Claudia Grothe

Sequential myelin protein expression during remyelination reveals fast and efficient repair after central nervous system demyelination

To understand the mechanisms of remyelination and the reasons for regeneration failure is one of the major challenges in multiple sclerosis research. This requires a good knowledge and reliable analysis of experimental models. This work was undertaken to characterize the pattern of myelin protein expression during experimental remyelination. Acute demyelination of the corpus callosum was induced by feeding of 0.3% cuprizone for 6u2003weeks, followed by a 10‐week remyelination period. We used a combination of Luxol fast blue (LFB) myelin staining, electron microscopy (EM) and immunohistochemistry for the myelin proteins 2′,3′‐cyclic nucleotide 3′ phosphodiesterase (CNPase), myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG). Early remyelination was detected by the re‐expression of CNPase, MBP and PLP as early as 4u2003days. MOG, as a marker for late differentiation of oligodendrocytes, was not detectable until 2u2003weeks of remyelination. EM data correlated well with the LFB myelin staining and myelin protein expression, with 50% of the axons being rapidly remyelinated within 2u2003weeks. While particularly MBP but also PLP and CNPase are re‐expressed very early before significant remyelination is observed by EM, the late marker MOG shows a lag behind the remyelination detected by EM. The presented data indicate that immunohistochemistry for various myelin proteins expressed early and late during myelin formation is a suitable and reliable method to follow remyelination in the cuprizone model. Furthermore, investigation of early remyelination confirms that the intrinsic repair programme is very fast and switched on within days.

Differentially promoted peripheral nerve regeneration by grafted schwann cells over-expressing different FGF-2 isoforms

Artificial nerve grafts are needed to reconstruct massive defects in the peripheral nervous system when autologous nerve grafts are not available in sufficient amounts. Nerve grafts containing Schwann cells display a suitable substrate for long-distance regeneration. We present here a comprehensive analysis of the in vivo effects of different isoforms of fibroblast growth factor-2 (FGF-2) on peripheral nerve regeneration across long gaps. FGF-2 isoforms were provided by grafted, genetically modified Schwann cells over-expressing 18-kDa-FGF-2 and 21-/23-kDa-FGF-2, respectively. Functional tests evaluated motor and sensory recovery. Additionally, morphometrical analyses of regenerated nerves were performed 3 and 6 months after grafting. Distinct regeneration promoting effects of the different FGF-2 isoforms were found. 18-kDa-FGF-2 mediated inhibitory effects on the grade of myelination of regenerating axons, whereas 21-/23-kDa-FGF-2 mediated early recovery of sensory functions and stimulation of long-distance myelination of regenerating axons. The results contribute to the development of new therapeutic strategies in peripheral nerve repair.

Fibroblast Growth Factor (FGF)-2 and FGF Receptor 3 Are Required for the Development of the Substantia Nigra, and FGF-2 Plays a Crucial Role for the Rescue of Dopaminergic Neurons after 6-Hydroxydopamine Lesion

Basic fibroblast growth factor (FGF-2) is involved in the development and maintenance of the nervous system. Exogenous administration of FGF-2 increased dopaminergic (DA) graft survival in different animal models of Parkinsons disease. To study the physiological function of the endogenous FGF-2 system, we analyzed the nigrostriatal system of mice lacking FGF-2, mice overexpressing FGF-2, and FGF-receptor-3 (FGFR3)-deficient mice both after development and after 6-hydroxydopamine lesion. FGFR3-deficient mice (+/−) displayed a reduced number of DA neurons compared with the respective wild type. Whereas absence of FGF-2 led to significantly increased numbers of DA neurons, enhanced amount of the growth factor in mice overexpressing FGF-2 resulted in less tyrosine hydroxylase expression and a reduced DA cell density. The volumes of the substantia nigra were enlarged in both FGF-2−/− and in FGF-2 transgenic mice, suggesting an important role of FGF-2 for the establishment of the proper number of DA neurons and a normal sized substantia nigra during development. In a second set of experiments, the putative relevance of endogenous FGF-2 after neurotoxin application was investigated regarding the number of rescued DA neurons after partial 6-OHDA lesion. Interestingly, the results after lesion were directly opposed to the results after development: significantly less DA neurons survived in FGF-2−/− mice compared with wild-type mice. Together, the results indicate that FGFR3 is crucially involved in regulating the number of DA neurons. The lack of FGF-2 seems to be (over)compensated during development, but, after lesion, compensation mechanisms fail. The transgenic mice showed that endogenous FGF-2 protects DA neurons from 6-OHDA neurotoxicity.

Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol

We present a fast protocol that can be used to obtain highly purified cultures of proliferating adult human and rat Schwann cells accessible for non-viral transfection methods. The use of enriched genetically modified adult Schwann cells is of interest in the context of autologous cell transplantation within nerve transplants for peripheral nerve repair. Cell preparation from pre-degenerated adult peripheral nerves is described, together with the use of melanocyte growth medium plus forskolin, fibroblast growth factor-2 (FGF-2), pituitary extract and heregulin as a selective, serum-free culture medium and a subsequent cell enrichment step (cold jet). Proliferating adult Schwann cells can be efficiently genetically modified using optimized, non-viral electroporation protocols. The protocol results in Schwann cell cultures that are more than 90–95% pure, and transfection efficiencies vary depending on the initial cell constitution from 20 to 40%. The procedure takes up to 21 d, depending on the length of the pre-degeneration period.

Chitosan tubes of varying degrees of acetylation for bridging peripheral nerve defects

Biosynthetic nerve grafts are desired as alternative to autologous nerve grafts in peripheral nerve reconstruction. Artificial nerve conduits still have their limitations and are not widely accepted in the clinical setting. Here we report an analysis of fine-tuned chitosan tubes used to reconstruct 10 mm nerve defects in the adult rat. The chitosan tubes displayed low, medium and high degrees of acetylation (DAI: ≈ 2%, DA: ≈ 5%, DAIII: ≈ 20%) and therefore different degradability and microenvironments for the regenerating nerve tissue. Short and long term investigations were performed demonstrating that the chitosan tubes allowed functional and morphological nerve regeneration similar to autologous nerve grafts. Irrespective of the DA growth factor regulation demonstrated to be the same as in controls. Analyses of stereological parameters as well as the immunological tissue response at the implantation site and in the regenerated nerves, revealed that DAI and DAIII chitosan tubes displayed some limitations in the support of axonal regeneration and a high speed of degradation accompanied with low mechanical stability, respectively. The chitosan tubes combine several pre-requisites for a clinical acceptance and DAII chitosan tubes have to be judged as the most supportive for peripheral nerve regeneration.

Rat embryonic motoneurons in long-term co-culture with Schwann cells—a system to investigate motoneuron diseases on a cellular level in vitro

Investigations of motoneuron diseases on a cellular and molecular level require long-term cultivation of primary cells. Here we present a new culture system in which matured motoneurons interact with their physiological partners like interneurons, astroglia and peripheral glia cells. This enables motoneuron-maturation for up to 3 weeks, while motoneurons consistently reached large diameters of their somata of 30-45 microm, occasionally more than 80 microm. Dissociated rat embryonic ventral spinal cord cells were enriched for motoneurons by density gradient centrifugation and seeded on a non-confluent mono-layer of highly enriched neonatal rat Schwann cells. Immunocytochemical visualization of neuron specific betaIII-tubulin in all neurons and of motoneuron specific non-phosphorylated neurofilament H/M, respectively, revealed that after 3 days in vitro >70% of all neurons were motoneurons. After 20 days in vitro, a motoneuron fraction of 12% was maintained. Motoneurons were susceptible to transient transfection with green fluorescent protein cDNA when liposomal transfection and an enhancer substance were combined. Synaptic connections enabled formation of spontaneously active neuronal networks which provide a culture model to study glutamate excitotoxicity and calcium deregulation on a molecular level. Both mechanisms are implied in the pathophysiology of amyotrophic lateral sclerosis, a neurodegenerative motoneuron disorder.

The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus

Spinal muscular atrophy is a neurodegenerative disease accompanied by a loss of motoneurons. Either mutations or deletions in the survival of motoneuron (SMN) gene are responsible for this defect. SMN is an assembly protein for RNA‐protein complexes in the nucleus and is also found in axons of neurons. However, it is unclear which dysfunctions of SMN are important for disease progression. In this study we analyzed the contributions of different SMN regions for localization and neuronal differentiation associated with outgrowth of neurites. Suppression of endogenous SMN protein levels significantly decreased the growth of neurites. Down‐regulation of the interacting protein gemin2 had the opposite effect. Surprisingly, selective overexpression of the SMN C‐terminal domain promoted neurite outgrowth similar to full‐length protein and could rescue the SMN knock‐down effects. The knock‐down led to a significant change in the G‐/F‐actin ratio, indicating a role for SMN in actin dynamics. Therefore, our data suggest a functional role for SMN in microfilament metabolism in axons of motoneurons.—van Bergeijk, J., Rydel‐Könecke, K., Grothe, C., Claus, P. The spinal muscular atrophy gene product regulates neurite outgrowth: importance of the C terminus. FASEB J. 21, 1492–1502 (2007)

Chitosan-film enhanced chitosan nerve guides for long-distance regeneration of peripheral nerves.

Biosynthetic nerve grafts are developed in order to complement or replace autologous nerve grafts for peripheral nerve reconstruction. Artificial nerve guides currently approved for clinical use are not widely applied in reconstructive surgery as they still have limitations especially when it comes to critical distance repair. Here we report a comprehensive analysis of fine-tuned chitosan nerve guides (CNGs) enhanced by introduction of a longitudinal chitosan film to reconstruct critical length 15xa0mm sciatic nerve defects in adult healthy Wistar or diabetic Goto-Kakizaki rats. Short and long term investigations demonstrated that the CNGs enhanced by the guiding structure of the introduced chitosan film significantly improved functional and morphological results of nerve regeneration in comparison to simple hollow CNGs. Importantly, this was detectable both in healthy and in diabetic rats (short term) and the regeneration outcome almost reached the outcome after autologous nerve grafting (long term). Hollow CNGs provide properties likely leading to a wider clinical acceptance than other artificial nerve guides and their performance can be increased by simple introduction of a chitosan film with the same advantageous properties. Therefore, the chitosan film enhanced CNGs represent a new generation medical device for peripheral nerve reconstruction.

Dopaminergic properties and function after grafting of attached neural precursor cultures

Generation of dopaminergic (DA) neurons from multipotent embryonic progenitors represents a promising therapeutical strategy for Parkinsons disease (PD). Aim of the present study was the establishment of enhanced cell culture conditions, which optimize the use of midbrain progenitor cells in animal models of PD. In addition, the progenitor cells were characterized during expansion and differentiation according to morphological and electrophysiological criteria and compared to primary tissue. Here, we report that CNS precursors can be expanded in vitro up to 40-fold and afterwards be efficiently differentiated into DA neurons. After 4-5 days under differentiation conditions, more than 70% of the neurons were TH+, equivalent to 30% of the total cell population. Calcium imaging revealed the presence of calcium-permeable AMPA receptors in the differentiated precursors which are capable to contribute to many developmental processes. The overall survival rate, degree of reinnervation and the behavioral performance after transplantation of 4 days in-vitro-differentiated cells were similar to results after direct grafting of E14 ventral mesencephalic cells, whereas after shorter or longer differentiation periods, respectively, less effects were achieved. Compared to the amount of in-vitro-generated DA neurons, the survival rate was only 0.8%, indicating that these cells are very vulnerable. Our results suggest that expanded and differentiated DA precursors from attached cultures can survive microtransplantation and integrate within the striatum in terms of behavioral recovery. However, there is only a short time window during in vitro differentiation, in which enough cells are already differentiated towards a DA phenotype and simultaneously not too mature for implantation. However, additional factors and/or genetical manipulation of these expanded progenitors will be required to increase their in vivo survival in order to improve both the ethical and the technical outlook for the use of fetal tissue in clinical transplantation.

Valproic Acid Promotes Neurite Outgrowth in PC12 Cells independent from Regulation of the Survival of Motoneuron Protein

Spinal muscular atrophy (SMA) is a neurodegenerative disorder of motoneurons. The disease is caused by deletions or mutations of the survival of motoneuron gene 1 (SMN1). The amount of protein expressed from the second gene, SMN2, correlated with the severity of the clinical phenotype. The histone deacetylase inhibitor valproic acid (VPA) has been shown to increase the total cellular amount of functional SMN protein and is therefore considered as a drug candidate for treatment of SMA. In this study, we analyzed the effects of VPA in PC12 cells, a model system for neuronal differentiation, with regard to neurite outgrowth and SMN expression. VPA promoted neurite outgrowth in PC12 cells. However, this effect did not correlate with upregulation of SMN protein levels, suggesting a SMN‐independent mechanism for VPA regulation of neurite outgrowth.