The cell cycle gene repressor DRM- complex modulates DNA damage response in Caenorhabditis elegans

Cancer is a frequent cause of death. Disturbed or inefficient DNA damage response including DNA repair, cell cycle control and apoptosis, can cause tumorigenesis. The central question for this thesis was to study the relation between DNA repair and cell cycle control. The DPL-1- RB- MuvB (DRM)- complex is a well-characterized gene repressor in the nematode Caenorhabditis elegans. Its main task is the regulation of the cell cycle by repression of different genes. Homologous complexes also exist in humans and Drosophila melanogaster. For this reason, the DRM- complex was investigated in Caenorhabditis elegans. DNA damage was applied to mutants for this complex to study the impact on larval development. Furthermore, these mutant worms were intercrossed with several mutants for DNA repair pathways to investigate the interplay of cell cycle regulation and DNA repair. Using multiple in-vivo approaches by applying different types of DNA damage to these worms and in-vitro studies with qPCR and western blot analyses provided evidence that DRM- complex mutant worms exhibit an improved response to DNA damage. This is not a consequence of accelerated cell cycle. Mutants with defects in different DNA repair pathways exhibit an arrest in larval development. Interestingly, this could be bypassed partially by intercross with mutants of the DRM- complex. In summary these data indicate that mutants defective for components of DRM- complex exhibit improved response to different DNA damaging agents and that multiple repair mechanisms are involved in this altered developmental response of DRM- complex mutants

Ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons

Although evidence obtained with the PC12 cell line has suggested a role for the ras oncogene proteins in the signal transduction of nerve growth factor-mediated fiber outgrowth, little is known about the signal transduction mechanisms involved in the neuronal response to neurotrophic factors in nontransformed cells. We report here that the oncogene protein T24-ras, when introduced into the cytoplasm of freshly dissociated chick embryonic neurons, promotes the in vitro survival and neurite outgrowth of nerve growth factor-responsive dorsal root ganglion neurons, brain-derived neurotrophic factor-responsive nodose ganglion neurons, and ciliary neuronotrophic factor-responsive ciliary ganglion neurons. The proto-oncogene product c-Ha-ras also promotes neuronal survival, albeit less strongly. No effect could be observed with truncated counterparts of T24-ras and c-Ha-ras lacking the 23 C-terminal amino acids including the membrane-anchoring, palmityl-accepting cysteine. These results suggest a generalized involvement of ras or ras-like proteins in the intracellular signal transduction pathway for neurotrophic factors

Synthesis and localization of ciliary neurotrophic factor in the sciatic nerve of the adult rat after lesion and during regeneration

Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to <5 % in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration

Rat ciliary neurotrophic factor (CNTF)

The structure of the rat ciliary neurotrophic factor (CNTF) gene and the regulation of CNTF mRNA levels in cultured glial cells were investigated. The rat mRNA is encoded by a simple two-exon transcription unit. Sequence analysis of the region upstream of the transcription start-site did not reveal a typical TATA-box consensus sequence. Low levels of CNTF mRNA were detected in cultured Schwann cells, and CNTF mRNA was not increased by a variety of treatments. Three-week-old astrocyte-enriched cell cultures from new-born rat brain contained easily detectable CNTF mRNA. In astrocyte-enriched cultures, upregulation of CNTF mRNA levels was observed after treatment with IFN-. CNTF mRNA levels were down-regulated in these cells by treatments that elevate intracellular cyclic AMP and by members of the fibroblast growth factor (FGF) family. The implications of these results for potential in vivo functions of CNTF are discusse

Muscle-derived factors that support survival and promote fiber outgrowth from embryonic chick spinal motor neurons in culture

The purpose of the experiments reported is to provide an unambiguous demonstration that embryonie skeletal muscle contains factors that act directly on embryonie spinal motor neurons both to support their survival and to stimulate the outgrowth of neurites. Cells of lumbar and brachial ventral spinal cords from 6-day-old chick embryos were separated by centrifugation in a two-step metrizamide gradient, and a motor neuron enriched fraction was obtained. Motor neurons were identified by retrogradely labeling with rhodamine isothiocyanate, and were enriched fourfold in the motor neuron fraction relative to unfractionated cells. In culture, the isolated motor neurons died within 3-4 days unless they were supplemented with embryonie chick skeletal muscle extract. Two functionally distinct entities separable by ammonium sulfate precipitation were responsible for the effects of muscle extracts on motor neurons. The 0-25% ammonium sulfate precipitate contained molecules that alone bad no effect on neuronal survival but when bound to polyornithine-coated culture substrata, stimulated neurite outgrowth and potentiated the survival activity present in muscle. Most of this activity was due to a laminin-like molecule being immunoprecipitated with antisera against laminin, and immunoblotting demonstrated the presence of both the A and B chains of laminin. A long-term survival activity resided in the 25-70% ammonium sulfate fraction, and its apparent total and specific activities were strongly dependent on the culture substrate. In contrast to the motor neurons, the cells from the other metrizamide fraction (including neuronal cells) could be kept in culture for a prolonged time without addition of exogenous factor(s)

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGF(delta/delta) mouse model of amyotrophic lateral sclerosis

Background: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGF(delta/delta) mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. Results: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGF(delta/delta) mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGF(delta/delta) mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGF(delta/delta) mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGF(delta/delta) mice, axon outgrowth is significantly reduced compared to wild-type littermates. Conclusions: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy

Trabekulektomie mit Mitomycin C in Kombination mit Bevacizumab - intravitreal, subkonjunktival, intracameral - retrospektive Evaluation des Einflusses von Bevacizumab auf die postoperative Wundheilungsmodulation, den Augendruck, operative Folgeeingriffe und die postoperative antiglaukomatöse Medikation

Zusammenfassung Ziel: Glaukom ist die zweithäufigste Erblindungsursache nach AMD (altersbedingte Makuladegeneration) weltweit. Gold-Standard bei konservativ nicht beherrschbarer Augeninnendruckerhöhung ist die Trabekulektomie (TET). Entscheidend für den Erfolg einer Trabekulektomie ist die Wundheilungsmodulation in den ersten vier bis sechs Wochen postoperativ. Die selektive Modulation der Wundheilung durch Anti-VEGF (Anti-Körper gegen Vascular Endothelial Growth Factor) z.B. durch Bevacizumab zeigt hierbei die effektivste Wirkung bei okulärer Narbenreduktion. In dieser retrospektiven Evaluation wurde der Einfluss von Bevacizumab in drei verschiedenen Applikationsformen – intravitreal (Gruppe 1), subkonjunktival (Gruppe 3), intracameral (Gruppe 4) – untereinander und gegenüber einer Kontrollgruppe ohne Bevacizumab (Gruppe 2) auf die postoperative Wundheilungsmodulation und die sich daraus ergebenden klinischen Parameter nach Trabekulektomie mit Mitomycin C untersucht. Methode: In einer retrospektiven Studie wurden 295 Trabekulektomien (TET) mit Mitomycin C in Kombination mit Bevacizumab von 256 Patienten mit einer Nachbeobachtungszeit bis zu einem Jahr (postoperativ bei Entlassung, 4-6 Wochen postoperativ, 6 ± 1 Monate postoperativ, 12 ± 1,5 Monate postoperativ) ausgewertet. Die Trabekulektomie erfolgte bei allen Patienten ausschließlich durch eine erfahrene Glaukomspezialistin unter standardisierten Bedingungen. Die einzelnen Gruppen setzen sich wie folgt zusammen: Gruppe 1: n=109 Patienten, Gruppe 2: n=101 Patienten, Gruppe 3: n=51 Patienten, Gruppe 4: n=34 Patienten. Folgende Daten wurden erhoben: Bestkorrigierter Visus, applanatorisch gemessener intraokularer Druck in mmHg (IOD), Anzahl der antiglaukomatösen Medikamente (Anzahl der Wirkstoffe), Anzahl der 5-Fluorouracil-Injektionen (5-FU) subkonjunktival, Sickerkissenmorphologiebeurteilung, Anzahl der postoperativen Komplikationen/Interventionen, relativer IOD-Erfolg (IOD ≥ 6 und ≤ 18 mmHg bei gleichzeitiger IOD-Reduktion mindestens 30 % im Vergleich zu präoperativ mit lokaler Medikation), absoluter Erfolg (IOD ≥ 6 und ≤ 18 mmHg bei gleichzeitiger IOD-Reduktion mindestens 30 % im Vergleich zu präoperativ ohne zusätzliche antiglaukomatöse Medikation und ohne zusätzliche notwendige operative Interventionen nach TET), Wundheilungsscore (Beurteilung von IOD-Erfolg, Sickerkissenstatus und post-OP Interventionen). Ergebnisse: Eine normale nicht überschießende Vaskularisation (Vaskularisation +) fand sich signifikant häufiger nach subkonjunktivaler Bevacizumabapplikation als nach intravitrealer Bevacizumabapplikation (p<0,05). Das Auftreten von Sickerkissen-Mikrozysten – ein positives Indikatormerkmal der Wundheilung – war ebenfalls nach subkonjunktivaler Bevacizumabapplikation (p<0,001) signifikant absolut am häufigsten, bei intravitrealer Bevacizumabapplikation geringer und bei intracameraler Applikation von Bevacizumab signifikant am niedrigsten. Korkenziehergefäßstatus und vor allem Tenonzysten waren als Indikatormerkmale bezüglich der Bevacizumabapplikationsform weniger wegweisend. Bezüglich Visus nach TET zeigten sich bei den drei verschiedenen Formen von Bevacizumabapplikationen keine signifikanten Unterschiede. Die Anzahl der zur Wundheilungsmodulation verabreichten 5-FU-Injektionen nach TET war nach intraoperativer subkonjunktivaler Bevacizumabinjektion (p<0,01) signifikant am niedrigsten. Die Glaukomwirkstoffanzahl nach TET konnte grundsätzlich bei allen Arten von Bevacizumabapplikation und auch in der Kontrollgruppe massiv reduziert werden. Dies zeigte eindrucksvoll die ausgeprägte IOD-senkende Wirkung einer Trabekulektomie. Nach subkonjunktivaler Bevacizumabapplikation waren sowohl im Kurzzeit- als auch im Langzeitverlauf die signifikant höchsten Raten an relativen IOD-Erfolgen (p<0,01) zu messen. Die Anzahl relevanter operativer Interventionen nach TET wurde nach subkonjunktivaler Bevacizumabapplikation und vor allem auch nach intracameraler Bevacizumabapplikation entscheidend gesenkt. Nach subkonjunktivaler Bevacizumabapplikation konnte die insgesamt höchste absolute Erfolgsrate (43,1%) im Laufe der Wundheilung im Kurzzeitverlauf erzielt werden, signifikant höher als nach intravitrealer Bevacizumabapplikation (p<0,01).Im Langzeitverlauf nach einem Jahr war nach subkonjunktivaler Bevacizumabapplikation die absolut höchste Erfolgsrate (72,2%) zu verzeichnen - höher als nach intravitrealer Bevacizumabapplikation und auch gegenüber durchgeführten Trabekulektomien ohne Bevacizumabapplikation. Die Ergebnisse des Wundheilungsscores implizieren eine gut modulierte Wundheilung mit darauf basierender effektiver Augeninnendrucksenkung vornehmlich bei subkonjunktivaler Bevacizumabapplikation (den Patienten in Gruppe 3 nach subkonjunktivaler Bevacizumabapplikation nach TET konnten insgesamt und zum Teil signifikant hohe Wundheilungsscorepunkte zugewiesen werden). Fazit: Die erfolgversprechendste Variante zur Wundheilungsmodulation hinsichtlich der untersuchten Parameter nach Trabekulektomie in der vorliegenden Studiengruppe war: TET mit Mitomycin C in Kombination mit subkonjunktivaler Bevacizumabapplikation

Whole transcriptome profiling reveals the RNA content of motor axons

Most RNAs within polarized cells such as neurons are sorted subcellularly in a coordinated manner. Despite advances in the development of methods for profiling polyadenylated RNAs from small amounts of input RNA, techniques for profiling coding and non-coding RNAs simultaneously are not well established. Here, we optimized a transcriptome profiling method based on double-random priming and applied it to serially diluted total RNA down to 10 pg. Read counts of expressed genes were robustly correlated between replicates, indicating that the method is both reproducible and scalable. Our transcriptome profiling method detected both coding and long non-coding RNAs sized >300 bases. Compared to total RNAseq using a conventional approach our protocol detected 70% more genes due to reduced capture of ribosomal RNAs. We used our method to analyze the RNA composition of compartmentalized motoneurons. The somatodendritic compartment was enriched for transcripts with post-synaptic functions as well as for certain nuclear non-coding RNAs such as 7SK. In axons, transcripts related to translation were enriched including the cytoplasmic non-coding RNA 7SL. Our profiling method can be applied to a wide range of investigations including perturbations of subcellular transcriptomes in neurodegenerative diseases and investigations of microdissected tissue samples such as anatomically defined fiber tracts

Effect of ciliary neurotrophic factor (CNTF) on motoneuron survival

We have demonstrated that the extensive degeneration of motoneurons in the rat facial nucleus after transection of the facial nerve in newborn rats can be prevented by local ciliary neurotrophic factor (CNTF) administration. CNTF differs distinctly from known neurotrophic molecules such as NGF, BDNF and NT-3 in both its molecular characteristics (CNTF is a cytosolic rather than a secretory molecule) and its broad spectrum of biological activities. CNTF is expressed selectively by Schwann cells and astrocytes of the peripheral and central nervous system, respectively, but not by target tissues of the great variety of CNTF -responsive neurons. CNTF mRNA is not detectable by Northern blot or PCR analysis during embryonic development and immediately after birth. However, during the second post-natal week, a more than 30-fold increase in CNTF mRNA and pro tein occurs in the sciatic nerve. Since the period of low CNTF levels in peripheral nerves coincides with that of high vulnerability of motoneurons (i.e. axonallesion results in degeneration of motoneuron cell bodies), insufficient availability of CNTF may be the reason for the rate of lesioninduced cell death of early post-natal motoneurons. Highly enriched embryonic chick motoneurons in culture are supported at survival rates higher than 60% by CNTF, even in single cell cultures, indicating that CNTF acts directly on motoneurons. In contrast to CNTF, the members of the neurotrophin gene family (NGF, BDNF and NT-3) do not support the survival of motoneurons in culture. However, aFGF and bFGF show distinct survival activities which are additive to those of CNTF, resulting in the survival of virtually all motoneurons cultured in the presence of CNTF and bFGF

Axotomy increases CNTF receptor mRNA in rat spinal cord

In order to study the role of ciliary neurotrophic factor (CNTF) and its receptor (CNTF-R) in the response of spinal cord neurons to axotomy, we measured the levels of CNTF mRNA in nerve and CNTF-R mRNA in spinal cord following transection of sciatic nerve, using reverse transcriptase PCR. We found CNTF mRNA levels in the nerve fell and that CNTF-R mRNA levels in spinal cord increased at both 1 and 7 days following transection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30830/1/0000492.pd