169 research outputs found
Targeting neurological disease with RNAi
No full textThe neuroscientific community rapidly adopted RNA interference techniques as an experimental tool for the dissection of gene function in vitro and in animal models of neurological disease in vivo. Here, we discuss recent advances in the biotechnical implementation of siRNA/shRNA-mediated gene silencing focusing on issues of design, delivery and putative detrimental effects. We then summarize the current use of RNAi in targeting neurological disease models and give an outlook on the implementation of this technique in clinical therapy
Axonal degeneration as a therapeutic target in the CNS
No full textDegeneration of the axon is an important step in the pathomechanism of traumatic, inflammatory and degenerative neurological diseases. Increasing evidence suggests that axonal degeneration occurs early in the course of these diseases and therefore represents a promising target for future therapeutic strategies. We review the evidence for axonal destruction from pathological findings and animal models with particular emphasis on neurodegenerative and neurotraumatic disorders. We discuss the basic morphological and temporal modalities of axonal degeneration (acute, chronic and focal axonal degeneration and Wallerian degeneration). Based on the mechanistic concepts, we then delineate in detail the major molecular mechanisms that underlie the degenerative cascade, such as calcium influx, axonal transport, protein aggregation and autophagy. We finally concentrate on putative therapeutic targets based on the mechanistic prerequisites
Galectin-1 expression in human glioma cells: modulation by ionizing radiation and effects on tumor cell proliferation and migration
No full textGalectins are evolutionarily conserved beta-galactoside-binding lectins which recognize specific glycoconjugates on the cell surface and the extracellular matrix. Accumulating evidence indicates that these proteins are involved in a variety of physiological and pathological processes including tumor growth and metastasis. Up-regulated expression of galectin-1 is a hallmark of a variety of malignant tumors. Here, we examined the expression of galectin-1 in glioma cell lines, the influence of ionizing irradiation and the intracellular and extracellular effects of this protein on tumor cell proliferation and migration. Galectin-1 was detected in both A172 and U118 glioma cells by immunoblot analysis. Ionizing irradiation induced a statistically significant up-regulation in glioma cell lines. RNA-interference-mediated silencing resulted in a significant suppression of the proliferation of the A172 cells, while the addition of recombinant galectin-1 had no effect. On the other hand, the migratory capacity of both cell lines was reduced after galectin-1 down-regulation, and up-regulated by the addition of exogenous galectin-1. Our results provide evidence of a role for galectin-1 in the regulation of glioma cell proliferation and migration. While an intracellular mechanism seemed to prevail in galectin-1-mediated regulation of tumor cell proliferation, the control of cell migration was exerted by both intracellular and extracellular mechanisms. In addition, this protein was up-regulated by ionizing radiation, indicating that the blockade of this protein should be performed before radiotherapy to avoid any undesired stimulating effects. Given the multifactorial role of galectin-1 in the regulation of tumor escape and metastasis, we conclude that targeting galectin-1 may have therapeutic benefits in the treatment of malignant glioma.Fil: Strik, Herwig M.. Universität Göttingen; AlemaniaFil: Schmidt, Katharina. Universität Göttingen; AlemaniaFil: Lingor, Paul. Universität Göttingen; AlemaniaFil: Tonges, Lars. Universität Göttingen; AlemaniaFil: Kugler, Wilfried. Universität Göttingen; AlemaniaFil: Nitsche, Mirko. Universität Göttingen; AlemaniaFil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Bähr, Mathias. Universität Göttingen; Alemani
Identification of new kinase clusters required for neurite outgrowth and retraction by a loss-of-function RNA interference screen
No full textDisruption of synaptic integrity, loss of connectivity and axodendritic degeneration are early and essential components of neurodegeneration. Although neuronal cell death mechanisms have been thoroughly investigated, less is known about the signals involved in axodendritic damage and the processes involved in regeneration. Here we conducted a genome-wide RNA interference-based forward genetic screen, using small interfering RNA targeting all human kinases, and identified clusters of kinases families essential for growth cone collapse, neurite retraction and neurite outgrowth. Of 59 kinases identified as positive regulators of neurite outgrowth, almost 50% were in the tyrosine kinase/tyrosine kinase-like (TK/TKL) receptor subgroups, underlining the importance of extracellular ligands in this process. Neurite outgrowth was inhibited by 66 other kinases, none of which were TK/TKL members, whereas 79 kinases inhibited lysophosphatidic acid-induced neurite retraction. Twenty kinases were involved in both inhibitory processes suggesting shared mechanisms. Within this group of 20 kinases, some (ULK1, PDK1, MAP4K4) have been implicated previously in axonal events, but others (MAST2, FASTK, CKM and DGUOK) have not. For a subset of kinases, the effect on neurite outgrowth was validated in rat primary cerebellar cultures. The ability to affect regeneration was further tested in a model of axodendritic lesion using primary rat midbrain cultures. Finally, we demonstrated that haploinsufficiency of two members of the AGC kinase subgroup, ROCK1 and PKN1, was able to suppress retinal degeneration in Drosophila model of class III Autosomal Dominant Retinitis Pigmentosa
Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units
No full textAbstractAdeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology
Early and Sustained Activation of Autophagy in Degenerating Axons after Spinal Cord Injury
No full textAxonal degeneration is one of the initial steps in many neurological disorders and has been associated with increased autophagic activity. Although there are increasing data on the regulation of autophagy proteins in the neuronal soma after spinal cord injury (SCI), their characterization in the axon is scarce. Here, we examined the regulation of autophagy during axonal degeneration in a rat model of SCI following a lesion at Th 8. We analyzed the morphological and ultrastructural changes in injured axons by immunohistochemical evaluation of autophagy-related proteins and electron microscopy at different time points following SCI. The expression of ULK1, Atg7 and Atg5 in damaged axons was rapidly upregulated within hours after SCI. The number of axonal LC3-positive autophagosomes was also rapidly increased after SCI and remained at an increased level for up to 6 weeks. Ultrastructural analysis showed early signs of axonal degeneration and increased autophagy. In conclusion, we show that autophagy is increased early and for a sustained period in degenerating axons after SCI and that it might be an important executive step involved in axonal degeneration. Therefore, autophagy may represent a promising target for future therapeutic interventions in the treatment of axonal degeneration in traumatic central nervous system disorders
Rifampicin inhibits neurodegeneration in the optic nerve transection model in vivo and after 1-methyl-4-phenylpyridinium intoxication in vitro
No full textRifampicin is an antibacterial drug which is highly effective in the treatment of tuberculosis and leprosy. It has been shown to exert antioxidative as well as anti-apoptotic effects. In this study, the neuroprotective effect of rifampicin was examined after 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic cell death in vitro, and on the survival of retinal ganglion cells after optic nerve transection in vivo. Rifampicin administration significantly increased the number of surviving dopaminergic neurons after MPP+ intoxication as compared to control cultures. No cytotoxic effects were noted even at final rifampicin concentrations of 100 muM. In the rifampicin-treated group, retinal ganglion cell survival was significantly increased after axotomy as compared with vehicle-treated and phosphate-buffered saline-treated control animals. These results suggest that rifampicin is able to prevent neuronal degeneration in cell death paradigms involving oxidative stress and activation of apoptotic pathways. It may thus play a role in the future treatments of neurodegenerative disorders
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