Andreas Ratzka

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.

Cooperation of Nuclear Fibroblast Growth Factor Receptor 1 and Nurr1 Offers New Interactive Mechanism in Postmitotic Development of Mesencephalic Dopaminergic Neurons

Background: Nurr1 and FGFR1 are integrative nuclear factors participating in postmitotic dopaminergic neuron development. Results: Both nuclear receptors show a functional interaction in co-immunoprecipitation, FRAP, ChIP, and luciferase gene reporter assay. Conclusion: Cooperation of nuclear FGFR1 and Nurr1 offers a new mechanism in transcriptional regulation and integration. Significance: This mechanism may channel diverse stimuli in developing and mature dopaminergic neurons, providing a potential therapeutic target. Experiments in mice deficient for Nurr1 or expressing the dominant-negative FGF receptor (FGFR) identified orphan nuclear receptor Nurr1 and FGFR1 as essential factors in development of mesencephalic dopaminergic (mDA) neurons. FGFR1 affects brain cell development by two distinct mechanisms. Activation of cell surface FGFR1 by secreted FGFs stimulates proliferation of neural progenitor cells, whereas direct integrative nuclear FGFR1 signaling (INFS) is associated with an exit from the cell cycle and neuronal differentiation. Both Nurr1 and INFS activate expression of neuronal genes, such as tyrosine hydroxylase (TH), which is the rate-limiting enzyme in dopamine synthesis. Here, we show that nuclear FGFR1 and Nurr1 are expressed in the nuclei of developing TH-positive cells in the embryonic ventral midbrain. Both nuclear receptors were effectively co-immunoprecipitated from the ventral midbrain of FGF-2-deficient embryonic mice, which previously showed an increase of mDA neurons and enhanced nuclear FGFR1 accumulation. Immunoprecipitation and co-localization experiments showed the presence of Nurr1 and FGFR1 in common nuclear protein complexes. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrated the Nurr1-mediated shift of nuclear FGFR1-EGFP mobility toward a transcriptionally active population and that both Nurr1 and FGFR1 bind to a common region in the TH gene promoter. Furthermore, nuclear FGFR1 or its 23-kDa FGF-2 ligand (FGF-223) enhances Nurr1-dependent activation of the TH gene promoter. Transcriptional cooperation of FGFR1 with Nurr1 was confirmed on isolated Nurr1-binding elements. The proposed INFS/Nurr1 nuclear partnership provides a novel mechanism for TH gene regulation in mDA neurons and a potential therapeutic target in neurodevelopmental and neurodegenerative disorders.

Electrical Stimulation Accelerates Axonal and Functional Peripheral Nerve Regeneration across Long Gaps

Short-term low-frequency electrical stimulation (ESTIM) of proximal peripheral nerve stumps prior to end-to-end coaptation or tubular bridging of small distances has been reported to increase preferential motor reinnervation and functional motor recovery in animal models and human patients undergoing carpal tunnel release surgery. We investigated the effects of ESTIM on regeneration across rat sciatic nerve gaps, which exceed distances that allow spontaneous regeneration. Three different reconstruction approaches were combined with ESTIM in the experimental groups. Nerve gaps (13 mm) were bridged using (I) nerve autotransplantation, (II) transplantation of differentially filled silicone tubes, or (III) transplantation of tubular grafts containing fibroblast growth factor-2 overexpressing Schwann cells (SCs) for gene therapy. The regeneration outcome was followed for up to 8 weeks, and functionally as well as histomorphometrically analyzed in comparison to non-stimulated control groups. Combining ESTIM with nerve autotransplantation significantly increased the nerve fiber density in the regenerated nerve, and the grade of functional recovery as detected by electrodiagnostic recordings from the gastrocnemius muscle. The combination of ESTIM with transplantation of naïve SCs increased the regeneration of gap-bridging nerve tissue. Although macroscopic tissue regeneration was not further improved after combining ESTIM with FGF-2(21/23-kD) gene therapy, the latter resulted in a high rate of regenerated nerves that functionally reconnected to the target muscle. Based on our results, brief ESTIM shows high potential to accelerate axonal as well as functional (motor and sensory) outcomes in the clinical setting of peripheral nerve gap reconstruction in human patients.

A novel nuclear FGF Receptor‐1 partnership with retinoid and Nur receptors during developmental gene programming of embryonic stem cells

FGF Receptor‐1 (FGFR1), a membrane‐targeted protein, is also involved in independent direct nuclear signaling. We show that nuclear accumulation of FGFR1 is a common response to retinoic acid (RA) in pluripotent embryonic stem cells (ESC) and neural progenitors and is both necessary and sufficient for neuronal‐like differentiation and accompanying neuritic outgrowth. Dominant negative nuclear FGFR1, which lacks the tyrosine kinase domain, prevents RA‐induced differentiation while full‐length nuclear FGFR1 elicits differentiation in the absence of RA. Immunoprecipitation and GST assays demonstrate that FGFR1 interacts with RXR, RAR and their Nur77 and Nurr1 partners. Conditions that promote these interactions decrease the mobility of nuclear FGFR1 and RXR in live cells. RXR and FGFR1 co‐associate with 5′‐Fluorouridine‐labeled transcription sites and with RA Responsive Elements (RARE). RA activation of neuronal (tyrosine hydroxylase) and neurogenic (fgf‐2 and fgfr1) genes is accompanied by increased FGFR1, Nur, and histone H3.3 binding to their regulatory sequences. Reporter‐gene assays show synergistic activations of RARE, NBRE, and NurRE by FGFR1, RAR/RXR, and Nurs. As shown for mESC differentiation, FGFR1 mediates gene activation by RA and augments transcription in the absence of RA. Cooperation of FGFR1 with RXR/RAR and Nurs at targeted genomic sequences offers a new mechanism in developmental gene regulation. J. Cell. Biochem. 113: 2920–2936, 2012.

FGF-2 deficiency does not influence FGF ligand and receptor expression during development of the nigrostriatal system.

Secreted proteins of the fibroblast growth factor (FGF) family play important roles during development of various organ systems. A detailed knowledge of their temporal and spatial expression profiles, especially of closely related FGF family members, are essential to further identification of specific functions in distinct tissues. In the central nervous system dopaminergic neurons of the substantia nigra and their axonal projections into the striatum progressively degenerate in Parkinsons disease. In contrast, FGF-2 deficient mice display increased numbers of dopaminergic neurons. In this study, we determined the expression profiles of all 22 FGF-ligands and 10 FGF-receptor isoforms, in order to clarify, if FGF-2 deficiency leads to compensatory up-regulation of other FGFs in the nigrostriatal system. Three tissues, ventral mesencephalon (VM), striatum (STR) and as reference tissue spinal cord (SC) of wild-type and FGF-2 deficient mice at four developmental stages E14.5, P0, P28, and adult were comparatively analyzed by quantitative RT-PCR. As no differences between the genotypes were observed, a compensatory up-regulation can be excluded. Moreover, this analysis revealed that the majority of FGF-ligands (18/22) and FGF-receptors (9/10) are expressed during normal development of the nigrostriatal system and identified dynamic changes for some family members. By comparing relative expression level changes to SC reference tissue, general alterations in all 3 tissues, such as increased expression of FGF-1, -2, -22, FgfR-2c, -3c and decreased expression of FGF-13 during postnatal development were identified. Further, specific changes affecting only one tissue, such as increased FGF-16 (STR) or decreased FGF-17 (VM) expression, or two tissues, such as decreased expression of FGF-8 (VM, STR) and FGF-15 (SC, VM) were found. Moreover, 3 developmentally down-regulated FGFs (FGF-8b, FGF-15, FGF-17a) were functionally characterized by plasmid-based over-expression in dissociated E11.5 VM cell cultures, however, such a continuous exposure had no influence on the yield of dopaminergic neurons in vitro.

Fibroblast growth factor 2 regulates adequate nigrostriatal pathway formation in mice

Fibroblast growth factor 2 (FGF‐2) is an important neurotrophic factor that promotes survival of adult mesencephalic dopaminergic (mDA) neurons and regulates their adequate development. Since mDA neurons degenerate in Parkinsons disease, a comprehensive understanding of their development and maintenance might contribute to the development of causative therapeutic approaches. The current analysis addressed the role of FGF‐2 in mDA axonal outgrowth, pathway formation, and innervation of respective forebrain targets using organotypic explant cocultures of ventral midbrain (VM) and forebrain (FB). An enhanced green fluorescent protein (EGFP) transgenic mouse strain was used for the VM explants, which allowed combining and distinguishing of individual VM and FB tissue from wildtype and FGF‐2‐deficient embryonic day (E)14.5 embryos, respectively. These cocultures provided a suitable model to study the role of target‐derived FB and intrinsic VM‐derived FGF‐2. In fact, we show that loss of FGF‐2 in both FB and VM results in significantly increased mDA fiber outgrowth compared to wildtype cocultures, proving a regulatory role of FGF‐2 during nigrostriatal wiring. Further, we found in heterogeneous cocultures deficient for FGF‐2 in FB and VM, respectively, similar phenotypes with wider fiber tracts compared to wildtype cocultures and shorter fiber outgrowth distance than cocultures completely deficient for FGF‐2. Additionally, the loss of target‐derived FGF‐2 in FB explants resulted in decreased caudorostral glial migration. Together these findings imply an intricate interplay of target‐derived and VM‐derived FGF signaling, which assures an adequate nigrostriatal pathway formation and target innervation. J. Comp. Neurol. 520:3949–3961, 2012.

Analysis of the Fibroblast Growth Factor System Reveals Alterations in a Mouse Model of Spinal Muscular Atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.

Topology of intrastriatal dopaminergic grafts determines functional and emotional outcome in neurotoxin-lesioned rats

Many Parkinsons disease (PD) patients suffer from anxiety disorders, which often precede the onset of classical motor symptoms. So far, there is no evidence from randomized, placebo-controlled trials for successful treatment of anxiety in patients with PD. Grafts of fetal nigral neurons are currently explored as a restorative cell therapy for PD. In PD animal models, intrastriatal transplantations of embryonic dopaminergic neurons have been shown to ameliorate behavioral defects. In our previous study we showed that expanded and differentiated neural progenitors improved drug-induced rotation behavior and posture balance as a more complex motor task. However, it is not clear whether grafting of these cells affected spontaneous locomotor activity and anxiety-like behavior in 6-OHDA lesioned rats. Therefore, we analyzed behavior of control, lesioned, sham-transplanted, and transplanted rats using open field (OF) and elevated plus maze (EPM). After unilateral 6-OHDA lesion of the medial forebrain bundle, we observed reduced locomotor activity in the EPM (wall-rearing, entries in closed arms) in lesioned and sham-transplanted rats, which correlated with the loss of dopaminergic neurons and apomorphine-induced rotation behavior. Furthermore, anxiety-like behavior in the EPM (entries and time in open arms) was increased in lesioned and sham-transplanted rats. Although exogenous cell replacement improved apomorphine-induced rotation behavior, locomotor activity and anxiety-like behavior was not reconstituted in transplanted rats. However, we provided evidence for an interaction of locomotor activity/anxiety-like behavior with graft localization in the host striatum. These results emphasize the crucial role of graft localization for benefit of restorative cell therapy for PD.

Neuronal firing activity and gene expression changes in the subthalamic nucleus after transplantation of dopamine neurons in hemiparkinsonian rats

Dopamine (DA) depletion in the nigrostriatal system leads to basal ganglia dysfunction both in Parkinsons disease (PD) and in 6-hydroxy dopamine (6-OHDA)-lesioned rats with neuronal hyperactivity in the subthalamic nucleus (STN), i.e. increased firing rate and burst activity, together with enhanced beta oscillatory activity. Moreover, intrastriatal transplantation of DA neurons has been shown to functionally re-innervate the host striatum and restore DA input. However, the effects of those transplanted cells on the STN are not well characterized. Therefore, we transplanted cells, derived from the ventral mesencephalon of E12 rat embryos, intrastriatally in the unilateral 6-OHDA-lesioned rat model of PD. We combined behavioral and histological findings with electrophysiological extracellular recordings in the STN, as well as qRT-PCR analyses of dopaminergic, GABAergic, and glutamatergic transporter and receptor genes in the striatum and the STN. Transplanted animals displayed improved rotational behavior after amphetamine injection by 50% in rats with small grafts (586±109 SEM dopamine cells), or even overcompensation by 116% in rats with large grafts (3486±548 SEM dopamine cells). Electrophysiological measurements revealed, that in rats with large grafts burst activity was not affected, while STN neuronal firing rate, as well as beta oscillatory activity was alleviated, whereas small grafts had less impact. Interestingly, both behavioral and electrophysiological measures were dependent on the number of surviving tyrosine hydroxylase positive cells. Although grafted rats displayed restored expression of the GABA synthesizing enzymes Gad65 and Gad67 in the striatum compared to naive rats, the grafts induced a decreased mRNA expression of dopamine receptor Drd2, glutamate receptors AMPA3, NMDA2A, and NMDA2B, and glutamate transporter Eaat3. Interestingly, the NMDA receptor subunit 2B and glutamate transporter Eaat3 were also less expressed in the STN of grafted animals compared to naive rats. In summary, DA grafts restore functional deficits and cause partial improvement of subthalamic neuronal activity. Incomplete recovery, however, may be due to decreased receptor gene expression induced by DA grafts in the striatum and in the STN.

Nanotechnology versus stem cell engineering: in vitro comparison of neurite inductive potentials.

Purpose Innovative nerve conduits for peripheral nerve reconstruction are needed in order to specifically support peripheral nerve regeneration (PNR) whenever nerve autotransplantation is not an option. Specific support of PNR could be achieved by neurotrophic factor delivery within the nerve conduits via nanotechnology or stem cell engineering and transplantation. Methods Here, we comparatively investigated the bioactivity of selected neurotrophic factors conjugated to iron oxide nanoparticles (np-NTFs) and of bone marrow-derived stem cells genetically engineered to overexpress those neurotrophic factors (NTF-BMSCs). The neurite outgrowth inductive activity was monitored in culture systems of adult and neonatal rat sensory dorsal root ganglion neurons as well as in the cell line from rat pheochromocytoma (PC-12) cell sympathetic culture model system. Results We demonstrate that np-NTFs reliably support numeric neurite outgrowth in all utilized culture models. In some aspects, especially with regard to their long-term bioactivity, np-NTFs are even superior to free NTFs. Engineered NTF-BMSCs proved to be less effective in induction of sensory neurite outgrowth but demonstrated an increased bioactivity in the PC-12 cell culture system. In contrast, primary nontransfected BMSCs were as effective as np-NTFs in sensory neurite induction and demonstrated an impairment of neuronal differentiation in the PC-12 cell system. Conclusion Our results evidence that nanotechnology as used in our setup is superior over stem cell engineering when it comes to in vitro models for PNR. Furthermore, np-NTFs can easily be suspended in regenerative hydrogel matrix and could be delivered that way to nerve conduits for future in vivo studies and medical application.