| 1. |
- Georgievska, Biljana, et al.
(författare)
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Dissociation between short-term increased graft survival and long-term functional improvements in Parkinsonian rats overexpressing glial cell line-derived neurotrophic factor.
- 2004
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Ingår i: European Journal of Neuroscience. - : Wiley. - 1460-9568 .- 0953-816X. ; 20:11, s. 3121-3130
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Tidskriftsartikel (refereegranskat)abstract
- The present study was designed to analyse whether continuous overexpression of glial cell line-derived neurotrophic factor (GDNF) in the striatum by a recombinant lentiviral vector can provide improved cell survival and additional long-term functional benefits after transplantation of fetal ventral mesencephalic cells in Parkinsonian rats. A four-site intrastriatal 6-hydroxydopamine lesion resulted in an 80–90% depletion of nigral dopamine cells and striatal fiber innervation, leading to stable motor impairments. Histological analysis performed at 4 weeks after grafting into the GDNF-overexpressing striatum revealed a twofold increase in the number of surviving tyrosine hydroxylase (TH)-positive cells, as compared with grafts placed in control (green fluorescent protein-overexpressing) animals. However, in animals that were allowed to survive for 6 months, the numbers of surviving TH-positive cells in the grafts were equal in both groups, suggesting that the cells initially protected at 4 weeks failed to survive despite the continued presence of GDNF. Although cell survival was similar in both grafted groups, the TH-positive fiber innervation density was lower in the GDNF-treated grafted animals (30% of normal) compared with animals with control grafts (55% of normal). The vesicular monoamine transporter-2-positive fiber density in the striatum, by contrast, was equal in both groups, suggesting that long-term GDNF overexpression induced a selective down-regulation of TH in the grafted dopamine neurons. Behavioral analysis in the long-term grafted animals showed that the control grafted animals improved their performance in spontaneous motor behaviors to approximately 50% of normal, whereas the GDNF treatment did not provide any additional recovery.
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| 2. |
- Georgievska, Biljana
(författare)
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GDNF gene delivery in an animal model of Parkinson's disease. Long-term effects on intact, injured and transplanted dopamine neurons using lentiviral gene transfer
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Parkinson's disease is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra, leading to a loss of dopamine in the target structure striatum and development of motor symptoms, such as bradykinesia, rigidity and tremor. New experimental treatment strategies for Parkinson's disease are aimed at either preventing the degeneration of the dopaminergic neurons, or at restoring dopamine in the striatum by fetal dopaminergic transplants. In this thesis work, we have evaluated the long-term effects of glial cell line-derived neurotrophic factor (GDNF) on intact, injured and transplanted dopaminergic neurons following GDNF gene delivery using a viral vector system based on lentiviruses. The results show that the lentiviral vectors provide an efficient transfer of the GDNF gene into the nigrostriatal dopamine system, resulting in a stable and long-lasting expression of the GDNF protein at high levels. The neuroprotective effects of lentiviral-mediated delivery of GDNF were evaluated in a rat model of Parkinson's disease and demonstrated that continuous overexpression of GDNF in the striatum provided an efficient protection of the nigral dopamine neurons, however, improvements in motor function were not observed. Instead, GDNF induced an aberrant sprouting of nigrostriatal fibers in areas outside of the striatum, and the phenotypic expression of tyrosine hydroxylase was reduced in the preserved dopaminergic terminals. We further evaluated the GDNF-induced downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system and showed that this effect was both time- and dose-dependent, and did not seem to have a detrimental effect on normal dopamine neurotransmission. The lentiviral vector was also used to study the long-term effects of GDNF on the survival and function of transplanted fetal dopamine neurons. GDNF initially increased the survival of the grafted dopamine neurons, however, the protected cells failed to survive long-term and the presence of GDNF around the grafts appeared to be detrimental to the transplant-induced recovery in these animals. Based on the observation that long-term and continuous GDNF delivery may have compromising effects on the functional outcome in either a neuroprotective or restorative paradigm, we developed a regulatable lentiviral vector system for controlled expression of GDNF in the rat brain. Efficient induction of GDNF gene expression was obtained following injection of the regulated lentiviral vector into the striatum, however, a significant basal expression was also observed, demonstrating the need to further improve the vector system for tight regulation in vivo. The findings of this thesis will have implications for the development of a GDNF gene therapy approach in Parkinson's disease.
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| 3. |
- Georgievska, Biljana, et al.
(författare)
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Overexpression of glial cell line-derived neurotrophic factor using a lentiviral vector induces time- and dose-dependent downregulation of tyrosine hydroxylase in the intact nigrostriatal dopamine system.
- 2004
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Ingår i: JNeurosci. - 1529-2401. ; 24:29, s. 6437-6445
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Tidskriftsartikel (refereegranskat)abstract
- The effects of continuous glial cell line-derived neurotrophic factor (GDNF) overexpression in the intact nigrostriatal dopamine (DA) system was studied using recombinant lentiviral (rLV) vector delivery of GDNF to the striatum or substantia nigra (SN) in the rat. Intrastriatal delivery of rLV-GDNF resulted in significant overexpression of GDNF in the striatum (2-4 ng/mg tissue) and anterograde transport of GDNF protein to the SN. Striatal rLV-GDNF delivery initially induced an increase in DA turnover (1-6 weeks), accompanied by significant contralateral turning in response to amphetamine, suggesting an enhancement of the DA system on the injected side. Starting 6 weeks after continuous GDNF delivery, we observed a selective downregulation of tyrosine hydroxylase (TH) protein (approximately 70%) that was maintained until the end of the experiment (24 weeks). A similar effect was observed when rLV-GDNF was injected into the SN. The magnitude of TH downregulation was related to the level of GDNF expression and was most pronounced in animals in which the striatal GDNF level exceeded 0.7 ng/mg tissue. The decreased TH protein levels were associated with similar reductions in the in vitro TH enzyme activity (approximately 70%); however, in vivo L-3,4-dihydroxyphenylalanine production rate and DA tissue levels were maintained at normal levels. The results indicate that downregulation of TH protein reflects a compensatory effect in response to continuous GDNF stimulation of the DA neurons mediated by a combination of overactivity at the DA synapse and a direct GDNF-induced action on TH gene expression. This compensatory mechanism is proposed to maintain long-term DA neuron function within the normal range.
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| 4. |
- Georgievska, Biljana, et al.
(författare)
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Regulated delivery of glial cell line-derived neurotrophic factor into rat striatum, using a tetracycline-dependent lentiviral vector.
- 2004
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Ingår i: Human Gene Therapy. - : Mary Ann Liebert Inc. - 1043-0342 .- 1557-7422. ; 15:10, s. 934-944
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Tidskriftsartikel (refereegranskat)abstract
- In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2S-M2, was developed for controlled expression of glial cell line-derived neurotrophic factor (GDNF) in the rat brain. Expression of the marker gene green fluorescent protein (GFP) and GDNF was tightly regulated in a dose-dependent manner in neural cell lines in vitro. Injection of high-titer lentiviral vectors into the rat striatum resulted in a 7-fold induction of GDNF tissue levels (1060 pg/mg tissue), when doxycycline (a tetracycline analog) was added to the drinking water. However, low levels of GDNF (150 pg/mg tissue) were also detected in animals that did not receive doxycycline, indicating a significant background leakage from the vector system in vivo. The level of basal expression was markedly reduced when a 10-fold lower dose of the tetracycline-regulated GDNF vector was injected into the striatum (3–11 pg/mg tissue), and doxycycline- induced GDNF tissue levels obtained in these animals were about 190 pg/mg tissue. Doxycycline-induced expression of GDNF resulted in a significant downregulation of the tyrosine hydroxylase (TH) protein in the intact striatum. Removal of doxycycline from the drinking water rapidly (within 3 days) turned off transgenic GDNF mRNA expression and GDNF protein levels in the tissue were completely reduced by 2 weeks, demonstrating the dynamics of the system in vivo. Accordingly, TH protein expression returned to normal by 2–8 weeks after removal of doxycycline, indicating that GDNF-induced downregulation of TH is a reversible event.
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| 5. |
- Jakobsson, Johan, et al.
(författare)
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Lesion-dependent regulation of transgene expression in the rat brain using a human glial fibrillary acidic protein-lentiviral vector.
- 2004
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Ingår i: European Journal of Neuroscience. - 1460-9568. ; 19:3, s. 761-765
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Tidskriftsartikel (refereegranskat)abstract
- The ability to regulate transgene expression will be crucial for development of gene therapy to the brain. The most commonly used systems are based on a transactivator in combination with a drug, e.g. the tetracycline-regulated system. Here we describe a different method of transgene regulation by the use of the human glial fibrillary acidic protein (GFAP) promoter. We constructed a lentiviral vector that directs transgene expression to astrocytes. Using toxin-induced lesions we investigated to what extent transgene expression could be regulated in accordance with the activation of the endogenous GFAP gene. In animals receiving excitotoxic lesions of the striatum we detected an eightfold increase of green fluorescent protein (GFP)-expressing cells. The vast majority of these cells did not divide, suggesting that the transgene was indeed regulated in a similar fashion as the endogenous GFAP gene. This finding will lead to the development of lentiviral vectors with autoregulatory capacities that may be very useful for gene therapy to the brain.
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| 6. |
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