AAV2/DJ-mediated alpha-synuclein overexpression in the rat substantia nigra as early stage model of Parkinson's disease.

Parkinson's disease (PD) is pathologically characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and alpha-synucleinopathy. We mimic the disease pathology with overexpression of either the human α-syn wildtype (α-syn-WT) or E46K mutant form (α-syn-E46K) in DA neurons of the SNpc in adult rats using AAV2/DJ as a viral vector for the first time. Transduction efficiency was compared to an equal virus titer expressing the green fluorescent protein (GFP). Motor skills of all animals were evaluated in the cylinder and amphetamine-induced rotation test over a total time period of 12 weeks. Additionally, stereological quantification of DA cells and striatal fiber density measurements were performed every 4 weeks after injection. Rats overexpressing α-syn-WT showed a progressive loss of DA neurons with 40% reduction after 12 weeks accompanied by a greater loss of striatal DA fibers. In contrast, α-syn-E46K led to this reduction after 4 weeks without further progress. Insoluble α-syn positive cytoplasmic inclusions were observed in both groups within DA neurons of the SNpc and VTA. In addition, both α-syn groups developed a characteristic worsening of the rotational behavior over time. However, only the α-syn-WT group reached statistically significant different values in the cylinder test. Summarizing these effects, we established a motor symptom animal model of PD by using AAV2/DJ in the brain for the first time. Thereby, overexpressing of α-syn-E46K mimicked a rather pre-symptomatic stage of the disease, while the α-syn-WT overexpressing animals imitated an early symptomatic stage of PD.

Immobile survival of motoneuron (SMN) protein stored in Cajal bodies can be mobilized by protein interactions.

Reduced levels of survival of motoneuron (SMN) protein lead to spinal muscular atrophy, but it is still unknown how SMN protects motoneurons in the spinal cord against degeneration. In the nucleus, SMN is associated with two types of nuclear bodies denoted as gems and Cajal bodies (CBs). The 23 kDa isoform of fibroblast growth factor-2 (FGF-2(23)) is a nuclear protein that binds to SMN and destabilizes the SMN-Gemin2 complex. In the present study, we show that FGF-2(23) depletes SMN from CBs without affecting their general structure. FRAP analysis of SMN-EGFP in CBs demonstrated that the majority of SMN in CBs remained mobile and allowed quantification of fast, slow and immobile nuclear SMN populations. The potential for SMN release was confirmed by in vivo photoconversion of SMN-Dendra2, indicating that CBs concentrate immobile SMN that could have a specialized function in CBs. FGF-2(23) accelerated SMN release from CBs, accompanied by a conversion of immobile SMN into a mobile population. Furthermore, FGF-2(23) caused snRNP accumulation in CBs. We propose a model in which Cajal bodies store immobile SMN that can be mobilized by its nuclear interaction partner FGF-2(23), leading to U4 snRNP accumulation in CBs, indicating a role for immobile SMN in tri-snRNP assembly.

Cooperation of nuclear fibroblast growth factor receptor 1 and Nurr1 offers new interactive mechanism in postmitotic development of mesencephalic dopaminergic neurons.

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-2(23)) 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.

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 Parkinson's 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.

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.

Fibroblast growth factor 2 regulates dopaminergic neuron development in vivo.

Fibroblast growth factor 2 (FGF-2) is a neurotrophic factor participating in regulation of proliferation, differentiation, apoptosis and neuroprotection in the central nervous system. With regard to dopaminergic (DA) neurons of substantia nigra pars compacta (SNpc), which degenerate in Parkinson's disease, FGF-2 improves survival of mature DA neurons in vivo and regulates expansion of DA progenitors in vitro. To address the physiological role of FGF-2 in SNpc development, embryonic (E14.5), newborn (P0) and juvenile (P28) FGF-2-deficient mice were investigated. Stereological quantification of DA neurons identified normal numbers in the ventral tegmental area, whereas the SNpc of FGF-2-deficient mice displayed a 35% increase of DA neurons at P0 and P28, but not at earlier stage E14.5. Examination of DA marker gene expression by quantitative RT-PCR and in situ hybridization revealed a normal patterning of embryonic ventral mesencephalon. However, an increase of proliferating Lmx1a DA progenitors in the subventricular zone of the ventral mesencephalon of FGF-2-deficient embryos indicated altered cell cycle progression of neuronal progenitors. Increased levels of nuclear FgfR1 in E14.5 FGF-2-deficient mice suggest alterations of integrative nuclear FgfR1 signaling (INFS). In summary, FGF-2 restricts SNpc DA neurogenesis in vivo during late stages of embryonic development.

Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of fibroblast growth factor 2.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss in brain and spinal cord. Mutations in the superoxide dismutase (SOD) 1 gene account for 10-20% of familial ALS patients. The ALS-mouse model over-expressing a mutant human SOD1 (G93A) gene closely mimics human ALS disease. The cause for the selective death of motoneurons is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility. Basic fibroblast growth factor 2 (FGF-2) plays a prominent role in the motor system. In order to evaluate a role of FGF-2 in ALS pathogenesis, double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene were generated. Both heterozygous and homozygous FGF-2 deficient mutant SOD1 mice showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels. Survival of the double mouse mutants was significantly prolonged for two weeks. Motoneuron numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1 animals, our results revealed a protective effect of FGF-2 reduction. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in muscle and spinal cord tissue of double mutant animals.

Expression patterns of sulfatase genes in the developing mouse embryo.

Mammalian sulfatase enzymes participate in various processes, such as hormone regulation, lysosomal degradation and modulation of several signaling pathways. The sulfatase gene family consists of 14 members in mice and 17 in humans. Mutations of at least eight members are associated with human disorders, with main disease manifestations in the nervous system and skeleton. Despite their biological significance, little is known about their expression during embryonic development, especially for the more recently discovered gene family members. By in situ hybridization, we compared the expression patterns of nine sulfatases: ArsB, ArsG, ArsI, ArsJ, Galns, Gns, Ids, Sulf1, and Sulf2 in midgestation mouse embryos. Of interest, overlapping expression domains of several sulfatases could be detected in the developing central nervous system, eye, skeleton, and inner organs. Moreover, novel expression patterns for ArsG in choroid plexus, ArsI in hypertrophic chondrocytes and ArsJ in joints were identified.

Characterization and differentiation potential of rat ventral mesencephalic neuronal progenitor cells immortalized with SV40 large T antigen.

Neuronal progenitor cells (NPCs) possess high potential for use in regenerative medicine. To overcome their limited mitotic competence, various immortalization strategies have been applied that allow their prolonged maintenance and expansion in vitro. Such immortalized cells can be used for the design and discovery of new cell-based therapies for neurodegenerative diseases, such as Parkinson's disease. We immortalized rat ventral mesencephalic NPCs by using SV40 large T antigen (SV40Tag). All cell clones displayed a two- to three-fold higher proliferation rate compared with the primary cells. In order to induce dopaminergic differentiation of generated cell clones, both glial-derived neurotrophic factor and di-butyryl cyclic adenosine monophosphate were applied. Treated cells were then characterized regarding the expression of dopaminergic lineage markers, differentiation of various cell populations, calcium imaging in the presence of kainate, and immunohistochemistry after intrastriatal transplantation. Treated cells displayed morphological maturation, and calcium imaging revealed neuronal properties in the presence of kainate. These cells also expressed low mRNA levels of the dopamine transporter and tyrosine hydroxylase (TH), although no TH-immunopositive neurons were found. Intrastriatal transplantation into the neurotoxin-lesioned rats did not induce further differentiation. As an alternative approach, we silenced SV40Tag with short interfering RNA, but this was not sufficient to trigger differentiation into dopaminergic neurons. Nevertheless, neuronal and glial cells were detected as shown by beta-tubulin type III and glial fibrillary acidic protein staining, respectively. SV40Tag cells are suitable for carrying out controlled genetic modifications as shown by overexpression of enhanced green fluorescence protein after efficient non-viral transfection.

Neuronal firing activity in the basal ganglia after striatal transplantation of dopamine neurons in hemiparkinsonian rats.

The loss of nigral dopaminergic neurons and the resulting dopamine (DA) depletion in the striatum (STR) lead to altered neuronal activity and enhanced beta activity in various regions of the basal ganglia (BG) motor loop in patients with Parkinson's disease and in rodents in the 6-hydroxydopamine (6-OHDA)-lesioned rat model. Intrastriatal DA graft implantation has been shown to re-innervate the host brain and restore DA input. Here, DA cell grafts were implanted into the STR of 6-OHDA-lesioned rats and the effect on neuronal activity under urethane anesthesia (1.4g/kg, injected intraperitoneally) was tested in the entopeduncular nucleus (EPN, the equivalent to the human globus pallidus internus), the output nucleus of the BG, and the globus pallidus (GP, the equivalent to the human globus pallidus externus), a key region in the indirect pathway. In animals, which were transplanted with cells derived from the ventral mesencephalon of embryonic day 12 rat embryos into the STR, the rotational behavior induced by DA agonists in 6-OHDA-lesioned rats was significantly improved. This was accompanied by alleviated EPN firing rate and reinstated patterns of neuronal activity in the GP and EPN. Analysis of oscillatory activity revealed enhanced beta activity in both regions, which was reduced after grafting. In summary these data indicate restoration of BG motor loop toward normal activity by DA graft integration.

Altered somatosensory cortex neuronal activity in a rat model of Parkinson's disease and levodopa-induced dyskinesias.

Several findings support the concept that sensorimotor integration is disturbed in Parkinson's disease (PD) and in levodopa-induced dyskinesias. In this study, we explored the neuronal firing activity of excitatory pyramidal cells and inhibitory interneurons in the forelimb region of the primary somatosensory cortex (S1FL-Ctx), along with its interaction with oscillatory activity of the primary motor cortex (MCtx) in 6-hydroxydopamine lesioned hemiparkinsonian (HP) and levodopa-primed dyskinetic (HP-LID) rats as compared to controls under urethane (1.4g/kg, i.p.) anesthesia. Further, gene expression patterns of distinct markers for inhibitory GABAergic neurons were analyzed in both cortical regions. While firing frequency and burst activity of S1FL-Ctx inhibitory interneurons were reduced in HP and HP-LID rats, measures of irregularity were enhanced in pyramidal cells. Further, enhanced coherence of distinct frequency bands of the theta/alpha, high-beta, and gamma frequency, together with enhanced synchronization of putative pyramidal cells and interneurons with MCtx oscillatory activity were observed. While GABA level was similar, gene expression levels of interneuron and GABAergic markers in S1FL-Ctx and MCtx of HP-LID rats differed to some extent. Our study shows that in a rat model of PD with dyskinesias, neuronal activity in putative interneurons was reduced, which was accompanied by high beta and gamma coherence between S1FL-Ctx and MCtx, together with changes in gene expression, indicating maladaptive neuroplasticity after long term levodopa treatment.

Recombination and gene conversion in a 170-kb genomic region of Arabidopsis thaliana.

Arabidopsis thaliana is a highly selfing plant that nevertheless appears to undergo substantial recombination. To reconcile its selfing habit with the observations of recombination, we have sampled the genetic diversity of A. thaliana at 14 loci of approximately 500 bp each, spread across 170 kb of genomic sequence centered on a QTL for resistance to herbivory. A total of 170 of the 6321 nucleotides surveyed were polymorphic, with 169 being biallelic. The mean silent genetic diversity (pi(s)) varied between 0.001 and 0.03. Pairwise linkage disequilibria between the polymorphisms were negatively correlated with distance, although this effect vanished when only pairs of polymorphisms with four haplotypes were included in the analysis. The absence of a consistent negative correlation between distance and linkage disequilibrium indicated that gene conversion might have played an important role in distributing genetic diversity throughout the region. We tested this by coalescent simulations and estimate that up to 90% of recombination is due to gene conversion.

Electrophysiological Characterization of eGFP-Labeled Intrastriatal Dopamine Grafts.

Substitution of degenerated dopaminergic (DA) neurons by intrastriatally transplanted ventral mesencephalon (VM)-derived progenitor cells has been shown to improve motor functions in parkinsonian patients and animal models, whereas investigations of electrophysiological properties of the grafted DA neurons have been rarely performed. Here we show electrophysiological properties of grafted VM progenitor cells at different time intervals up to 12 weeks after transplantation measured in acute brain slices using eGFP-Flag transfection to identify the graft. We were able to classify typical DA neurons according to the biphasic progression (voltage "sag") to hyperpolarizing current injections. Two types of DA-like neurons were classified. Whereas type 1 neurons were characterized by delayed action potentials after hyperpolarization and irregular spontaneous firing, type 2 neurons displayed burst firing after hyperpolarization, spontaneous bursts, and regular firing. Comparison to identified DA neurons in vitro indicates a high integration of the intrastriatally grafted neurons, since in vitro cultures displayed regular firing spontaneously, whereas grafted identified DA neurons showed irregular firing. Additionally, type 1 and type 2 neurons exhibited a slight increase in the spontaneous firing frequency over time intervals after grafting, which might reflect a progressive integration of the grafted DA neurons. Our results provide evidence of the differentiation of grafted VM progenitor cells into mature integrated DA neurons, which are shown to replace the missing DA neurons functionally early after grafting.

Striatal Transplantation of Human Dopaminergic Neurons Differentiated From Induced Pluripotent Stem Cells Derived From Umbilical Cord Blood Using Lentiviral Reprogramming.

Human induced pluripotent stem cells (hiPSCs) are promising sources for regenerative therapies like the replacement of dopaminergic neurons in Parkinson's disease. They offer an unlimited cell source that can be standardized and optimized to produce applicable cell populations to gain maximal functional recovery. In the present study, human cord blood-derived iPSCs (hCBiPSCs) were differentiated into dopaminergic neurons utilizing two different in vitro protocols for neural induction: (protocol I) by fibroblast growth factor (FGF-2) signaling, (protocol II) by bone morphogenetic protein (BMP)/transforming growth factor (TGF-ß) inhibition. After maturation, in vitro increased numbers of tyrosine hydroxylase (TH)-positive neurons (7.4% of total cells) were observed by protocol II compared to 3.5% in protocol I. Furthermore, 3 weeks after transplantation in hemiparkinsonian rats in vivo, a reduced number of undifferentiated proliferating cells was achieved with protocol II. In contrast, proliferation still occurred in protocol I-derived grafts, resulting in tumor-like growth in two out of four animals 3 weeks after transplantation. Protocol II, however, did not increase the number of TH(+) cells in the striatal grafts of hemiparkinsonian rats. In conclusion, BMP/TGF-ß inhibition was more effective than FGF-2 signaling with regard to dopaminergic induction of hCBiPSCs in vitro and prevented graft overgrowth in vivo.

Adult hemiparkinsonian rats do not benefit from tactile stimulation.

Tactile stimulation (TS) applied to adult rats after cortical injury (medial frontal cortex aspiration or sensorimotor pial stripping stroke model) has been previously shown to ameliorate behavioral impairments and to improve morphological parameters like dendritic length of prefrontal cortical neurons (Gibb et al., 2010). The purpose of this study was to examine the effect of TS on healthy and hemiparkinsonian adult rats. Therefore, the animals received TS for 14 days and 15 min three times daily. At different time points rats were tested in various behavioral tests (amphetamine-induced rotation, cylinder test, staircase test). Finally, rats were sacrificed, their brains removed, and processed for Golgi-Cox analyses, tyrosine hydroxylase immunohistochemistry and quantitative RT-PCR. We found that the striatal 6-OHDA lesion itself induced a long-term increase of astroglial Fgf2 transcript levels, but was not further increased by TS. In contrast TS applied to healthy rats elicited a transient short-term increase of Fgf2 in the striatum and Bdnf, Grin1, and Fgf2 in the hippocampus. Moreover, behavioral and histological analyses do not support a beneficial effect of TS for hemiparkinsonian rats, applied for two weeks starting one day after partial striatal 6-OHDA lesion.

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.

Regulation of neuronal differentiation by proteins associated with nuclear bodies.

Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23)) is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-2(23) blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23)-dependent transcription. Our results indicate that FGF-2(23) and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation.

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.

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 Parkinson's 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.

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.