyric acid (GABA) and serotonin (5HT), respectively, have been used to evaluate the potential of cell-based release of antinociceptive agents near the lumbar dorsal (horn) spinal sensory cell centers to relieve neuropathic pain after PNS (partial nerve and diabetes-related injury) and CNS (spinal cord injury) damage in rat models. Both cell lines transplants potently and permanently reverse behavioral hypersensitivity without inducing tumors or other complications after grafting. Functioning as cellular minipumps for antinociception, human neuronal precursors, like these NT2-derived cell lines, would likely provide a useful adjuvant or replacement for current pharmacological treatments for neuropathic pain.

Subarachnoid Transplant of the Human Neuronal hNT2.19 Serotonergic Cell Line Attenuates Behavioral Hypersensitivity without Affecting Motor Dysfunction after Severe Contusive Spinal Cord Injury.

Transplant of cells which make biologic agents that can modulate the sensory and motor responses after spinal cord injury (SCI) would be useful to treat pain and paralysis. To address this need for clinically useful human cells, a unique neuronal cell line that synthesizes and secretes/releases the neurotransmitter serotonin (5HT) was isolated. Hind paw tactile allodynia and thermal hyperalgesia induced by severe contusive SCI were potently reversed after lumbar subarachnoid transplant of differentiated cells, but had no effect on open field motor scores, stride length, foot rotation, base of support, or gridwalk footfall errors associated with the SCI. The sensory effects appeared 1 week after transplant and did not diminish during the 8-week course of the experiment when grafts were placed 2 weeks after SCI. Many grafted cells were still present and synthesizing 5HT at the end of the study. These data suggest that the human neuronal serotonergic hNT2.19 cells can be used as a biologic minipump for receiving SCI-related neuropathic pain, but likely requires intraspinal grafts for motor recovery.

Clinical feasibility for cell therapy using human neuronal cell line to treat neuropathic behavioral hypersensitivity following spinal cord injury in rats.

Management of neuropathic pain remains problematic; however, cell therapy to treat the effects of pain on the sensory system after spinal cord injury (SCI) could be a useful approach. Since many clinical trials ultimately do not succeed, use of cell therapy will require that safety and efficacy issues be addressed early in preclinical rat studies. We used the human neuronal cell line hNT2.17, which secretes the inhibitory neurotransmitters gamma-aminobutyric acid and glycine, in an excitotoxic SCI pain model after intraspinal injection of quisqualic acid into rats. One week after lumbar transplant of these cells, behavioral hypersensitivity was permanently reversed. Antinociceptive grafts displayed an optimal transplant time that included moderate effectiveness with chronic SCI and late graft placement and that required a minimal course of cyclosporine A 2 weeks after transplant for durable reversal of painlike behaviors. In addition, grafts did not need to be placed near the SCI level to be effective. These data suggest not only that these cells are safe and efficacious but also that they could be an effective clinical tool for treating SCI-associated neuropathic pain.

The combination of human neuronal serotonergic cell implants and environmental enrichment after contusive SCI improves motor recovery over each individual strategy.

A human neuronal cell line, hNT2.19, which secretes serotonin (5-HT) after differentiation, was used as a transplant source to improve motor dysfunction following severe contusive spinal cord injury (SCI). Also, environmental enrichment (EE) was added to the interspinal transplant treatment paradigm. Motor testing was performed weekly before and following SCI, with and without EE and/or cell transplant conditions. Motor recovery was maximal when both cell transplant and EE were used. Individual treatment paradigms also significantly improved foot rotation and reduced footfall errors but not stride length or base of support dysfunction. This recovery of motor function after SCI suggests that the combinatory use of serotonergic hNT2.19 cell grafts plus EE is a meaningful strategy to modestly improve motor dysfunction that accompanies contusive SCI.

Social and environmental enrichment improves sensory and motor recovery after severe contusive spinal cord injury in the rat.

Neuropathic pain and motor dysfunction are difficult problems following spinal cord injury (SCI). Social and environmental enrichment (SEE), which models much of the clinical rehabilitation environment for post-SCI persons, is the focus of the current investigation which examines the effects of multiple-housing and the addition of climbing spaces, improved bedding and crawl toys on the sensory and motor recovery following a severe contusive SCI. Efficacy was determined with sensory testing, open-field motor behavioral testing, lesion volume analysis and quantification of brain-derived neurotrophic factor (BDNF) in the lumbar spinal cord with and without SEE provided during the recovery period. Sensory and motor testing were performed weekly for 12 weeks following SCI. SEE significantly and permanently reversed cutaneous allodynia, but not thermal hyperalgesia, to near normal levels. The gross locomotor performance (BBB [Basso, Beattie, and Bresnahan] motor scores) significantly improved about two points. In addition, the BBB subscale scores were significantly improved nearly seven points by the end of the study. SEE also significantly improved foot rotation to normal levels and reduced gridwalk footfall errors nearly 50%, but had no effect on stride length or base of support dysfunctions. SEE significantly increased the total volume of a thoracic segment of cord encompassing the injury site at 12 weeks, by reducing cavitation and increasing both the volume of grey and white matter spared, compared to SCI alone. When BDNF levels were examined in the injured lumbar spinal cord, SEE significantly returned BDNF levels to near-normal. These data suggest that immediate use of SEE after contusive SCI is able to improve overall spinal cell survival and prevent much of the sensory and motor dysfunction that accompanies contusive SCI.

Optimizing the transplant dose of a human neuronal cell line graft to treat SCI pain in the rat.

Neuropathic pain is a prevalent and difficult problem in the setting of spinal cord injury (SCI). The use of cellular transplant therapy to treat this pain has been successful with the use of a human neuronal cell line, hNT2.17 [M.J. Eaton, S.Q. Wolfe, M.A. Martinez, M. Hernandez, C. Furst, J. Huang, B.R. Frydel, O. Gomez-Marin, Subarachnoid transplant of a human neuronal cell line attenuates chronic allodynia and hyperalgesia after excitotoxic SCI in the rat, J. Pain 8 (2007) 33-50]. Intrathecal transplant of these cells potently reverses behavioral hypersensitivity after excitotoxic spinal cord injury in the rat model. This study focuses on delineating the optimal dose of these cell grafts in the same model. Two weeks after intraspinal injection of quisqualic acid (QUIS) with subsequent behavioral hypersensitivity, terminally differentiated hNT2.17 cells were transplanted into 300 g Wistar-Furth rats in a logarithmic variation of doses: 10(6), 10(5) and 10(3) cells. Behavioral hypersensitivity testing was performed weekly for 6 weeks following transplant. The dose of 10(6) cells (or approximately 3 million/kg) potently and permanently reversed both cutaneous allodynia (CA) and thermal hyperalgesia (TH). Reduced transplant doses of the hNT2.17 cell line did not permanently reverse behavioral hypersensitivity, suggesting that there is an optimal dose that can be used as a clinical tool to treat SCI-associated neuropathic pain.

Subarachnoid transplant of a human neuronal cell line attenuates chronic allodynia and hyperalgesia after excitotoxic spinal cord injury in the rat.

UNLABELLED: The relief of neuropathic pain after spinal cord injury (SCI) remains daunting, because pharmacologic intervention works incompletely and is accompanied by multiple side effects. Transplantation of human cells that make specific biologic agents that can potentially modulate the sensory responses that are painful would be very useful to treat problems such as pain. To address this need for clinically useful human cells, the human neuronal NT2 cell line was used as a source to isolate a unique human neuronal cell line that synthesizes and secretes/releases the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine. This new cell line, hNT2.17, expresses an exclusively neuronal phenotype, does not incorporate bromodeoxyuridine during differentiation, and does not express the tumor-related proteins fibroblast growth factor 4 and transforming growth factor-alpha during differentiation after 2 weeks of treatment with retinoic acid and mitotic inhibitors. The transplant of predifferentiated hNT2.17 cells was used in the excitotoxic SCI pain model, after intraspinal injection of the mixed AMPA/metabotropic receptor agonist quisqualic acid (QUIS). When hNT2.17 cells were transplanted into the lumbar subarachnoid space, tactile allodynia and thermal hyperalgesia induced by the injury were quickly and potently reversed. Control cell transplants of nonviable hNT2.17 cells had no effect on the hypersensitivity induced by QUIS. The effects of hNT2.17 cell grafts appeared 1 week after transplants and did not diminish during the 8-week course of the experiment when grafts were placed 2 weeks after SCI. Immunohistochemistry and quantification of the human grafts were used to ensure that many grafted cells were still present and synthesizing GABA at the end of the study. These data suggest that the human neuronal hNT2.17 cells can be used as a "biologic minipump" for antinociception in models of SCI and neuropathic pain. PERSPECTIVE: This study describes the initial characterization and use of a human-derived cell line to treat neuropathic pain that would be suitable for clinical application, once further tested for safety and approved by the Food and Drug Administration. A dose of these human cells could be delivered with a spinal tap and affect the intrathecal spinal environment for sensory system modulation.

Cell and molecular approaches to the attenuation of pain after spinal cord injury.

Recent experimental research to treat spinal cord injury (SCI) pain has greatly increased our understanding of how such chronic pain might be modulated in the human population. Neuropathic pain is caused by the structural and biochemical changes associated with the peripheral and central nervous system damage associated with nervous system trauma, often leading to an imbalance in endogenous excitatory and inhibitory spinal systems that modulate sensory processing. But current pharmacological therapies are often ineffective over time for the greater number of patients. Although there are a variety of useful surgical and pharmacologic interventions (including electric stimulation, implantable mechanical pumps and a myriad of drugs for pain relief) cell and molecular technologies are a new frontier in pain medicine. These other potential therapeutic agents of pain are based on current and developing treatment strategies elucidated from recent research, especially concerning central spinal sensitization, and the spinal mechanisms that are thought to be the origin and ongoing cause of chronic pain, even when the injury is peripheral in location. Newly developing translational strategies such as molecular agents, viral-mediated gene transfer or cellular transplants to treat chronic pain are being evaluated in a variety of peripheral and central injury models. They seek to address both the causes of neuropathic pain, to interfere with its development and maintenance over time, and give the injured person with pain an improved quality-of-life that allows them to better deal with the larger tasks of daily life and the strenuous rehabilitation that might also improve motor function after SCI.

Monoclonal antibodies to distinct regions of human myelin proteolipid protein simultaneously recognize central nervous system myelin and neurons of many vertebrate species.

Myelin proteolipid protein (PLP), the major protein of mammalian CNS myelin, is a member of the proteolipid gene family (pgf). It is an evolutionarily conserved polytopic integral membrane protein and a potential autoantigen in multiple sclerosis (MS). To analyze antibody recognition of PLP epitopes in situ, monoclonal antibodies (mAbs) specific for different regions of human PLP (50-69, 100-123, 139-151, 178-191, 200-219, 264-276) were generated and used to immunostain CNS tissues of representative vertebrates. mAbs to each region recognized whole human PLP on Western blots; the anti-100-123 mAb did not recognize DM-20, the PLP isoform that lacks residues 116-150. All of the mAbs stained fixed, permeabilized oligodendrocytes and mammalian and avian CNS tissue myelin. Most of the mAbs also stained amphibian, teleost, and elasmobranch CNS myelin despite greater diversity of their pgf myelin protein sequences. Myelin staining was observed when there was at least 40% identity of the mAb epitope and known pgf myelin proteins of the same or related species. The pgf myelin proteins of teleosts and elasmobranchs lack 116-150; the anti-100-123 mAb did not stain their myelin. In addition to myelin, the anti-178-191 mAb stained many neurons in all species; other mAbs stained distinct neuron subpopulations in different species. Neuronal staining was observed when there was at least approximately 30% identity of the PLP mAb epitope and known pgf neuronal proteins of the same or related species. Thus, anti-human PLP epitope mAbs simultaneously recognize CNS myelin and neurons even without extensive sequence identity. Widespread anti-PLP mAb recognition of neurons suggests a novel potential pathophysiologic mechanism in MS patients, i.e., that anti-PLP antibodies associated with demyelination might simultaneously recognize pgf epitopes in neurons, thereby affecting their functions.

Cell therapy for neuropathic pain in spinal cord injuries.

Cell therapy to treat neuropathic pain after spinal cord injury (SCI) is in its infancy. However, the development of cellular strategies that would replace or be used as an adjunct to existing pharmacological treatments for neuropathic pain have progressed tremendously over the past 20 years. The earliest cell therapy studies for pain relief tested adrenal chromaffin cells from rat or bovine sources, placed in the subarachnoid space, near the spinal cord pain- processing pathways. These grafts functioned as cellular minipumps, secreting a cocktail of antinociceptive agents around the spinal cord for peripheral nerve injury, inflammatory or arthritic pain. These initial animal, and later clinical, studies suggested that the spinal intrathecal space was a safe and accessible location for the placement of cell grafts. However, one major problem was the lack of a homogeneous, expandable cell source to supply the antinociceptive agents. Cell lines that can be reversibly immortalised are the next phase for the development of a practical, homogenous cell source. These technologies have been modelled with a variety of murine cell lines, derived from embryonic adrenal medulla or CNS brainstem, in which cells are transplanted, which downregulate their proliferative, oncogenic phenotype either before or after transplant. An alternative approach for existing human cell lines is the use of neural or adrenal precursors, in which the antinociceptive properties are induced by in vitro treatment with molecules that move the cells to an irreversible neural or chromaffin, and non-oncogenic, phenotype. Although such human cell lines are at an early stage of investigation, their clinical antinociceptive potential is significant given the daunting problem of difficult-to-treat neuropathic SCI pain.

Common animal models for spasticity and pain.

Animal models of spasticity and pain have allowed for the elucidation of possible mechanisms and the evaluation of potential therapeutic interventions for these serious clinical problems. Each model mirrors the clinical appearance of many features of the syndrome, but few reproduce the myriad patient reports of either intensity or relevant contributing factors, especially in models of chronic neuropathic pain. Often these models have been used to predict the potency and efficacy of pharmacologic agents that work in human pain states. Pain models have relied on measurements of the shifts in behavioral hypersensitivity to tactile and thermal stimuli, tests that are not used quantitatively in human patients. Even with the multiple peripheral and central models of spasticity and pain used in animals, only a few actually test human conditions: namely, diabetic neuropathy, chemotherapy, and immunotherapy for tumors. However, all these models have allowed for the comparison of certain behavioral, cellular, biochemical, and molecular mechanisms with human patient populations. Here we review the few extant models of spasticity, nerve injury, and central injury models of pain, and describe their features and use.

Interferon-beta counteracts inflammatory mediator-induced effects on brain endothelial cell tight junction molecules-implications for multiple sclerosis.

To elucidate mechanisms of endothelial cell (EC) dysfunction in CNS inflammatory responses and beneficial effects of interferon-beta (IFN-gamma) in multiple sclerosis (MS), we analyzed effects of individual and combinations of soluble inflammatory mediators on the intracellular localization of the EC tight junction-associated molecules zonula occludens-1 and -2 (ZO-1 and ZO-2) in human brain ECs. The cytoplasm in the majority of cells in control EC cultures was clear; ZO-1 and ZO-2 were localized peripherally near sites of cell contact and associated with submembranous cytoplasmic filaments. H2O2 induced reversible time- and concentration-dependent translocation of ZO-1 and ZO-2 to a random distribution within EC cytoplasm and retraction of EC borders. For low concentrations, these effects were accompanied by less prominent submembranous filaments but not by evidence of cytotoxicity, increased cell death or altered amounts of ZO-1. Tumor necrosis factor-beta induced similar alterations but interferon-y did not. Co-treatment with either cytokine increased H2O2 effects whereas IFN-beta reversed H2O2-induced effects. In control white matter samples, EC cytoplasm was clear and ZO-1 was located on cell borders. In inflammatory/demyelinating lesions, EC ZO-1 was diffuse, indicating that the alterations induced in vitro mimic those in active MS lesions. These findings suggest that in MS patients, IFN-beta treatment may counteract inflammatory mediator effects on CNS EC tight junction molecules, thereby preserving EC barrier function.

Intralesion transplantation of serotonergic precursors enhances locomotor recovery but has no effect on development of chronic central pain following hemisection injury in rats.

The effects of intralesion grafts of serotonergic precursors on locomotor recovery and development of chronic pain were assessed after chronic spinal cord hemisection injury (SCI) in rats. Serotonin- and brain-derived neurotrophic factor-secreting (RN46A-B14) and RN46A-vector-only cells were transplanted into the site of T13 lateral hemisection 10 days following injury in immunosuppressed animals, and locomotor and pain related behaviors were assessed weekly for 28 days. There were significant improvements in the degree of spontaneous locomotor recovery, but no significant difference was found in the magnitude of development of mechanical allodynia or thermal hyperalgesia in any transplant group. From these results, we conclude that intraparenchymal engraftment of RN46A-B14 cells is largely ineffective in influencing somatosensory outcomes after SCI, in contrast with the efficacy of dorsal intrathecal placement.