Elena N. Kozlova

Central neuron–glial and glial–glial interactions following axon injury

Axon injury rapidly activates microglial and astroglial cells close to the axotomized neurons. Following motor axon injury, astrocytes upregulate within hour(s) the gap junction protein connexin-43, and within one day glial fibrillary acidic protein (GFAP). Concomitantly, microglial cells proliferate and migrate towards the axotomized neuron perikarya. Analogous responses occur in central termination territories of peripherally injured sensory ganglion cells. The activated microglia express a number of inflammatory and immune mediators. When neuron degeneration occurs, microglia act as phagocytes. This is uncommon after peripheral nerve injury in the adult mammal, however, and the functional implications of the glial cell responses in this situation are unclear. When central axons are injured, the glial cell responses around the affected neuron perikarya appears to be minimal or absent, unless neuron degeneration occurs. Microglia proliferate, and astrocytes upregulate GFAP along central axons undergoing anterograde, Wallerian, degeneration. Although microglia develop into phagocytes, they eliminate the disintegrating myelin very slowly, presumably because they fail to release molecules which facilitate phagocytosis. During later stages of Wallerian degeneration, oligodendrocytes express clusterin, a glycoprotein implicated in several conditions of cell degeneration. A hypothetical scheme for glial cell activation following axon injury is discussed, implying the injured neurons initially interact with adjacent astrocytes. Subsequently, neighbouring resting microglia are activated. These glial reactions are amplified by paracrine and autocrine mechanisms, in which cytokines appear to be important mediators. The specific functional properties of the activated glial cells will determine their influence on neuronal survival, axon regeneration, and synaptic plasticity. The control of the induction and progression of these responses are therefore likely to be critical for the outcome of, for example, neurotrauma, brain ischemia and chronic neurodegenerative diseases.

Metastasis‐associated Mts1 (S100A4) protein is selectively expressed in white matter astrocytes and is up‐regulated after peripheral nerve or dorsal root injury

The S100 family of calcium binding proteins has been shown to be involved in a variety of physiological functions, such as regulation of enzyme function, cell motility, modification of extracellular matrix, and cell proliferation. Several members of the S100 family are expressed in the nervous system, but their functional roles are still largely obscure. The Mts1 gene codes for the S100A4 protein, which has been implicated in the control of cell proliferation and metastasis activity of tumor cells. We have used immunohistochemistry to examine the expression pattern of the Mts1 protein in the adult rat spinal cord and how this expression is influenced by peripheral nerve or dorsal root injury. Mts1 immunoreactivity (IR) was present only in white matter astrocytes in the intact spinal cord. Sciatic nerve as well as dorsal root injury induced a marked and prolonged up‐regulation of Mts1‐IR in astrocytes in the region of the dorsal funiculus containing the central processes of the injured primary sensory neurons. These findings suggest that Mts1 plays a unique physiological role in white matter astrocytes as well as in the response of astrocytes to degeneration of myelinated axons. GLIA 27:249–258, 1999.

Human dorsal root ganglion neurons from embryonic donors extend axons into the host rat spinal cord along laminin-rich peripheral surroundings of the dorsal root transitional zone.

Following dorsal root crush, the lesioned axons regenerate in the peripheral compartment of the dorsal root, but stop at the boundary between the peripheral and the central nervous system, the dorsal root transitional zone. We have previously shown that fibres from human fetal dorsal root ganglia grafted to adult rat hosts are able to grow into the spinal cord, but were not able to specify the route taken by the ingrowing fibres. In this study we have challenged the dorsal root transitional zone astrocyte boundary with human dorsal root ganglion transplants from 5–8-week-old embryos. By tracing immunolabelled human fibres in serial sections, we found that fibres consistently grow around the dorsal root transitional zone astrocytes in laminin-rich peripheral surroundings, and extend into the host rat spinal cord along blood vessels, either into deep or superficial laminae of the dorsal horn, or into the dorsal funiculus. Human fibres that did not have access to blood vessels grew on the spinal cord surface. These findings indicate, that in spite of a substantial growth capacity by axons from human embryonic dorsal root ganglion cells as well as their tolerance to non-permissive factors in the mature mammalian CNS, these axons are still sensitive to the repellent effects of astrocytes of the mature dorsal root transitional zone. Furthermore, this axonal ingrowth is consistently associated with laminin-expressing structures until the axons reach the host spinal cord.

Allografted fetal dorsal root ganglion neuronal survival in the guinea pig cochlea.

Neural grafting is a potential strategy to help restore auditory function following loss of spiral ganglion cells. As a first step towards the reconstruction of a neural pathway from the cochlea to the brainstem, we have examined the survival of fetal dorsal root ganglion (DRG) neurons allografted into the cochlea of adult guinea pigs. In some animals implantation of DRGs was combined with a local infusion of neurotrophic substances whereas in others auditory sensory receptors were chemically destroyed prior to DRG implantation by injection of the ototoxin neomycin into the middle ear. The results show that many transplanted DRG neurons attached close to the cochlear spiral ganglion neurons. The survival of the implant was significantly increased by treatment with neurotrophic factors, but not reduced by the absence of auditory sensory structures. This study shows that implanted sensory neurons can survive heterotopic grafting immediately adjacent to the eighth cranial nerve, thereby providing a basis for further studies of the anatomical and functional influence of neural grafts in the inner ear.

Neural crest stem cells increase beta cell proliferation and improve islet function in co-transplanted murine pancreatic islets

Aims/hypothesisLong-term graft survival after islet transplantation to patients with type 1 diabetes is insufficient, necessitating the development of new strategies to enhance transplant viability. Here we investigated whether co-transplantation of neural crest stem cells (NCSCs) with islets improves islet survival and function in normoglycaemic and diabetic mice.MethodsIslets alone or together with NCSCs were transplanted under the kidney capsule to normoglycaemic or alloxan-induced diabetic mice. Grafts were analysed for size, proliferation, apoptosis and insulin release. In diabetic recipients blood glucose levels were examined before and after graft removal.ResultsIn mixed transplants NCSCs actively migrated and extensively associated with co-transplanted pancreatic islets. Proliferation of beta cells was markedly increased and transplants displayed improved insulin release in normoglycaemic mice compared with those receiving islet-alone transplants. Mixed grafts survived successfully and partially restored normoglycaemia in alloxan-induced diabetic mice.Conclusions/interpretationCo-grafting of NCSCs with pancreatic islets improved insulin release in mixed transplants and enhanced beta cell proliferation, resulting in increased beta cell mass. This co-transplantation model offers an opportunity to restore neural–islet interactions and improve islet functions after transplantation.

Functional connections are established in the deafferented rat spinal cord by peripherally transplanted human embryonic sensory neurons

Functionally useful repair of the mature spinal cord following injury requires axon growth and the re‐establishment of specific synaptic connections. We have shown previously that axons from peripherally grafted human embryonic dorsal root ganglion cells grow for long distances in adult host rat dorsal roots, traverse the interface between the peripheral and central nervous system, and enter the spinal cord to arborize in the dorsal horn. Here we show that these transplants mediate synaptic activity in the host spinal cord. Dorsal root ganglia from human embryonic donors were transplanted in place of native adult rat ganglia. Two to three months after transplantation the recipient rats were examined anatomically and physiologically. Human fibres labelled with a human‐specific axon marker were distributed in superficial as well as deep laminae of the recipient rat spinal cord. About 36% of the grafted neurons were double labelled following injections of the fluorescent tracers MiniRuby into the sciatic and Fluoro‐Gold into the lower lumbar spinal cord, indicating that some of the grafted neurons had grown processes into the spinal cord as well as towards the denervated peripheral targets. Electrophysiological recordings demonstrated that the transplanted human dorsal roots conducted impulses that evoked postsynaptic activity in dorsal horn neurons and polysynaptic reflexes in ipsilateral ventral roots. The time course of the synaptic activation indicated that the human fibres were non‐myelinated or thinly myelinated. Our findings show that growing human sensory nerve fibres which enter the adult deafferentated rat spinal cord become anatomically and physiologically integrated into functional spinal circuits.

Regulation of Boundary Cap Neural Crest Stem Cell Differentiation After Transplantation

Success of cell replacement therapies for neurological disorders will depend largely on the optimization of strategies to enhance viability and control the developmental fate of stem cells after transplantation. Once transplanted, stem/progenitor cells display a tendency to maintain an undifferentiated phenotype or differentiate into inappropriate cell types. Gain and loss of function experiments have revealed key transcription factors which drive differentiation of immature stem/progenitor cells toward more mature stages and eventually to full differentiation. An attractive course of action to promote survival and direct the differentiation of transplanted stem cells to a specific cell type would therefore be to force expression of regulatory differentiation molecules in already transplanted stem cells, using inducible gene expression systems which can be controlled from the outside. Here, we explore this hypothesis by employing a tetracycline gene regulating system (Tet‐On) to drive the differentiation of boundary cap neural crest stem cells (bNCSCs) toward a sensory neuron fate after transplantation. We induced the expression of the key transcription factor Runx1 in Sox10‐expressing bNCSCs. Forced expression of Runx1 strongly increased transplant survival in the enriched neurotrophic environment of the dorsal root ganglion cavity, and was sufficient to guide differentiation of bNCSCs toward a nonpeptidergic nociceptive sensory neuron phenotype both in vitro and in vivo after transplantation. These findings suggest that exogenous activation of transcription factors expression after transplantation in stem/progenitor cell grafts can be a constructive approach to control their survival as well as their differentiation to the desired type of cell and that the Tet‐system is a useful tool to achieve this. STEM CELLS 2009;27:1592–1603

Role of intracellular S100A4 for migration of rat astrocytes

S100A4 is a member of the EF‐hand family of calcium‐binding proteins, first identified in tumor cells, and implicated in tumor invasion and metastasis. Intracellular upregulation of S100A4 is associated with increased motility of tumor cells. Extracellular application of S100A4 increases the motility of glioma cells in vitro. We showed previously that astrocytes in spinal cord and brain white matter also express S100A4. This expression is markedly increased in reactive white matter astrocytes after injury. Here, we have explored how changes in intracellular S100A4 affect migration of astrocytes. We produced cultures of white matter, S100A4 expressing astrocytes, and developed a small interfering (si) RNA approach to specifically eliminate S100A4 expression in these cells, and compared the migration of astrocytes expressing S100A4 with astrocytes transfected with S100A4 siRNA. As a “positive control” we used S100A4 expressing C6 glioma cells. In contrast to malignant cells, S100A4 expressing astrocytes increased their migration capacity after S100A4 siRNA treatment. At the same time, and in parallel with increased migration, white matter astrocytes increased their expression of metalloproteinases MMP‐9 and MT1‐MMP. The addition of MMP‐2/MMP‐9 inhibitor resulted in a significant inhibition of migration in S100A4 siRNA‐treated astrocytes. These findings indicate that S100A4 has a stabilizing function in reactive white matter astrocytes, a function that may contribute to the development of a rigid, growth‐inhibitory glial scar.

Metastasis-associated S100A4 (Mts1) protein is expressed in subpopulations of sensory and autonomic neurons and in Schwann cells of the adult rat

S100A4 (Mts1) is a member of a family of calcium‐binding proteins of the EF‐hand type, which are widely expressed in the nervous system, where they appear to be involved in the regulation of neuron survival, plasticity, and response to injury or disease. S100A4 has previously been demonstrated in astrocytes of the white matter and rostral migratory stream of the adult rat. After injury, S100A4 is markedly up‐regulated in affected central nervous white matter areas as well as in the periventricular area and rostral migratory stream. Here, we show that S100A4 is expressed in a subpopulation of dorsal root, trigeminal, geniculate, and nodose ganglion cells; in a subpopulation of postganglionic sympathetic and parasympathetic neurons; in chromaffin cells of the adrenal medulla; and in satellite and Schwann cells. In dorsal root ganglia, S100A4‐positive cells appear to constitute a subpopulation of small ganglion neurons, a few of which coexpressed calcitonin gene‐related peptide (CGRP) and Griffonia simplicifolia agglutinin (GSA) isolectin B4 (B4). S100A4 protein appears to be transported from dorsal root ganglia to the spinal cord, where it is deposited in the tract of Lissauer. After peripheral nerve or dorsal root injury, a few S100A4‐positive cells coexpress CGRP, GSA, or galanin. Peripheral nerve or dorsal root injury induces a marked up‐regulation of S100A4 expression in satellite cells in the ganglion and in Schwann cells at the injury site and in the distal stump. This pattern of distribution partially overlaps that of the previously studied S100B and S100A6 proteins, indicating a possible functional cooperation between these proteins. The presence of S100A4 in sensory neurons, including their processes in the central nervous system, suggests that S100A4 is involved in propagation of sensory impulses in specific fiber types. J. Comp. Neurol. 473:233–243, 2004.

Mts1 protein expression in the central nervous system after injury

We recently showed that Mts1 is expressed in white matter astrocytes in the rat brain and spinal cord from the first postnatal day. Its expression level declined in the adult CNS, but its topographical localization was maintained. Only white matter astrocytes in the cerebellum did not express Mts1. After dorsal root or sciatic nerve injury, we observed a marked upregulation of Mts1 in the area of the dorsal funiculus undergoing Wallerian degeneration. Here we show that upregulation of Mts1 is a consistent feature of astrocytes in white matter undergoing Wallerian degeneration. In addition, Mts1 is upregulated in astrocytes outlining the lesion site of a penetrating injury to the forebrain, or cerebellum. Gray matter astrocytes did not express Mts1, even after direct injury. In injured brain, we consistently noted a close relationship between Mts1‐expressing astrocytes and ED1‐positive microglia/macrophages, which are known to be highly motile cells. Mts1 was expressed in the periventricular area and the rostral migratory stream, i.e., sites of ongoing neuroplasticity in adulthood, and was upregulated in these areas after injury. These data suggest that Mts1‐expressing astrocytes play a significant role in degenerative events in the mature white matter, interact with phagocytic microglia/macrophages and regulate cell migration and differentiation in areas of the adult brain with a high degree of plasticity. GLIA 37:337–348, 2002.