Papers by Bisera Georgievska
Neuroscience, 2006
Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in anim... more Functional recovery following intrastriatal transplantation of fetal dopaminergic neurons in animal models of Parkinson's disease is, at least in part, dependent on the number of surviving dopaminergic neurons and the degree of graft-derived dopaminergic reinnervation of the host striatum. In the present study, we analyzed whether continuous exposure of glial cell line-derived neurotrophic factor (GDNF) to mature dopaminergic grafts could further boost the functional outcome of widespread intrastriatal dopaminergic grafts. Rats with dopamine-denervating lesions received multiple intrastriatal transplants of fetal dopaminergic cells and graft-induced behavioral effects were analyzed in drug-induced and spontaneous motor behaviors. At three months after grafting, animals received intrastriatal injections of recombinant lentiviral vectors encoding for either human GDNF or the green fluorescent protein. Continuous exposure of GDNF to the grafts did not boost the functional recovery beyond what was observed in the control animals. Rather, in some of the spontaneous motor behaviors, animals in the GDNF-group showed deterioration as compared with control animals, and this negative effect of GDNF was associated with a down-regulation of the tyrosine hydroxylase enzyme. Based on these and our earlier results, we propose that intrastriatal administration of GDNF at the time of or shortly after grafting is highly effective in initially promoting the cell survival and fiber outgrowth from the grafts. However, once the grafts are mature, GDNF's ability to boost dopaminergic neurotransmission follows the same dynamics as for the native nigral dopaminergic neurons, which appears to be dependent on the concentration of GDNF. Since rather low doses of glial cell line-derived neurotrophic factor at nanogram levels appear to saturate these effects, it may be critical to adjust GDNF levels using tightly regulated gene expression systems.
NeuroReport, 2007
We investigated the feasibility of viral vector-mediated expression and axonal transport of the g... more We investigated the feasibility of viral vector-mediated expression and axonal transport of the glial cell-line-derived neurotrophic factor, a potential antiepileptic agent, to the hippocampus and the piriform cortex, areas involved in the induction and spread of seizure activity. Glial cell-line-derived neurotrophic factor overexpression was induced by injections of recombinant vectors derived from serotype 2 adeno-associated virus or lentivirus. We found that recombinant adeno-associated viral vector was able to effectively transduce mitral cells of the olfactory bulb and pyramidal cells of CA1, resulting in transport of glial cell-line-derived neurotrophic factor to the piriform cortex and to the contralateral CA1 area, respectively. These data suggest that the recombinant adeno-associated viral vector vector system is an optimal alternative for therapeutic glial cell-line-derived neurotrophic factor gene transduction and transport of the protein to the epileptogenic brain areas.
Molecular Therapy, 2005
Abstract Functional effects of ventral mesencephalic (VM) cell transplants in models of Parkinson... more Abstract Functional effects of ventral mesencephalic (VM) cell transplants in models of Parkinson's disease are limited by the survival and fiber outgrowth of the grafted cells. In a recent study, we used a recombinant lentiviral vector (rLV) encoding glial cell line-derived ...
European Journal of Neuroscience, 2004
The present study was designed to analyse whether continuous overexpression of glial cell line-de... more 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.
Journal of Neurochemistry, 2014
Aggregation of amyloid beta (Ab) peptides and the subsequent neural plaque formation is a central... more Aggregation of amyloid beta (Ab) peptides and the subsequent neural plaque formation is a central aspect of Alzheimer's disease. Various strategies to reduce Ab load in the brain are therefore intensely pursued. It has been hypothesized that reducing Ab peptides in the periphery, that is in organs outside the brain, would be a way to diminish Ab levels and plaque load in the brain. In this report, we put this peripheral sink hypothesis to test by investigating how selective inhibition of Ab production in the periphery using a b-secretase (BACE)1 inhibitor or reduced BACE1 gene dosage affects Ab load in the brain. Selective inhibition of peripheral BACE1 activity in wildtype mice or mice over-expressing amyloid precursor protein (APPswe transgenic mice; Tg2576) reduced Ab levels in the periphery but not in the brain, not even after chronic treatment over several months. In contrast, a BACE1 inhibitor with improved brain disposition reduced Ab levels in both brain and periphery already after acute dosing. Mice heterozygous for BACE1, displayed a 62% reduction in plasma Ab40, whereas brain Ab40 was only lowered by 11%. These data suggest that reduction of Ab in the periphery is not sufficient to reduce brain Ab levels and that BACE1 is not the rate-limiting enzyme for Ab processing in the brain. This provides evidence against the peripheral sink hypothesis and suggests that a decrease in Ab via BACE1 inhibition would need to be carried out in the brain.
Neuroreport, 2002
We used a recombinant lentiviral vector (rLV) for gene delivery of GDNF to the striatum, and asse... more We used a recombinant lentiviral vector (rLV) for gene delivery of GDNF to the striatum, and assessed its neuroprotective e¡ects in the intrastriatal 6 -hydroxydopamine (6 -OHDA) lesion model.The level of GDNF expression obtained with the rLV-GDNF vector was dose-related and ranged between 0.9^9.3 ng/mg tissue in the transduced striatum, as determined by ELISA, and 0.2^3.0 ng/mg tissue were detected in the ipsilateral substantia nigra (SN), due to anterograde transport of the GDNF protein. GDNF expression was apparent at 4 days and maintained for ! 8 months after injection. Striatal delivery of rLV-GDNF e⁄ciently protected the nigral dopamine (DA) neurons and their projection, against the 6 -OHDA lesion (65^77% of intact side). Sprouting of the lesioned axons was observed along the nigrostriatal pathway, precisely corresponding to the areas containing anterogradely transported GDNF.NeuroReport13:75^82
FEBS Journal, 2014
A major hallmark of Alzheimer&amp... more A major hallmark of Alzheimer's disease (AD) is the deposition of amyloid-β (Aβ) peptides in amyloid plaques. Aβ peptides are produced by sequential cleavage of the amyloid precursor protein by the β amyloid cleaving enzyme (BACE) and the γ-secretase (γ-sec) complex. Pharmacological treatments that decrease brain levels of in particular the toxic Aβ42 peptide are thought to be promising approaches for AD disease modification. Potent and selective BACE1 inhibitors as well as γ-sec modulators (GSMs) have been designed. Pharmacological intervention of secretase function is not without risks of either on- or off-target adverse effects. One way of improving the therapeutic window could be to combine treatment on multiple targets, using smaller individual doses and thereby minimizing adverse effect liability. We show that combined treatment of primary cortical neurons with a BACE1 inhibitor and a GSM gives an additive effect on Aβ42 level change compared with the individual treatments. We extend this finding to C57BL/6 mice, where the combined treatment results in reduction of brain Aβ42 levels reflecting the sum of the individual treatment efficacies. These results show that pharmacological targeting of two amyloid precursor protein processing steps is feasible without negatively interfering with the mechanism of action on individual targets. We conclude that targeting Aβ production by combining a BACE inhibitor and a GSM could be a viable approach for therapeutic intervention in AD modification.
Journal of Neuroscience, 2005
The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) for Parkinson's d... more The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) for Parkinson's disease is likely to depend on sustained delivery of the appropriate amount to the target areas. Recombinant adeno-associated viral vectors (rAAVs) expressing GDNF may be a suitable delivery system for this purpose. The aim of this study was to define a sustained level of GDNF that does not affect the function of the normal dopamine (DA) neurons but does provide anatomical and behavioral protection against an intrastriatal 6-hydroxydopamine (6-OHDA) lesion in the common marmoset. We found that unilateral intrastriatal injection of rAAV resulting in the expression of high levels of GDNF (14 ng/mg of tissue) in the striatum induced a substantial bilateral increase in tyrosine hydroxylase protein levels and activity as well as in DA turnover. Expression of low levels of GDNF (0.04 ng/mg of tissue), on the other hand, produced only minimal effects on DA synthesis and only on the injected side. In addition, the low level of GDNF provided ϳ85% protection of the nigral DA neurons and their projections to the striatum in the 6-OHDA-lesioned hemisphere. Furthermore, the anatomical protection was accompanied by a complete attenuation of sensorimotor neglect, head position bias, and amphetamine-induced rotation. We conclude that when delivered continuously, a low level of GDNF in the striatum (approximately threefold above baseline) is sufficient to provide optimal functional outcome.
Proceedings of the National Academy of Sciences, 1999
A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic ag... more A number of in vitro studies have shown that activation of muscarinic receptors by cholinergic agonists stimulates the nonamyloidogenic, alpha-secretase-processing pathway of amyloid precursor protein (APP). To determine whether increased cholinergic neurotransmission can modify the APP processing in vivo, we administered a muscarinic receptor agonist (RS86) to normal or aged rats and rats with severe basal forebrain cholinergic deficits (induced by 192 IgG-saporin). The levels of the cell-associated APP in neocortex, hippocampus, and striatum, as well as the secreted form of APP (APPs) in cerebrospinal fluid, were examined by Western blots. Additionally, we investigated the association between the altered APP levels and behavioral deficits caused by cholinergic lesions. We found that treatment with muscarinic receptor agonist resulted in decreased APP levels in neocortex and hippocampus and increased levels of APPs in cerebrospinal fluid. Regulation of APP processing by the muscarinic agonist treatment occurred not only in normal rats, but also in aged and cholinergic denervated rats that model this aspect of Alzheimer's disease. Interestingly, we found that elevation of APP in neocortex correlated with the cognitive deficits in water-maze testing of rats with cholinergic dysfunction. These data indicate that increased cholinergic neurotransmission can enhance nonamyloidogenic APP processing in intact and lesioned rats and that APP may be involved in cognitive performance.
Proceedings of the National Academy of Sciences, 2002
Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a t... more Intrastriatal delivery of the tyrosine hydroxylase gene by viral vectors is being explored as a tool for local delivery of L-dopa in animals with lesions of the nigrostriatal pathway. The functional effects reported using this approach have been disappointing, probably because the striatal L-dopa levels attained have been too low. In the present study, we have defined a critical threshold level of L-dopa, 1.5 pmol͞mg of tissue, that has to be reached to induce any significant functional effects. Using new generation high-titer recombinant adeno-associated virus vectors, we show that levels of striatal L-dopa production exceeding this threshold can be obtained provided that tyrosine hydroxylase is coexpressed with the cofactor synthetic enzyme, GTP-cyclohydrolase-1. After striatal transduction with this combination of vectors, substantial functional improvement in both drug-induced and spontaneous behavior was observed in rats with either complete or partial 6hydroxydopamine lesions of the nigrostriatal pathway. However, complete reversal of motor deficits occurred only in animals in which part of the striatal dopamine innervation was left intact. Spared nigrostriatal fibers thus may convert L-dopa to dopamine and store and release dopamine in a more physiologically relevant manner in the denervated striatum to mediate better striatal output-dependent motor function. We conclude that intrastriatal L-dopa delivery may be a viable strategy for treatment and control of adverse side effects associated with oral L-dopa therapy such as on-off fluctuations and drug-induced dyskinesias in patients with Parkinson's disease.
Nature Neuroscience, 2004
Ten years ago, a glial cell line-derived neurotrophic factor (GDNF) that has prominent actions on... more Ten years ago, a glial cell line-derived neurotrophic factor (GDNF) that has prominent actions on nigral dopaminergic neurons, both in vitro and in animal models of Parkinson disease (PD), was discovered. A recently published open-label clinical trial now reports that long-term intracerebral delivery of GDNF may also markedly improve symptoms in patients with PD. Here we review the experimental data underlying the current clinical trial and discuss the types of structural and functional changes induced by GDNF that may provide symptomatic benefit in PD patients. Data obtained in rodent and primate models of PD highlight the importance of how and where the factor is administered, supporting the view that GDNF has to be delivered locally in the brain parenchyma, at the receptive target site, to provide therapeutic benefit in PD patients.
Journal of Neurochemistry, 2013
Abnormal tau phosphorylation resulting in detachment of tau from microtubules and aggregation are... more Abnormal tau phosphorylation resulting in detachment of tau from microtubules and aggregation are critical events in neuronal dysfunction, degeneration, and neurofibrillary pathology seen in Alzheimer's disease. Glycogen synthase kinase-3β (GSK3β) is a key target for drug discovery in the treatment of Alzheimer's disease and related tauopathies because of its potential to abnormally phosphorylate proteins and contribute to synaptic degeneration. We report the discovery of AZD1080, a potent and selective GSK3 inhibitor that demonstrates peripheral target engagement in Phase 1 clinical studies. AZD1080 inhibits tau phosphorylation in cells expressing human tau and in intact rat brain. Interestingly, subchronic but not acute administration with AZD1080 reverses MK-801-induced deficits, measured by long-term potentiation in hippocampal slices and in a cognitive test in mice, suggesting that reversal of synaptic plasticity deficits in dysfunctional systems requires longer term modifications of proteins downstream of GSK3β signaling. The inhibitory pattern on tau phosphorylation reveals a prolonged pharmacodynamic effect predicting less frequent dosing in humans. Consistent with the preclinical data, in multiple ascending dose studies in healthy volunteers, a prolonged suppression of glycogen synthase activity was observed in blood mononuclear cells providing evidence of peripheral target engagement with a selective GSK3 inhibitor in humans.
Human Gene Therapy, 2004
In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-depen... more In this study, a tetracycline-regulated lentiviral vector system, based on the tetracycline-dependent transactivator rtTA2(S)-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.
Gene Therapy, 2006
Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the... more Lentiviral vectors are promising tools for CNS gene transfer since they efficiently transduce the cells of the nervous system in vivo. In this study, we have investigated the transduction efficiency of lentiviral vectors pseudotyped with Ross River virus glycoprotein (RRV-G) (RRV-G-pseudotyped lentiviral vectors (RRV-LV)). The RRV is an alphavirus with an extremely broad host range, including the cells of the central nervous system. Previous studies have shown that lentiviral vectors can be efficiently pseudotyped with this envelope protein and have demonstrated promising features of such vectors, including the possibility to establish stable producer cell lines. After injection of RRV-LV expressing green fluorescent protein into different structures in the rat brain we found efficient transduction of both neurons and glial cells. By using two cell-type-specific promoters, neuron-specific enolase and human glial fibrillary acidic protein, we demonstrated cellspecific transgene expression in the desired cell type. Ross River virus glycoprotein-pseudotyped lentiviral vectors also transduced human neural progenitor cells in vitro, showing that receptors for the RRV-G are present on human neural cells. Gene Therapy (2006) 13, 966-973.
Experimental Neurology, 2002
The effects of sustained (up to 9 months) striatal overexpression of glial cell line derived neur... more The effects of sustained (up to 9 months) striatal overexpression of glial cell line derived neurotrophic factor (GDNF) on lesioned nigrostriatal dopamine (DA) neurons was studied using a recombinant lentiviral (rLV) vector to deliver GDNF into the striatum 4 weeks prior to the creation of an intrastriatal 6-hydroxydopamine lesion. The results of the amphetamine-induced rotation suggested an initial partial protection followed by a complete recovery, whereas the spontaneous motor behaviors remained impaired. There was a clear protection of the nigral tyrosine hydroxylase (TH)-positive neurons in the rLV-GDNF group compared to rats injected with the control vector encoding green fluorescent protein (GFP) (70 and 20% of the intact side, respectively). However, the striatal TH؉ fiber density was equally reduced (to 20% of the intact side) in both groups. Further morphological analyses indicated that the nigrostriatal projections of the DA neurons were indeed preserved in the GDNF group. The axonal projections were visualized using two independent methods: First, retrograde labeling of the nigral cell bodies by intrastriatal Fluoro-Gold injections showed that the majority of rescued cells in the GDNF group had preserved axonal projections to striatum. Second, injections of a recombinant adenoassociated viral vector expressing GFP into the nigra was used to anterogradely fill the DA neurons and their projections with GFP protein. GFP immunostaining clearly demonstrated that the fibers of the nigral DA cells were preserved along the nigrostriatal pathway and innervated large parts of the striatum, but did not express TH at detectable levels. In addition, fiber sprouting was observed in the globus pallidus, entopeduncular nucleus, and substantia nigra, corresponding to areas where GDNF protein was released. The lack of functional recovery in the spontaneous motor behaviors may, at least in part, be ex-plained by this extensive aberrant fiber sprouting in the downstream striatal target nuclei and/or decreased synthesis of dopamine in the striatum. © 2002 Elsevier Science (USA)
Uploads
Papers by Bisera Georgievska