TGF-ß is an inducer of ZEB1-dependent mesenchymal transdifferentiation in glioblastoma that is associated with tumor invasion.

Different molecular subtypes of glioblastoma (GBM) have been recently identified, of which the mesenchymal subtype is associated with worst prognoses. Here, we report that transforming growth factor-ß (TGF-ß) is able to induce a mesenchymal phenotype in GBM that involves activation of SMAD2 and ZEB1, a known transcriptional inducer of mesenchymal transition in epithelial cancers. TGF-ß exposure of established and newly generated GBM cell lines was associated with morphological changes, enhanced mesenchymal marker expression, migration and invasion in vitro and in an orthotopic mouse model. TGF-ß-induced mesenchymal differentiation and invasive behavior was prevented by chemical inhibition of TGF-ß signaling as well as small interfering RNA (siRNA)-dependent silencing of ZEB1. Furthermore, TGF-ß-responding and -nonresponding GBM neurospheres were identified in vitro. Interestingly, nonresponding cells displayed already high levels of pSMAD2 and ZEB1 that could not be suppressed by inhibition of TGF-ß signaling, suggesting the involvement of yet unknown mechanisms. These different GBM neurospheres formed invasive tumors in mice as well as revealed mesenchymal marker expression in immunohistochemical analyses. Moreover, we also detected distinct zones with overlapping pSMAD2, elevated ZEB1 and mesenchymal marker expression in GBM patient material, suggestive of the induction of local, microenvironment-dependent mesenchymal differentiation. Overall, our findings indicate that GBM cells can acquire mesenchymal features associated with enhanced invasive potential following stimulation by secretory cytokines, such as TGF-ß. This property of GBM contributes to heterogeneity in this tumor type and may blur the boundaries between the proposed transcriptional subtypes. Targeting TGF-ß or downstream targets like ZEB1 might be of potential benefit in reducing the invasive phenotype of GBM in a subpopulation of patients.

Differentiation of non-mesencephalic neural stem cells towards dopaminergic neurons.

Neural stem cells (NSCs), either isolated from fetal or adult human brain or derived from induced pluripotent stem cells, are now considered major candidates for in vitro generation of transplantable dopaminergic (DA) neurons and modeling of Parkinson's disease. It is generally thought that in vitro differentiation of neural stem cells into meso-diencephalic dopaminergic neurons, requires recapitulation of dopaminergic differentiation pathway normally occurring in the ventral mesencephalon during embryogenesis. This dopaminergic pathway is partially activated by a combination of the extracellular induction factors Sonic Hedgehog (Shh), Fibroblast Growth Factor 8 (FGF8) and Wnt1 that trigger specific intracellular transcription cascades. In vitro mimicking of these embryonic ventral mesencephalic conditions has been successful for dopaminergic differentiation of embryonic stem cells and ventral mesencephalic NSCs. Dopaminergic differentiation of non-mesencephalic NSCs (nmNSCs), however, is considered arduous. Here we examine whether Shh, FGF8 and Wnt1 can activate typical dopaminergic transcription factors, such as Lmx1a, Msx1 and Otx2 in nmNSCs. We found that Shh, FGF8 and Wnt1 induced the expression of Lmx1a and Otx2 in nmNSCs resulting in the differentiation of up to 39% of the nmNSCs into neurons expressing Pitx3. However, only a low number ( approximately 13%) of these cells became more DA-like neurons also expressing tyrosine hydroxylase (TH). The histone deacetylase (HDAC)-inhibitor trichostatin A combined with Shh, FGF8 and Wnt1 caused orchestrated induction of Lmx1a, Otx2, Msx1 plus the early DA transcription factor En1. Now significantly increased numbers of TH ( approximately 22%) and Pitx3 ( approximately 33%) neurons were observed. Most of these cells coexpressed the DA markers DAT and Vmat2. Taken together, we demonstrate that nmNSCs indeed can be differentiated towards DA-like neurons, but this differentiation is far from complete in comparison to ventral mesencephalic NSCs and embryonic stem cells; most likely, the nmNSCs lack the proper "primed" epigenetic state of these cells for DA differentiation facilitating the induction of DA specific transcription factors.

Effects of histone deacetylation inhibition on neuronal differentiation of embryonic mouse neural stem cells.

Neural stem cells (NSCs) are multipotent cells that have the capacity for self-renewal and for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. The molecular mechanisms regulating gene transcription resulting in NSC differentiation and cell lineage specification are slowly being unraveled. An important mechanism in transcriptional regulation is modulation of chromatin by histone acetylation and deacetylation, allowing or blocking the access of transcriptional factors to DNA sequences. The precise involvement of histone acetyltransferases and histone deacetylases (HDACs) in the differentiation of NSCs into mature functional neurons is still to be revealed. In this in vitro study we have investigated the effects of the HDAC inhibitor trichostatin A (TSA) on the differentiation pattern of embryonic mouse NSCs during culture in a minimal, serum-free medium, lacking any induction or growth factor. We demonstrated that under these basic conditions TSA treatment increased neuronal differentiation of the NSCs and decreased astrocyte differentiation. Most strikingly, electrophysiological recordings revealed that in our minimal culture system only TSA-treated NSC-derived neurons developed normal electrophysiological membrane properties characteristic for functional, i.e. excitable and firing, neurons. Furthermore, TSA-treated NSC-derived neurons were characterized by an increased elongation and arborization of the dendrites. Our study shows that chromatin structure modulation by HDACs plays an important role in the transcriptional regulation of the neuronal differentiation of embryonic NSCs particularly as far as the development of functional properties are concerned. Manipulation of HDAC activity may be an important tool to generate specific neuronal populations from NSCs for transplantation purposes.

Cryopreservation of porcine fetal ventral mesencephalic tissue for intrastriatal transplantation in Parkinson's disease.

In this study we examined the efficacy of cryopreserving porcine fetal mesencephalic tissue. After microscopical dissection of the ventral mesencephalon (VM) from E28 pig fetuses, the collection of explants was randomly divided into two equal parts. One part was directly prepared as cell suspension. The other part was stored in hibernation medium for less than 2 days and then cryopreserved as tissue fragments and stored in liquid nitrogen. After 2 weeks up to 1 year, these tissue fragments were thawed and processed as cell suspensions. After cell counting and assessment of viability, these cell suspensions were used to examine survival, morphology, and neurite formation of the dopaminergic neurons in cell culture as well as after intrastriatal implantation in 6-OHDA-lesioned rats. Comparison of cryopreserved with fresh VM cell suspensions showed no significant difference with respect to cell viability and the average number of living cells per VM explant. The morphology of cultured dopaminergic neurons after cryopreservation was identical to that of fresh cells. After intrastriatal implantation, survival and outgrowth of cryopreserved dopaminergic neurons as well as functional effects did not differ from those of fresh cells. In conclusion, the cryopreservation technique we used proves to be a reliably effective method for storing porcine fetal VM tissue.

Ischemia-induced neuronal expression of the microglia attracting chemokine Secondary Lymphoid-tissue Chemokine (SLC).

Recently, it has been demonstrated that Secondary Lymphoid-tissue Chemokine (SLC) is constitutively expressed in secondary lymphoid organs and controls the homing of naive T-cells and mature dendritic cells. By screening cDNA isolated from ischemic mouse brain, we found expression of SLC mRNA 6 h up to 4 days after the onset of ischemia. In situ hybridization combined with immunohistochemistry showed neurons expressing SLC mRNA in the ischemic area of the cortex. SLC mRNA expression was also found in cultured neurones after various treatments known to induce neuronal death, but not in cultured glial cells. Stimulation with SLC induced intracellular calcium transients and chemotaxis in cultured microglia. Since mRNA encoding CXCR3, an alternative receptor for SLC, but no CCR7 mRNA was found in microglia, we suggest that the effects of SLC on microglia are mediated by CXCR3. This assumption was corroborated by cross-desensitization experiments using IP-10 as a ligand for CXCR3. The inducible expression of SLC in neurones acting on microglia suggests a new and important role of SLC in the neuroimmune system. We propose that SLC is part of a neurone-microglia signaling system which is related to pathological conditions of the brain like ischemia.

Neurotrophins and trk-receptors in adult rat spinal motoneurons: differences related to cell size but not to 'slow/fast' specialization.

We have studied the mRNA expression of the neurotrophins brain-derived neurotrophic factor (BDNF), NT-3 and NT-4 and of their receptors trkB and trkC in individual retrogradely labeled lumbar spinal motoneurons of the adult rat, using quantitative non-radioactive in situ hybridization (ISH). We measured soma size and the optical density of the ISH reaction. We analyzed mRNA expression patterns in predominantly 'slow' or 'fast' motoneurons by comparing retrogradely labelled cells that innervated the slow m.soleus (Sol) with those innervating the fast m.extensor digitorum longus (EDL). Within individual motoneuron pools, there was a considerable variation in the intensity of mRNA expression encoding for BDNF, NT-3 and NT-4, as well as for the receptors trkB and trkC. The expression of trkC mRNA was significantly higher in small, presumed fusimotor neurons than in the larger alpha-motoneurons. Surprisingly, none of the variations in the level of motoneuronal mRNA expression for trkB, BDNF, NT-3 and NT-4 were related to soma size and no differences were found between EDL and Sol motoneurons. Apparently, the considerable variations in neurotrophin and neurotrophin receptor mRNA expression represent other parameters of motoneuronal specialization than those related to the well-established 'fast' vs. 'slow' categories of motoneurons.

Formation of a full complement of cranial proprioceptors requires multiple neurotrophins.

Inactivation of neurotrophin-3 (NT3) completely blocks the development of limb proprioceptive neurons and their end organs, the muscle spindles. We examined whether cranial proprioceptive neurons of the trigeminal mesencephalic nucleus (TMN) require NT3, brain-derived neurotrophic factor (BDNF) or neurotrophin-4 (NT4) for their development. Complements of TMN neurons and masticatory muscle spindles were decreased by 62% in NT3 null mutants, 33% in BDNF null mutants, and 10% in NT4 null mutant mice at birth. The extent of proprioceptive deficiencies differed among different masticatory muscles, particularly in NT3 null mice. Masticatory muscles of embryonic mice heterozygous for the NT3(lacZneo) or BDNF(lacZ) reporter genes expressed both NT3 and BDNF, consistent with target-derived neurotrophin support of TMN neurons. Although more than 90% of TMN neurons expressed TrkB as well as TrkC receptor proteins by immunocytochemistry in wild-type newborns, TrkC or TrkB null mice exhibited only partial proprioceptive deficiencies similar to those present in NT3 or BDNF;NT4 null mice. Thus, in terms of the survival outcome, two main subpopulations of TMN neurons may exist during embryogenesis, one dependent on TrkC/NT3 functioning and the other utilizing TrkB/BDNF signaling. The differential dependence of TMN neurons on neurotrophins may reflect differential accessibility of the neurons to limiting amounts of NT3, BDNF, or NT4 in target tissues, especially if the tissue distribution or levels of BDNF, NT3, and NT4 were dynamically regulated both spatially and temporally.

Contraction-induced muscle fiber damage is increased in soleus muscle of streptozotocin-diabetic rats and is associated with elevated expression of brain-derived neurotrophic factor mRNA in muscle fibers and activated satellite cells.

The expression of brain-derived neurotrophic factor (BDNF) is elevated in the soleus muscle of streptozotocin-diabetic rats. To determine whether this diabetes-induced elevation was associated with or enhanced by muscle activity we have induced high-intensity muscle contraction by electrically stimulating the sciatic nerve. In 6-week diabetic rats, intense contraction of the soleus muscle resulted in a two- to four-fold elevation of BDNF mRNA and increased plasma levels of creatine kinase that were associated with severe focal muscle fiber damage and concomitant satellite cell activation. Focal muscle fiber damage and concomitant satellite cell activation were also observed in the soleus muscle of nonstimulated diabetic rats, but to a much lesser extent. No effects of muscle contraction, i.e., experimentally induced or during normal daily activity, on muscle fiber structure or BDNF mRNA expression were seen in diabetic extensor digitorum longus (EDL) muscle. Using a nonradioactive in situ hybridization technique for electron microscopy, the elevated expression of BDNF mRNA in the diabetic soleus muscle was localized within muscle fibers as well as activated satellite cells. This study shows that diabetic soleus muscle, in contrast to diabetic EDL and to soleus and EDL muscle of normal animals, is highly susceptible to contraction-induced damage. Intense contraction and the associated muscle fiber damage in the diabetic soleus muscle result in an upregulation of BDNF mRNA in muscle fibers and activated satellite cells, which may be involved in the restoration and/or maintenance of nerve/muscle integrity.

Development of fusimotor innervation correlates with group Ia afferents but is independent of neurotrophin-3.

Fusimotor neurons, group Ia afferents and muscle spindles are absent in mutant mice lacking the gene for neurotrophin-3 (NT3). To partition the effect of Ia afferent or spindle absence from that of NT3 deprivation on fusimotor neuron development, we examined the fusimotor system in a mutant mouse (NesPIXpNT3) that lacks Ia afferents and spindles, but has normal or elevated tissue levels of NT3 during embryogenesis. Fusimotor fibers were absent in lumbar ventral spinal roots, and limb muscles were devoid of Ia afferents and spindles in adult NesPIXpNT3 mice. In contrast, no deficiency in motoneuron numbers was observed in the trigeminal nucleus which contains cell bodies of motor axons innervating muscles of mastication. Spindles and Ia afferents were also present in the masticatory muscles. Thus, the development and/or survival of fusimotor neurons correlates with the presence of Ia afferents and/or spindles, and not with the amount of NT3 in the spinal cord or muscle.

Neurotrophin-3 and trkC in muscle are non-essential for the development of mouse muscle spindles.

Neurotrophin-3 (NT3) or TrkC null mutant mice were examined for the presence of muscle spindles. Muscles of mastication, but not limbs, contained spindles in newborn and adolescent mutants. The intramuscular distribution and morphological properties of spindles in mutant masticatory muscles were indistinguishable from those of wild-type spindles. Intrafusal fibers of NT3- or trkC-deficient spindles-expressed the slow-tonic isoform of myosin heavy chains, characteristic of wild-type spindles. Sensory nerve endings were observed in spindles of mutants by electron microscopy. Thus, NT3 or trkC, which is expressed in wild-type spindles, may serve functions other than those related to spindle assembly. Presumably, proprioceptive neurons innervating jaw muscles are dependent on factors other than NT3 for survival and maintenance.

Coculture of rat embryonic proprioceptive sensory neurons and myotubes.

With the aim to study the cellular mechanism underlying the process of muscle spindle regeneration, dorsal root ganglia (DRG) neurons derived from 16-day rat embryos were cocultured with developing myotubes in a compartmentalized culture device. To accomplish the selective survival and neurite formation of the proprioceptive subpopulation, the neurotrophic factor, neurotrophin-3, was added to the culture medium. It appeared that the proprioceptive DRG neurons could develop specialized, Ia afferent terminal-like contacts with myotubes. However, these interactions were scarce and did not result in the induction of differentiation of the contacted myotubes into intrafusal fibers as normally occurs during in vivo development. The present coculture setup apparently lacks appropriate regulatory factors essential for the proper matching of sensory axons and intrafusal fiber precursors and the induction of a functional sensory myoneural connection.