Role Of S100A4 Protein In Sensory Neurite Outgrowth And Injury-induced Migration Of White Matter Astrocytes

Astrocytes are specialized to meet local functional requirements in different areas of the central nervous system (CNS). Whereas gray matter astrocytes mainly co-operate with neurons in synaptic networking white matter astrocytes are primarily involved in the maintenance of secure impulse propagation over long distance, projecting myelinated and non-myelinated axons. Astrocytes play a crucial role in CNS injury and disease, e.g., in the formation of a glial scar, and restoration of a disrupted glial limitans and blood-brain barrier. Reactive white matter astrocytes are key players in the development of a non-permissive environment to neurite growth, in the development of a glial scar following injury, and appear to be critical for efficient remyelination by oligodendrocytes following demyelination. White, but not gray matter astrocytes in the rat express the calcium-binding protein S100A4, which is strongly up-regulated exclusively in white matter after injury. The role of S100A4 protein in the CNS remains largely unknown.Part I : Sensory neurite outgrowth on white matter astrocytes is influenced by intracellular and extracellualr S100A4 proteinPrevious studies have shown that extracellular S100A4 is a potent stimulator of neurite outgrowth from embryonic hippocampal neurons and a neuroprotectant and neurite outgrowth stimulator of embryonic mesencephalic and early postnatal cerebellar neurons in vitro. However, these results were obtained in pure neuronal cultures, in the absence of neuron-astroglial interactions. Therefore, we wanted to explore whether such interactions alter the neuronal response to extracellular S100A4 in our coculture system, which more closely reflects the in vivo situation. Therefore, in addition to exploring how DRG cell neurites interact with white matter astrocytes, we have addressed the issue of how extracellular S100A4 influences DRG cell neurite outgrowth on S100A4-expressing white matter astrocytes.1. DRG Cells on Poly-L-LysineWe first determined the extent to which adult DRG cells developed neurites on poly-L-lysine in the absence of astrocytes. Adult DRGs showed good survival under these culture conditions but did not develop neurites during the 24-hr observation period.2. DRG Cell Neurite Outgrowth on Control siRNA-Treated Astrocyte CulturesIn the next series of experiments, we determined whether adult DRG cells developed neurites when cultured on white matter S100A4-expressing astrocytes. We examined the time course of neurite outgrowth on white matter astrocytes treated with control siRNA. These astrocytes expressed high levels of S100A4 but did not secrete S100A4. Six hours after coculture with S100A4-expressing astrocytes, dissociated DRG cells had not extended fibers on astrocytes. Twelve hours after coculture, neurite growth from DRG cells was observed on S100A4-expressing astrocytes. After 18 hr of coculture with S100A4-expressing astrocytes, neurite growth from DRGs was increased. After 24 hr of coculture, strong neurite growth was recorded on S100A4-positive astrocytes. These findings show that cultured white matter astrocytes support neurite outgrowth from adult DRG cells.3. DRG Cell Neurite Outgrowth on S100A4 siRNA-Treated Astrocyte CulturesTo explore the role of intracellular S100A4, we examined DRG cell neurite outgrowth on white matter astrocytes treated with S100A4 siRNA. Three days after this treatment, the expression of S100A4 is almost completely eliminated. At this time, dissociated adult DRG cells were placed on the astrocytes. Six hours after coculture with S100A4-silenced astrocytes, no neurite outgrowth from DRG neurons was recorded. Twelve hours after coculturing, extensive neurite growth from dissociated DRG cells was recorded. Eighteen hours after coculture, the neurite growth from DRG neurons was increased on S100A4-negative astrocytes, with an additional increase 24 hr after coculturing of DRG neurites. The quantitative analysis showed that neurite outgrowth at 12, 18. and 24 hr was significantly greater in S100A4 siRNA-treated compared with control siRNA-treated cultures. These findings indicate that down-regulation of S100A4 expression in white matter astrocytes promotes neurite outgrowth from adult DRG cells. 4. DRG Cells on S100A4-Treated S100A4-Expressing Astrocyte CulturesTo determine whether extracellular S100A4 protein influences DRG neurite outgrowth on white matter astrocytes, we prepared white matter astrocyte cultures and added recombinant S100A4 protein (5 μg/ml) to these cultures 2 hr before dissociated adult DRG cells were seeded on top of the astrocytes. This concentration has previously been shown to provide maximal neurite outgrowth in vitro. Six hours later, no neurite growth was recorded. Twelve hours after coculture, long neurites extended from the DRG neurons. At 18 hr in culture, extreme neurite outgrowth was observed on S100A4-expressing astrocytes. After 24 hr in culture, the growth of DRG fibers was still extensive. The quantitative analysis showed significantly greater neurite outgrowth in S100A4-treated cultures compared with control siRNA cultures at 12, 18, and 24 hr as well as S100A4 siRNA-treated cultures at 18 hr. Thus, in contrast to intracellular S100A4, extracellular S100A4 stimulates neurite outgrowth.Taken together, our findings show that DRG cell neurite extension is supported by S100A4-expressing as well as S100A4-silenced white matter astrocytes, with the latter being the most efficient ones. Furthermore, extracellular administration of S100A4 protein strongly supported DRG cell neurite growth on S100A4-expressing white matter astrocytes. Part II : Intracellular calcium-bingding protein S100A4 influences injury-induced migration of white matter astrocytesStudies have demonstrated that down-regulation of S100A4 in white matter astrocytes increases their migratory capacity in vitro. These findings suggest that S100A4 may play a role in how white matter astrocytes respond to pathological events, such as loss of normal tissue integrity. Here, we have addressed this issue in vitro by comparing the behavior of S100A4 positive and negative white matter astrocytes to a localized injury (scratch) in the culture dish, as well as in vivo, using mice lacking the S100A4 gene, and the corresponding wild type strain.1. White matter astroglial culturesAstrocytes prepared from the corpus callosum expressed high levels of S100A4. Three days after transfection with S100A4 siRNA, the expression of S100A4 was reduced by about 90%. Control siRNA transfected cultures showed no down-regulation of S100A4 protein expression. Twenty-four hours after the scratch injury, the astrocytes in control siRNA- treated cultures showed an up-regulation of S100A4 in the vicinity of the injury place, as well as marked process extension toward the injured area. Forty-eight hours after the scratch injury, astrocytes expressing high levels of S100A4 extended their fibers toward the injury from both sides of the injury gap, but without closing it. Following S100A4 siRNA treatment, S100A4 immunoreactivity (IR) was undetectable in the cultures at 24 and 48 h after scratch injury. Astrocytes had migrated to the injury gap at 24 h after the scratch injury, and after 48 h they completely occupied the gap of the scratch injury. The quantitative analysis confirmed the marked increase in the number of astrocytes present in the middle of the injury area at 24 as well as 48 h following treatment with S100A4 siRNA compared to control treated cultures.2. Ethidium bromide injectionsEthidium bromide (EB) injection resulted in extensive demyelination and disappearance of glial cells in the dorsal funiculus on the side of injection as well as to a small extent on the contralateral side in (+/+) and (-/-) mice. The lesion sites showed similar extensions in the transverse and longitudinal directions in both strains (not shown). Four days after injury the astrocyte reaction appeared similar in (+/+) and (-/-) mice, with the exception of some accumulation of astrocytes along the margins of the injury in (+/+) mice. Seven days after EB injection, GFAP immunoreactivity (IR) showed hypertrophied astrocytes at the border of the demyelinated area, in (+/+) mice. At this stage, GFAP-IR cells were already present within the demyelinated area in (-/-) mice.The results show that astrocyte migration into the demyelinated area is promoted in S100A4 (-/-) compared to (+/+) mice, in which a pronounced glial scar was formed. These data indicate that S100A4 reduces the migratory capacity of reactive white matter astrocytes in the injured CNS and is involved in glial scar formation after injury.