Engineering polysialic acid on Schwann cells using polysialyltransferase gene transfer or purified enzyme exposure for spinal cord injury transplantation.

Polysialic acid (PolySia) is a critical post-translational modification on the neural cell adhesion molecule (NCAM, a.k.a., CD56), important for cell migration and axon growth during nervous system development, plasticity and repair. PolySia induction on Schwann cells (SCs) enhances their migration, axon growth support and ability to improve functional recovery after spinal cord injury (SCI) transplantation. In the current investigation two methods of PolySia induction on SCs, lentiviral vector transduction of the mouse polysialytransferase gene ST8SIA4 (LV-PST) or enzymatic engineering with a recombinant bacterial PST (PSTNm), were examined comparatively for their effects on PolySia induction, SC migration, the innate immune response and axon growth after acute SCI. PSTNm produced significant PolySia induction and a greater diversity of surface molecule polysialylation on SCs as evidenced by immunoblot. In the scratch wound assay, PSTNm was superior to LV-PST in the promotion of SC migration and gap closure. At 24 h after SCI transplantation, PolySia induction on SCs was most pronounced with LV-PST. Co-delivery of PSTNm with SCs, but not transient cell exposure, led to broader induction of PolySia within the injured spinal cord due to polysialylation upon both host cells and transplanted SCs. The innate immune response after SCI, measured by CD68 immunoreactivity, was similar among PolySia induction methods. LV-PST or PSTNm co-delivery with SCs provided a similar enhancement of SC migration and axon growth support above that of unmodified SCs. These studies demonstrate that LV-PST and PSTNm provide comparable acute effects on SC polysialation, the immune response and neurorepair after SCI.

Preclinical Efficacy and Safety of a Human Embryonic Stem Cell-Derived Midbrain Dopamine Progenitor Product, MSK-DA01.

Parkinson's disease is characterized by the loss of dopaminergic neurons in the substantia nigra leading to disabling deficits. Dopamine neuron grafts may provide a significant therapeutic advance over current therapies. We have generated midbrain dopamine neurons from human embryonic stem cells and manufactured large-scale cryopreserved dopamine progenitors for clinical use. After optimizing cell survival and phenotypes in short-term studies, the cell product, MSK-DA01, was subjected to an extensive set of biodistribution, toxicity, and tumorigenicity assessments in mice under GLP conditions. A large-scale efficacy study was also performed in rats with the same lot of cells intended for potential human use and demonstrated survival of the grafted cells and behavioral amelioration in 6-hydroxydopamine lesioned rats. There were no adverse effects attributable to the grafted cells, no obvious distribution outside the brain, and no cell overgrowth or tumor formation, thus paving the way for a future clinical trial.

Improved stem cell-derived motoneuron survival, migration, sprouting, and innervation with enhanced expression of polysialic acid.

Motoneurons (MNs) derived from mouse embryonic stem cells (ESCs) begin to express low levels of polysialic acid (PSA) at the time when they acquire an ability to migrate and extend neurites. PSA is known to promote cell migration and process outgrowth/guidance in the developing nervous system. To test if experimentally enhanced expression of PSA would augment these cellular events, the PSA-synthesizing polysialyltransferase was introduced into ESCs. In culture, the resulting higher PSA expression specifically increased neurite outgrowth and cell migration from differentiated embryoid bodies. In addition, the MN population obtained after sorting for HB9::GFP expression showed enhanced survival as well as extensive neurite outgrowth. Following transplantation of ESC-derived MNs into an adult sciatic nerve devoid of endogenous axons, the PSA augmentation increased the numbers of axons growing toward the denervated muscles. Migration of some transplanted cells inside the nerve toward muscle was also enhanced. Moreover, higher PSA expression selectively affected target innervation. It produced greater numbers of neuromuscular junctions in a predominantly fast twitch muscle and had no effect in a slow twitch muscle. These findings suggest that engineering of PSA expression in ESC could serve as an enhancement for MN cell therapy.

Changes in polysialic acid expression on myeloid cells during differentiation and recruitment to sites of inflammation: role in phagocytosis.

Polysialic acid (polySia) is a unique linear homopolymer of α2,8-linked sialic acid that has been studied extensively as a posttranslational modification of neural cell adhesion molecule in the central nervous system. Only two proteins are known to be polysialylated in cells of the immune system: CD56 on human natural killer cells and murine bone marrow (BM) leukocytes, and neuropilin-2 (NRP-2) on dendritic cells (DCs). We tested the hypothesis that polySia expression is regulated during maturation and migration of leukocytes and plays a role in functional activity. Using wild-type and NCAM(-/-) mice, we show that BM neutrophils express only polysialylated CD56, whereas a subset of BM monocytes expresses polysialylated CD56 and/or another polysialylated protein(s). We demonstrate that polysialylated CD56 expression is progressively down-regulated in wild-type monocytes and monocyte-derived cells during migration from BM through peripheral blood to pulmonary and peritoneal sites of inflammation. Freshly isolated monocyte-derived peritoneal macrophages are devoid of polySia yet re-express polySia on NRP-2 and an additional protein(s) after maintenance in culture. Removal of polySia from these cells enhances phagocytosis of Klebsiella pneumoniae, suggesting that down-regulation of polySia on macrophages facilitates bacterial clearance. Using wild-type and NRP-2(-/-) mice, we demonstrate that NRP-2 and an additional protein(s) are polysialylated by ST8 SiaIV in BM-derived DCs. We conclude that polySia expression in monocyte-derived cells is dynamically regulated by ST8 SiaIV activity and by expression of carrier proteins during recruitment to sites of inflammation and influences cellular interactions with microbes, contributing to innate and adaptive immune responses.

Enhancement of polysialic acid expression improves function of embryonic stem-derived dopamine neuron grafts in Parkinsonian mice.

There has been considerable progress in obtaining engraftable embryonic stem (ES) cell-derived midbrain dopamine neurons for cell replacement therapy in models of Parkinson's disease; however, limited integration and striatal reinnervation of ES-derived grafts remain a major challenge for future clinical translation. In this paper, we show that enhanced expression of polysialic acid results in improved graft efficiency in correcting behavioral deficits in Parkinsonian mice. This result is accompanied by two potentially relevant cellular changes: greater survival of transplanted ES-derived dopamine neurons and robust sprouting of tyrosine hydroxylase-positive processes into host tissue. Because the procedures used to enhance polysialic acid are easily translated to other cell types and species, this approach may represent a general strategy to improve graft integration in cell-based therapies.

Depletion of polysialic acid from neural cell adhesion molecule (PSA-NCAM) increases CA3 dendritic arborization and increases vulnerability to excitotoxicity.

Chronic immobilization stress (CIS) shortens apical dendritic trees of CA3 pyramidal neurons in the hippocampus of the male rat, and dendritic length may be a determinant of vulnerability to stress. Expression of the polysialylated form of neural cell adhesion molecule (PSA-NCAM) in the hippocampal formation is increased by stress, while PSA removal by Endo-neuraminidase-N (endo-N) is known to cause the mossy fibers to defasciculate and synapse ectopically in their CA3 target area. We show here that enzymatic removal of PSA produced a remarkable expansion of dendritic arbors of CA3 pyramidal neurons, with a lesser effect in CA1. This expansion eclipsed the CIS-induced shortening of CA3 dendrites, with the expanded dendrites of both no-stress-endo-N and CIS-endo-N rats being longer than those in no-stress-control rats and much longer than those in CIS-control rats. As predicted by the hypothesis that endo-N-induced dendritic expansion might increase vulnerability to excitotoxic challenge, systemic injection with kainic acid, showed markedly increased neuronal degeneration, as assessed by fluorojade B histochemistry, in rats that had been treated with endo-N compared to vehicle-treated rats throughout the entire hippocampal formation. PSA removal also exacerbated the CIS-induced reduction in body weight and abolished effects of CIS on NPY and NR2B mRNA levels. These findings support the hypothesis that CA3 arbor plasticity plays a protective role during prolonged stress and clarify the role of PSA-NCAM in stress-induced dendritic plasticity.

Enzymatic engineering of polysialic acid on cells in vitro and in vivo using a purified bacterial polysialyltransferase.

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST(Nm)) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST(Nm) synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST(Nm) and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST(Nm) requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST(Nm) was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST(Nm)-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST(Nm) occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST(Nm)-based approach may provide a valuable alternative to PST gene therapy.

Extensive cell migration, axon regeneration, and improved function with polysialic acid-modified Schwann cells after spinal cord injury.

Schwann cell (SC) implantation after spinal cord injury (SCI) promotes axonal regeneration, remyelination repair, and functional recovery. Reparative efficacy, however, may be limited because of the inability of SCs to migrate outward from the lesion-implant site. Altering SC cell surface properties by overexpressing polysialic acid (PSA) has been shown to promote SC migration. In this study, a SCI contusion model was used to evaluate the migration, supraspinal axon growth support, and functional recovery associated with polysialyltransferase (PST)-overexpressing SCs [PST-green fluorescent protein (GFP) SCs] or controls (GFP SCs). Compared with GFP SCs, which remained confined to the injection site at the injury center, PST-GFP SCs migrated across the lesion:host cord interface for distances of up to 4.4 mm within adjacent host tissue. In addition, with PST-GFP SCs, there was extensive serotonergic and corticospinal axon in-growth within the implants that was limited in the GFP SC controls. The enhanced migration of PST-GFP SCs was accompanied by significant growth of these axons caudal to lesion. Animals receiving PST-GFP SCs exhibited improved functional outcome, both in the open-field and on the gridwalk test, beyond the modest improvements provided by GFP SC controls. This study for the first time demonstrates that a lack of migration by SCs may hinder their reparative benefits and that cell surface overexpression of PSA enhances the ability of implanted SCs to associate with and support the growth of corticospinal axons. These results provide further promise that PSA-modified SCs will be a potent reparative approach for SCI. © 2012 Wiley Periodicals, Inc.

Neural cell adhesion molecule and its polysialic acid moiety exhibit opposing and linked effects on neuropathic hyperalgesia.

Spinal lamina II, where nociceptive C-fibers terminate, expresses high amounts of the polysialylated form of neural cell adhesion molecule (PSA-NCAM). While enzymatic removal of the PSA moiety from NCAM did not affect normal sensitivity to thermal stimuli, it exacerbated nerve injury-induced neuropathic hyperalgesia. The genetic removal of the NCAM core protein also did not alter thermal sensitivity. However in the presence of a peripheral nerve injury, NCAM-null mutants exhibited a complete suppression of thermal hyperalgesia. This strong NCAM mutant phenotype appears to involve the long form of NCAM's cytoplasmic domain, in that it is duplicated by selective genetic deletion of the NCAM-180 isoform. PSA appears therefore to provide a mechanism for modulation of chronic sensory overload, by means of attenuation of the activity of the NCAM-180 isoform, which reduces nociceptive transmission.

Physical and functional cooperation of neural cell adhesion molecule and ß1-integrin in neurite outgrowth induction.

Neural cell adhesion molecule (NCAM) and ß1-integrin are both involved in cell differentiation, with changes in the expression of these two molecules correlating with changes in the malignancy of tumor cells. There is a known functional correlation between NCAM and ß1-integrin in adhesion and also neurite outgrowth in tumor cells. In the present study, we used immunostaining and immunoprecipitation studies to demonstrate that isoform 120 of NCAM associates physically as well as functionally with ß1-integrin in the induction of neurite outgrowth in SH-SY5Y-human neuroblastoma cells. The interaction between these two molecules is mandatory for neurite outgrowth. NCAM blockage completely inhibits the effects of ß1-integrin on neurite outgrowth. These findings further our understanding of the interactions between NCAMs and integrins in malignancy.

Removal of polysialylated neural cell adhesion molecule increases morphine analgesia and interferes with tolerance in mice.

Neurons that express high levels of polysialylated neural cell adhesion molecule (PSA-NCAM) in adult spinal substantia gelatinosa also express the µ-opioid receptor. While PSA removal from NCAM by spinal intrathecal injection of endoneuraminidase-N (endo-N) did not detectably change opioid receptor expression, morphine-induced analgesia was significantly increased. This analgesic strengthening was detected as early as 15 min after endo-N treatment and persisted for at least 7 days. In addition, the tolerance that develops with chronic morphine treatment was overcome in the absence of PSA. Interestingly, the same effects on analgesia and tolerance were also produced by selective deletion of the NCAM-180 isoform.

Brain-derived neurotrophic factor levels influence the balance of migration and differentiation of subventricular zone cells, but not guidance to the olfactory bulb.

New progenitor cells in the subventricular zone (SVZ) migrate rostrally and differentiate into interneurons in the olfactory bulb (OB) throughout life. Brain-derived neurotrophic factor (BDNF) may influence the normal progression of this migration. In the present study, mouse SVZ explant cultures were used to investigate how BDNF modulates the behavior of these migrating progenitors. Concentrations of BDNF in the physiological range (e.g. 1ng/mL) stimulated migration, whereas doses of 10 ng/mL or higher induced SVZ cell differentiation and reduced migration. Pharmacological inhibition of the mitogen-activated protein kinase (MAPK) pathway blocked the BDNF-induced differentiation of SVZ progenitors, indicating that differentiation of SVZ progenitors in response to high-dose BDNF is initiated through MAPK. Physiological concentrations of BDNF, like the presence of polysialic acid in the tissue, stimulated migration of cells from the explant without affecting the speed at which this occurs. Interestingly, in vivo immunohistochemical and molecular analysis showed similar levels of BDNF in both the SVZ and OB; that is, there was no positive gradient attracting SVZ cells towards the OB. Our data show that SVZ cells respond differently to different concentrations of BDNF.

WITHDRAWN: Use of PSA-NCAM in Repair of the Central Nervous System.

Polysialic acid (PSA) is a highly hydrated polymer whose presence at the cell surface can reduce cell interactions, and thereby increase tissue and cellular plasticity. Given its ability to create a permissive environment for cell migration and axonal growth, the potential of engineered over-expression of PSA to promote tissue repair has been explored in the adult CNS. Several promising results have been obtained that suggest that PSA engineering may become a valuable therapeutic tool.

Reorganization of dorsal root ganglion neurons following chronic sciatic nerve constriction injury: correlation with morphine and lidocaine analgesia.

Chronic constriction injury of the sciatic nerve is an animal model for neuropathic pain. In this model, the analgesic potency of systemic morphine was significantly diminished in nerve-injured mice (ED(50) 19.4 mg/kg) compared with sham-operated mice (ED(50) 3.3 mg/kg) using a unilateral hot plate withdrawal test, with a similar reduction in sensitivity of intrathecal morphine. The sciatic nerve injury resulted in a reorganization of the dorsal root ganglion (DRG) neurons. Immunohistochemically, the chronic constriction injury triggered a withdrawal of C-fibers from the ipsilateral dorsal horn of the spinal cord. Although A-beta terminals centrally sprouted into Lamina II of the dorsal horn of the spinal cord, the peripheral A-beta fibers in the skin retracted from the epidermis to deeper layers of the dermis. To explore the functional significance of these dermal changes, we examined the topical morphine and lidocaine analgesia following chronic sciatic nerve constriction. Both morphine and lidocaine retained topical activity following chronic sciatic nerve injury, but their analgesic dose-response curves were shifted to the right when compared to sham-operated mice. Thus, the chronic nerve constriction injury model is associated with pathological changes in distribution of the central and peripheral axons of the dorsal root ganglion neurons that correspond to a decreased pharmacological sensitivity to topical analgesic agents.

Use of polysialic acid in repair of the central nervous system.

Polysialic acid (PSA), a large cell-surface carbohydrate that regulates cell interactions, is used during vertebrate development to promote precursor cell migration and axon path-finding. The induction of PSA expression in damaged adult CNS tissues could help them to rebuild by creating conditions permissive for architectural remodeling. This possibility has been explored in two contexts, the regeneration of axons and the recruitment of endogenous neural precursors to a lesion. Glial scars that form at CNS injury sites block axon regeneration. It has been found that transfection of scar astrocytes by a viral vector encoding polysialyltransferase leads to sustained expression of high levels of PSA. With this treatment, a substantial portion of severed corticospinal tract axon processes were able to grow through a spinal injury site. In the studies of precursor cell migration to a cortical lesion, it was found that induced PSA expression in a path extending from the subventricular zone to a lesion near the cortical surface increased recruitment of BrdU/nestin-positive cells along the path and into the injury site. These displaced precursors were able to differentiate in a regionally appropriate manner. These findings suggest that induced PSA expression can be used as a strategy for promoting tissue repair involving both replacement of cells and rebuilding of neural connections.

Polysialic acid-induced plasticity reduces neuropathic insult to the central nervous system.

Under chronic conditions of neuropathic pain, nociceptive C terminals are lost from their target region in spinal lamina II, leading to reduced thermal hyperalgesia. This region of the spinal cord expresses high levels of polysialic acid (PSA), a cell surface carbohydrate known to weaken cell-cell interactions and promote plasticity. Experimental removal of PSA from the spinal cord exacerbates hyperalgesia and results in retention of C terminals, whereas it has no effect on plasticity of touch Abeta fibers and allodynia. We propose that expression of PSA at this stress pathway relay point could serve to protect central circuitry from chronic sensory overload.

Polysialic acid regulates cell contact-dependent neuronal differentiation of progenitor cells from the subventricular zone.

Expression of polysialic acid (PSA) promotes migration of progenitor cells from the subventricular zone (SVZ) to the olfactory bulb, where they differentiate into interneurons. This differentiation has been found to coincide with a loss of PSA. Moreover, specific removal of PSA from the mouse SVZ by endoneuraminidase-N was found to cause premature differentiation, as evidenced by neurite outgrowth and tyrosine hydroxylase synthesis in vivo and by expression of neurofilament-L and beta III-tubulin in SVZ explant cultures. This differentiation involved activation of mitogen-activated protein kinase through p59fyn and was blocked by its inhibition. The effects of PSA removal were found to be cell contact-dependent and to be reduced by anti-neural cell adhesion molecule antibodies. These findings indicate that PSA expression regulates the fate of SVZ precursors by two contact-dependent mechanisms, the previously reported reduction in cell-cell adhesion that allows cell translocation, and the postponement of cell differentiation that otherwise would be induced by signals generated through surface molecule-mediated cell-cell interactions.

Removal of polysialic acid induces aberrant pathways, synaptic vesicle distribution, and terminal arborization of retinotectal axons.

Developing chick retinotectal projections extend rostrally in the superficial stratum opticum of the tectum until they reach their appropriate target zone. They then penetrate, arborize, and form synapses within distinct tectal retinorecipient layers. In this study, we show that the polysialylated neural cell adhesion molecule is expressed both on the membrane of these developing projections and in the stratum opticum and retinorecipient layers during the period of optic innervation. On this basis, the role of polysialic acid was analyzed with respect to both trajectory and arborization in the tectum, using confocal imaging of DiI-labeled retinotectal fibers in whole-mount tecta of embryos pretreated with a polysialic acid-specific degrading enzyme, endoneuraminidase N. The removal of polysialic acid caused several distinct abnormalities, including random dorsal/ventral meandering of fibers in the stratum opticum, a distorted branching and extension of arbors in the retinorecipient layers, and inappropriate synaptic vesicle accumulation in pretarget areas. These findings indicate that the unique ability of polysialic acid to regulate different types of cell interactions is an essential component of axon behavior during multiple steps of tectal target innervation.