Andras Lakatos

Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes

Transplanted olfactory ensheathing cells (OECs) are able to remyelinate demyelinated axons and support regrowth of transected axons after transplantation into the adult CNS. Transplanted Schwann cells (SCs) share these repair properties but have limitations imposed on their behavior by the presence of astrocytes (ACs). Because OECs exist alongside astrocytes in the olfactory bulb, we have hypothesized that they have advantages over SCs in transplant‐mediated CNS repair due to an increased ability to integrate and migrate within an astrocytic environment. In this study, we have tested this hypothesis by comparing the interactions between astrocytes and either SCs or OECs, using a range of in vitro assays. We have shown that (1) astrocytes and SCs segregate into defined non‐overlapping domains in co‐culture, whereas astrocytes and OECs freely intermingle; (2) both SCs and OECs will migrate across astrocyte monolayers, but only OECs will migrate into an area containing astrocytes; (3) SCs spend less time in contact with astrocytes than do OECs; and (4) astrocytes undergo hypertrophy when in contact with SCs, but not with OECs. Expression of N‐cadherin has been implicated as a key mediator of the failure of SCs to integrate with astrocytes. However, we found no differences in the intensity of N‐cadherin immunoreactivity between SCs and OECs, suggesting that it is not the adhesion molecule that accounts for the observed differences. In addition, the number of astrocytes expressing chondroitin sulfate proteoglycans (CSPG) is increased when astrocytes are co‐cultured with Schwann cells compared with the number when astrocytes are grown alone or with OECs. Taken together, these data support the hypothesis that OECs will integrate more extensively than Schwann cells in astrocytic environments and are therefore better candidates for transplant‐mediated repair of the damaged CNS. GLIA 32:214–225, 2000.

Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than schwann cells following transplantation into adult cns white matter

Both Schwann cells and olfactory ensheathing cells (OECs) create an environment favorable to axon regeneration when transplanted into the damaged CNS. However, transplanted cells can also exert an effect on the host tissue that will influence the extent to which regenerating axons can grow beyond the transplanted area and reenter the host environment. In this study equivalent numbers of Lac-Z-labeled Schwann cells and OECs have been separately transplanted into normal white matter of adult rat spinal cord and the host astrocyte response to each compared. Schwann cell transplantation resulted in a greater area of increased glial fibrillary acidic protein (GFAP) expression compared to that associated with OEC transplantation. This was accompanied by a greater increase in the expression of axon growth inhibitory chrondroitin sulfate proteoglycans (CSPGs) following Schwann cell transplantation compared to OEC transplantation. However, no differences were detected in the increased expression of the specific CSPG neurocan following transplantation of the two cell types. These results mirror differences in the interactions between astrocytes and either Schwann cells or OECs observed in tissue culture models and reveal one aspect of the complex biology of creating regeneration-promoting environments by cell transplantation where transplanted OECs have favorable properties compared to transplanted Schwann cells.

Superparamagnetic Iron Oxide-Labeled Schwann Cells and Olfactory Ensheathing Cells Can Be Traced In Vivo by Magnetic Resonance Imaging and Retain Functional Properties after Transplantation into the CNS

Schwann cell (SC) and olfactory ensheathing cell (OEC) transplantation has been shown experimentally to promote CNS axonal regeneration and remyelination. To advance this technique into a clinical setting it is important to be able to follow the fates of transplanted cells by noninvasive imaging. Previous studies, using complex modification processes to enable uptake of contrast agents, have shown that cells labeled in vitro with paramagnetic contrast agents transplanted into rodent CNS can be visualized using magnetic resonance imaging (MRI). Here we show that SCs and OECs efficiently internalize dextran-coated superparamagnetic iron oxide (SPIO) from the culture medium by fluid phase pinocytosis. After transplantation into focal areas of demyelination in adult rat spinal cord both transplanted SPIO-labeled SCs and OECs produce a signal reduction using T2-weighted MRI in anesthetized rats that persists for up to 4 weeks. Although signal reduction was discernable after transplantation of unlabelled cells, this is nevertheless distinguishable from that produced by transplanted labeled cells. The region of signal reduction in SPIO-labeled cell recipients correlates closely with areas of remyelination. Because the retention of functional integrity by labeled cells is paramount, we also show that SPIO-labeled SCs and OECs are able to myelinate normally after transplantation into focal areas of demyelination. These studies demonstrate the feasibility of noninvasive imaging of transplanted SCs and OECs and represent a significant step toward the clinical application of promising experimental approaches.

Cryopreserved cells isolated from the adult canine olfactory bulb are capable of extensive remyelination following transplantation into the adult rat CNS

Naturally occurring spinal cord injury in dogs provides a potentially powerful intermediate model for testing the efficacy of therapeutic strategies developed in experimental rodent models before phase 1 trials in human patients. A particularly promising strategy involves transplantation of olfactory ensheathing cells (OECs) that both promote axon regeneration and generate new myelin sheaths. As a first step in developing OEC transplantation in the canine intermediate model we describe the isolation, purification, and characterization of OECs from adult dog olfactory bulb. We also show that the canine OEC behaves in a manner similar to its rodent counterpart following transplantation into demyelinating lesions in rat spinal cord and that these properties are retained following cryopreservation.

Clinical canine spinal cord injury provides an opportunity to examine the issues in translating laboratory techniques into practical therapy.

Study design:Review.Objectives:To highlight the value of investigating the effects of putative therapeutic interventions in clinical spinal cord injury (SCI) in domestic dogs.Setting:England, UK.Methods:Many experimental interventions in laboratory rodents have been shown to ameliorate the functional deficits caused by SCI; the challenge now is to determine whether they can be translated into useful clinical techniques. Important differences between clinical SCI in human patients and that in laboratory rodents are in the size of the spinal cord and heterogeneity of injury severity. A further key issue is whether the statistical difference in outcome in the laboratory will translate into a useful difference in clinical outcome. Here, we stress the value of investigating the effects of putative therapies in clinical SCI in domestic dogs. The causes of injury, ability to categorise the severity and methods available to measure outcome are very similar between canine and human patients. Furthermore, postmortem tissue more rapidly becomes available from dogs because of their short lifespan than from human patients.Results:The role that investigation of canine SCI might play is illustrated by our preliminary trials on intraspinal transplantation of olfactory glial cells for severe SCI.Conclusions:This canine translational model provides a means of ‘filtering’ putative treatments before human application.Sponsorship:Our work described here was supported by the International Spinal Research Trust.

Increasing local levels of neuregulin (glial growth factor-2) by direct infusion into areas of demyelination does not alter remyelination in the rat CNS

Glial growth factor‐2 (GGF‐2) is a neuronally derived isoform of neuregulin shown in vitro to promote proliferation and survival of oligodendrocytes, the myelinating cells of the CNS. Enhanced remyelination has been demonstrated in vivo following systemic delivery of human recombinant GGF‐2 (rhGGF‐2) in experimental autoimmune encephalomyelitis (EAE). However, it is uncertain whether this is the result of direct effects of rhGGF‐2 on cells of the oligodendrocyte lineage or due to modulation of the immune or inflammatory response. If this enhanced remyelination was due to direct effects of rhGGF‐2 on cells of the oligodendrocyte lineage then one would expect rhGGF‐2 to induce a similar proremyelinating response in nonimmune, gliotoxin models of demyelination. Using a gliotoxin model of demyelination we were therefore able to ascertain the in vivo effect of rhGGF‐2 following local CNS delivery in a model that is not confounded by the concurrent presence of an immune‐mediated process. No significant alteration in the rate or character of remyelination was evident following local delivery as compared to controls, and indeed nor following systemic delivery in the gliotoxin model. The results of this study therefore indicate that both direct infusion and systemic delivery of rhGGF‐2 do not alter remyelination in a nonimmune, gliotoxin model of demyelination. This suggests that the proremyelinating effects of systemically delivered rhGGF‐2 in EAE are unlikely to be due to direct effects on the oligodendrocyte lineage, but may be mediated by rhGGF‐2 inducing an environment more favourable to remyelination, possibly through modulation of the immune response.

Astrocyte response to motor neuron injury promotes structural synaptic plasticity via STAT3-regulated TSP-1 expression

The role of remote astrocyte (AC) reaction to central or peripheral axonal insult is not clearly understood. Here we use a transgenic approach to compare the direct influence of normal with diminished AC reactivity on neuronal integrity and synapse recovery following extracranial facial nerve transection in mice. Our model allows straightforward interpretations of AC–neuron signalling by reducing confounding effects imposed by inflammatory cells. We show direct evidence that perineuronal reactive ACs play a major role in maintaining neuronal circuitry following distant axotomy. We reveal a novel function of astrocytic signal transducer and activator of transcription-3 (STAT3). STAT3 regulates perineuronal astrocytic process formation and re-expression of a synaptogenic molecule, thrombospondin-1 (TSP-1), apart from supporting neuronal integrity. We demonstrate that, through this new pathway, TSP-1 is responsible for the remote AC-mediated recovery of excitatory synapses onto axotomized motor neurons in adult mice. These data provide new targets for neuroprotective therapies via optimizing AC-driven plasticity.

Expression of the POU-Domain Transcription Factors SCIP/Oct-6 and Brn-2 Is Associated with Schwann Cell but Not Oligodendrocyte Remyelination of the CNS

The class III POU-domain transcription factor SCIP/Oct-6 is expressed by promyelinating Schwann cells and, in tissue culture, by oligodendrocyte progenitors (OPs), but is down-regulated in both cells types as they differentiate. Although the expression of SCIP/Oct-6 has been examined in peripheral nerve remyelination, its expression in CNS remyelination has not been addressed. Using a toxin model of demyelination, in which the demyelinated axons are remyelinated in an age-dependent manner by both oligodendrocytes and Schwann cells, we have compared the expression of SCIP/Oct-6 mRNA with that of an OP marker (PDGF-alphaR), a marker of myelinating oligodendrocytes (PLP), and markers of myelinating Schwann cells (P(0) and Krox-20) by in situ hybridization. We have found that the expression of SCIP/Oct-6 mRNA precedes that of P(0) and Krox-20 mRNA expression, but bears little correlation with the expression profiles of either PDGF-alphaR or PLP mRNA. Moreover, there is a spatial correlation between the expression SCIP/Oct-6 mRNA and that of P(0) but not of PDGF-alphaR. These results indicate that SCIP/Oct-6 expression following CNS demyelination is associated with Schwann cell and not oligodendrocyte remyelination. We have also shown that another POU-domain transcription factor, Brn-2, is expressed during CNS remyelination, but that like SCIP/Oct-6, it too has an expression profile indicating that it is associated with the Schwann cell component of remyelination. In addition, we show that Brn-2 expression in Schwann cells is not restricted to CNS remyelination but is also expressed in a similar manner to SCIP/Oct-6 during Schwann cell myelination of neonatal peripheral nerves and regenerating transected adult nerve and in cultured Schwann cells following induction of elevated cAMP levels.

Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS

Summary Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

New Aspects of the Neuroendocrine Role of PACAPa

The presence of PACAP was revealed in the anterior pituitary with RIA, HPLC, and with the demonstration of its mRNA. The level of PACAP mRNA in the anterior pituitary is the highest during the proestrous LH surge. In our immunohistochemical studies we were able to demonstrate PACAP immunoreactive cells in the anterior pituitary. The shape and the distribution of PACAP immunoreactive cells were very similar to that of the gonadotropes; however, the number of PACAP cells was less than that of LH cells. Additionally, another PACAP-positive cell population with small diameter appeared in the proestrous stage, during pregnancy and lactation. Double labeling revealed that the major part of large PACAP cells exhibited LH immunoreactivity and those with a small diameter contained PRL. It is not clear whether the pituitary- or the hypothalamic-born PACAP, or both, influence pituitary LH and PRL secretion. I.c.v. administration of PACAP just prior to the critical period in the proestrous stage inhibited the expected ovulation and blocked the proestrus LH and PRL surge, although i.v. administration of PACAP had no effect. PACAP antiserum did not interfere with ovulation when i.c.v. or i.v. injection was used. Our results support the view that PACAP has a role in the control of LH and PRL secretion during the estrous cycle, pregnancy, and lactation. The inhibitory effect of PACAP on ovulation is mediated through the hypothalamus.