Poster Sessions CP14: Transplantation and Neurological Repair. GDNF and VMC cotransplantation helps in restoration of neurobehavioral function in Parkinson's disease

During transplantation of VMC in Parkinson's disease their degeneration is very high due to lack of trophic support and mismatch conditions. To overcome this problem, Glial cell line‐derived Neurotrophic Factor (GDNF) known to increase the functional viability and regeneration of dopaminergic cells. In the present study an attempt has been made to validate the role of GDNF cotransplanted with fetal VMC in functional restoration in rat model of Parkinson's disease. A significant restoration was observed in apomorphine induced rotation in rats co transplanted with GDNF and VMC (66%) as compare to VMC alone (42%). Apomorphine induced locomotor activity was restored by 67, 38% in cotransplanted and VMC alone transplanted rats, respectively. Level of dopamine and 3,4 dihydroxy‐phenyl acetic acid (DOPAC) in the striatum were significantly restored by 67 and 62, 42 and 33% in cotransplanted and VMC alone transplanted rats, respectively. A significant restoration was observed in striatum dopamine receptors by 69% in rats cotransplanted with VMC & GDNF, and 45% in those transplanted with VMC alone. GDNF alone transplantation did not show significant restoration in either of the parameters. Functional viabilty of dopaminergic neurons was further confirmed by Tyrosine Hydroxylase (TH) immunopositivity in striatal region where a significantly high expression was observed in cotransplanted animals when compared with VMC alone.Results of the present study suggests that cotransplantation of GDNF and VMC may help in better functional restoration in 6‐OHDA lesioned rat model of Parkinson's disease studied at 4 weeks post transplantation.     

Intrastriatal Transplantation of GDNF-engineered BMSCs and its neuroprotection in Lactacystin-induced Parkinsonian Rat Model

The potential value of glial cell line-derived neurotrophic factor (GDNF) in treating Parkinson's disease (PD) remains controversial. In order to evaluate the therapeutic effect of GDNF-engineered bone marrow stromal cells (BMSCs) in parkinsonian rat model, GDNF-BMSCs and LacZ-BMSCs were transplanted into striatum and followed by Lactacystin lesioning at median forebrain bundles 1 week later. We observed that the intrastriatal transplantation of GDNF-BMSCs could significantly rescue the dopaminergic neurons from lactacystin-induced neurotoxicity with regard to behavioral recovery, tyrosine hydroxylase level in nigra and striatum, and striatal dopamine level. We interpret the outcomes that intrastriatal transplantation of GDNF-BMSCs might be beneficial in the treatment of PD.     

Striatal GDNF administration increases tyrosine hydroxylase phosphorylation in the rat striatum and substantia nigra

Glial cell line-derived neurotrophic factor (GDNF) improves motor dysfunction associated with aging in rats and non-human primates, in animal models of Parkinson's disease, and may improve motoric function in patients with advanced Parkinson's disease. These improvements are associated with increased dopamine function in the nigrostriatal system, but the molecular events associated with this increase are unknown. In these studies, 100 micro g of GDNF was injected into the striatum of normal aged (24-month-old) male Fischer 344 rats. The protein levels and phosphorylation of TH, ERK1/2, and related proteins were determined by blot-immunolabeling of striatum and substantia nigra harvested 30 days after injection. In GDNF-treated rats, TH phosphorylation at Ser31 increased approximately 40% in striatum and approximately 250% in the substantia nigra. In the substantia nigra, there was a significant increase in ERK1 phosphorylation. In striatum, there was a significant increase in ERK2 phosphorylation. Microdialysis studies in striatum showed that both amphetamine- and potassium-evoked dopamine release in GDNF recipients were significantly increased. These data show that GDNF-induced increases in dopamine function are associated with a sustained increase in TH phosphorylation at Ser31, which is greatest in the substantia nigra and maintained for at least one month following a single striatal administration of GDNF. These findings, taken from the nigrostriatal system of normal aged rats, may help explain the long lasting effects of GDNF on dopamine function and prior studies supporting that a major effect of GDNF involves its effects on dopamine storage and somatodendritic release of dopamine in the substantia nigra.     

Long-term glial cell line-derived neurotrophic factor overexpression in the intact nigrostriatal system in rats leads to a decrease of dopamine and increase of tetrahydrobiopterin production

Parkinson's disease (PD) is characterized by the progressive degeneration of the nigrostriatal dopaminergic system. Brain delivery of glial cell line-derived neurotrophic factor (GDNF) has been shown to protect and restore the dopaminergic pathway in various animal models of PD. However, GDNF overexpression in the dopaminergic pathway leads to a time-dependent down-regulation of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis. In order to elucidate GDNF-mediated biochemical effects on dopaminergic neurons, we overexpressed GDNF in the intact rat striatum using a lentiviral vector-mediated gene transfer technique. Long-term GDNF overexpression led to increased GTP cyclohydrolase I (GTPCH I) activity and tetrahydrobiopterin (BH4) levels. Further, we observed a down-regulation of TH enzyme activity in morphologically intact striatal dopaminergic nerve terminals, as well as a significant decrease of dopamine levels in striatal tissue samples. These results indicate that long-term GDNF delivery is a major factor affecting dopamine biosynthesis via a direct or indirect modulation of TH and GTPCH I and further underscore the importance of assessing both GDNF dose and delivery duration prior to clinical application in order to circumvent potentially adverse pharmacological effects on the biosynthesis of dopamine.     

Partial recovery of dopaminergic pathway after graft of adult mesenchymal stem cells in a rat model of Parkinson's disease

Cellular therapy with adult stem cells appears as an opportunity for treatment of Parkinson's disease. To validate this approach, we studied the effects of transplantation of rat adult bone-marrow mesenchymal stem cells in a rat model of Parkinson's disease. Animals were unilaterally lesioned in the striatum with 6-hydroxydopamine. Two weeks later, group I did not undergo grafting, group II underwent sham grafting, group III was intra-striatal grafted with cells cultured in an enriched medium and group IV was intra-striatal grafted with cells cultured in a standard medium. Rotational amphetamine-induced behavior was measured weekly until animals were killed 6 weeks later. One week after graft, the number of rotations/min was stably decreased by 50% in groups III and IV as compared with groups I and II. At 8 weeks post-lesion, the density of dopaminergic markers in the nerve terminals and cell bodies, i.e. immunoreactive tyrosine hydroxylase, membrane dopamine transporter and vesicular monoamine transporter-2 was significantly higher in group III as compared with group I. Moreover, using microdialysis studies, we observed that while the rate of pharmacologically induced release of dopamine was significantly reduced in lesioned versus intact striatum in no grafted rats, it was similar in both sides in animals transplanted with mesemchymal stem cells. These data demonstrate that graft of adult mesemchymal stem cells reduces behavioral effects induced by 6-hydroxydopamine lesion and partially restores the dopaminergic markers and vesicular striatal pool of dopamine. This cellular approach might be a restorative therapy in Parkinson's disease.     

Co-transplantation of carotid body and ventral mesencephalic cells as an alternative approach towards functional restoration in 6-hydroxydopamine-lesioned rats: implications for Parkinson's disease

Exogenous administration of various neurotrophic factors has been shown to protect neurons in animal model of Parkinson's disease (PD). Several attempts are being made to search a tissue source simultaneously expressing many of these neurotrophic factors. Carotid body (CB) contains oxygen-sensitive glomus cells rich in dopamine (DA) and expresses glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor and neurotrophin-3. We have attempted to study the functional restoration following co-transplantation of CB cells and ventral mesencephalic cells (VMC) in a 6-hydroxydopamine-lesioned rat model of PD. A significant recovery (p < 0.001) in d-amphetamine-induced circling behavior (80%) and spontaneous locomotor activity (85%) was evident in co-transplanted animals at 12 weeks post-transplantation as compared to lesioned animals. Similarly, a significant (p < 0.001) restoration was observed in DA-D(2) receptor binding (77%), striatal DA (87%) and 3,4-dihydroxyphenylacetic acid (DOPAC) (85%) levels and nigral DA (75%) and DOPAC (74%) levels. Functional recovery was accompanied by tyrosine hydroxylase (TH) expression and quantification of TH-positive cells by image analysis revealed a significant restoration in TH-immunoreactive (IR) fiber density in striatum, as well as TH-IR neurons in substantia nigra pars compacta in co-transplanted animals over VMC-transplanted animals. The result suggests that co-transplantation of CB cells along with VMC provides better and long-term functional restoration in the rat model of PD, possibly by supporting the survival of newly grafted cells as well as remaining host DA neurons.     

Transplantation of polymer encapsulated neurotransmitter secreting cells: effect of the encapsulation technique

Deficits associated with neurological diseases may be improved by the transplantation within the brain lesioned target structure of polymer encapsulated cells releasing the missing neurotransmitter. Surrounding cells with a permselective membrane of appropriate molecular weight cut-off allows inward diffusion of nutrients and outward diffusion of neurotransmitters, but prevents immunoglobulins or immune cells from reaching the transplant. This technique therefore allows transplantation of post-mitotic cells across species. It also permits neural grafting of transformed cell lines since the polymer capsule prevents the formation of tumors by physically sequestering the transplanted tissue. In the present study, we compared the ability of dopamine-secreting cells, encapsulated by 2 different methods, to reverse experimental Parkinson's disease, a neurodegenerative disease characterized by motor disturbances due to a lack of dopamine within the striatum following degeneration of the dopaminergic nigro-striatal pathway. PC12 cells were loaded in polyelectrolyte-based microcapsules or thermoplastic-based macrocapsules and maintained in vitro or transplanted in a rat experimental Parkinson model for 4 weeks. Chemically-induced depolarization increased the in vitro release of dopamine from macrocapsules over time, while no increase in release was observed from microcapsules. Encapsulated PC12 cells were able to reduce lesion-induced rotational asymmetry in rats for at least 4 weeks, regardless of the encapsulation technique used. With both encapsulation methods, PC12 cell viability was greater in vivo than in vitro which suggests that the striatum releases trophic factors for PC12 cells. More brain tissue damage was observed with microcapsules than macrocapsules, possibly the result of the difficulty of manipulating the more fragile microcapsules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compensation in pre-synaptic dopaminergic function following nigrostriatal damage in primates

Clinical symptoms of Parkinson's disease only become evident after 70-80% reductions in striatal dopamine. To investigate the importance of pre-synaptic dopaminergic mechanisms in this compensation, we determined the effect of nigrostriatal damage on dopaminergic markers and function in primates. MPTP treatment resulted in a graded dopamine loss with moderate to severe declines in ventromedial striatum (approximately 60-95%) and the greatest reductions (approximately 95-99%) in dorsolateral striatum. A somewhat less severe pattern of loss was observed for striatal nicotinic receptor, tyrosine hydroxylase and vesicular monoamine transporter expression. Declines in striatal dopamine uptake and transporter sites were also less severe than the reduction in dopamine levels, with enhanced dopamine turnover in the dorsolateral striatum after lesioning. The greatest degree of adaptation occurred for nicotine-evoked [(3)H]dopamine release from striatal synaptosomes, which was relatively intact in ventromedial striatum after lesioning, despite > 50% declines in dopamine. This maintenance of evoked release was not due to compensatory alterations in nicotinic receptor characteristics. Rather, there appeared to be a generalized preservation of release processes in ventromedial striatum, with K(+)-evoked release also near control levels after lesioning. These combined compensatory mechanisms help explain the finding that Parkinson's disease symptomatology develops only with major losses of striatal dopamine.     

Differentiation and migration of long term expanded human neural progenitors in a partial lesion model of Parkinson's disease

Human neural progenitor cells (HNPCs) can be expanded in large numbers for significant periods of time to provide a reliable source of neural cells for transplantation in neurodegenerative disorders such as Parkinson's disease (PD). In the present study, HNPCs isolated from embryonic cortex were expanded as neurospheres in cell culture for 10 months. Just prior to transplantation, a proportion of the HNPCs were treated in a "predifferentiation" protocol in combination with the neurotropic factor NT4, in order to yield significant numbers of neurons. For transplantation, either undifferentiated HNPCs, or predifferentiated HNPCs were transplanted into the substantia nigra of a rat model of Parkinson's disease. At 12 weeks, there was good survival with proliferation of transplanted HNPCs occurring after transplantation but ceasing before the animals were sacrificed. Transplants of predifferentiated cells contained a higher proportion of neurons. The presence of a lesion in the striatum had a significant influence on the migration of transplanted cells from the substantia nigra into the striatum. There was no significant behavioural recovery or effect of transplanted HNPCs on the loss of dopaminergic cells from the host brain. In conclusion, HNPCs may provide a source of cells for use in the treatment of Parkinson's disease.     

Increased levels of ubiquitin in the 6-OHDA-lesioned striatum of rats

Multiple genetic deficits have linked impaired ubiquitin-conjugation pathways to various forms of familiar Parkinson's disease. We therefore examined the possible role of 6-hydroxydopamine, a dopaminergic neurotoxin used in Parkinson's disease experimental models, in causing protein degradation and its association with the ubiquitin proteasome system. Using unilaterally 6-hydroxydopamine-denervated rats and mass spectrometry profiling directly on brain tissue sections, we here report for the first time an increased level of unconjugated ubiquitin specifically in the dorsal striatum of the dopamine depleted hemisphere. No similar changes were found in the intact hemisphere or in the ventral striatum of the dopamine depleted hemisphere. The lesioning of the dopamine innervation to the striatum was confirmed by a strongly reduced dopamine transporter binding in the striatum, indicating an abundant loss of dopamine neurons. These results suggest that denervation of dopamine neurons per se is implicated in the regulation of ubiquitin pathways, at least in a classical animal model of Parkinson's disease. This study adds additional information regarding the involvement of the ubiquitin-proteasome system in Parkinson's disease.     

Qualitative and quantitative re-evaluation of epidermal growth factor-ErbB1 action on developing midbrain dopaminergic neurons in vivo and in vitro: target-derived neurotrophic signaling (Part 1)

Although epidermal growth factor (EGF) receptor (ErbB1) is implicated in Parkinson's disease and schizophrenia, the neurotrophic action of ErbB1 ligands on nigral dopaminergic neurons remains controversial. Here, we ascertained colocalization of ErbB1 and tyrosine hydroxylase (TH) immunoreactivity and then characterized the neurotrophic effects of ErbB1 ligands on this cell population. In mesencephalic culture, EGF and glial-derived neurotrophic factor (GDNF) similarly promoted survival and neurite elongation of dopaminergic neurons and dopamine uptake. The EGF-promoted dopamine uptake was not inhibited by GDNF-neutralizing antibody or TrkB-Fc, whereas EGF-neutralizing antibody fully blocked the neurotrophic activity of the conditioned medium that was prepared from EGF-stimulated mesencephalic cultures. The neurotrophic action of EGF was abolished by ErbB1 inhibitors and genetic disruption of erbB1 in culture. In vivo administration of ErbB1 inhibitors to rat neonates diminished TH and dopamine transporter (DAT) levels in the striatum and globus pallidus but not in the frontal cortex. In parallel, there was a reduction in the density of dopaminergic varicosities exhibiting intense TH immunoreactivity. In agreement, postnatal erbB1-deficient mice exhibited similar decreases in TH levels. Although neurotrophic supports to dopaminergic neurons are redundant, these results confirm that ErbB1 ligands contribute to the phenotypic and functional development of nigral dopaminergic neurons.     

Effect of neurotransplantation on the content of catecholamines in the rat brain structures after heavy craniocerebral trauma

It was found that heavy craniocerebral trauma (CCT) in rats results in a drop in the dopamine and noradrenaline content in the damaged cerebral hemisphere, striatum, and hypothalamus. Transplantation of embryonic neural tissue after CCT in rats favors restoration of the level of catecholamines. Thirty days after the transplantation, the level of noradrenaline and dopamine in damaged structures mentioned above becomes similar to that in intact animals, or exhibits a tendency to be restored.     

Loss of alpha-conotoxinMII- and A85380-sensitive nicotinic receptors in Parkinson's disease striatum

Multiple nicotinic receptors are present in rodent and monkey striatum, with a selective localization of alpha-conotoxinMII-sensitive sites in the striatum and preferential declines in their numbers after nigrostriatal damage. Here we report the presence of 125I-alpha-conotoxinMII and alpha-conotoxinMII-sensitive 125I-epibatidine nicotinic receptors in human control and Parkinson's disease striatum. 125I-alpha-ConotoxinMII bound to control striatum with the characteristics of a nicotinic receptor ligand although the number of sites was approximately fivefold lower than in rodent and monkey. Competition analyses of alpha-conotoxinMII with 125I-epibatidine showed that toxin-sensitive sites comprised approximately 15% of nicotinic receptors in human striatum. In Parkinson's disease caudate, there was a approximately 50% decline in 125I-alpha-conotoxinMII sites with a similar decline in the dopamine transporter. In putamen, there were substantially greater losses of the dopamine transporter (80-90%) but only 50-60% decreases in 125I-alpha-conotoxinMII sites with corresponding declines in alpha-conotoxinMII-sensitive 125I-epibatidine sites, 125I-epibatidine (multiple) sites and 125I-A85380 (beta2-containing) nicotinic receptors. The greater loss of the transporter compared with nicotinic sites suggests that only a subpopulation of nicotinic receptors is located pre-synaptically on striatal dopaminergic neurons in man. Correlation analyses between changes in nicotinic receptors and the dopamine transporter in Parkinson's disease striatum suggest that alpha-conotoxinMII-sensitive 125I-epibatidine sites (low-affinity sites), 125I-A85380 and 125I-epibatidine sites are localized in part to dopaminergic terminals. In summary, these results show that alpha-conotoxinMII-sensitive sites are present in human striatum and that there are high- and low-affinity subtypes which are both decreased in Parkinson's disease.     

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.     

Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions

To model human neural-cell-fate specification and to provide cells for regenerative therapies, we have developed a method to generate human neural progenitors and neurons from human embryonic stem cells, which recapitulates human fetal brain development. Through the addition of a small molecule that activates canonical WNT signaling, we induced rapid and efficient dose-dependent specification of regionally defined neural progenitors ranging from telencephalic forebrain to posterior hindbrain fates. Ten days after initiation of differentiation, the progenitors could be transplanted to the adult rat striatum, where they formed neuron-rich and tumor-free grafts with maintained regional specification. Cells patterned toward a ventral midbrain (VM) identity generated a high proportion of authentic dopaminergic neurons after transplantation. The dopamine neurons showed morphology, projection pattern, and protein expression identical to that of human fetal VM cells grafted in parallel. VM-patterned but not forebrain-patterned neurons released dopamine and reversed motor deficits in an animal model of Parkinson's disease.     

Bromocriptine Markedly Suppresses Levodopa-Induced Abnormal Increase of Dopamine Turnover in the Parkinsonian Striatum

Bromocriptine, a dopamine agonist, is commonly used in combination with levodopa for the treatment of Parkinson's disease (PD). To investigate the theoretical basis of such combination therapy, we examined the effects of bromocriptine administered alone or in combination with levodopa on dopamine turnover in the striatum of hemi-parkinsonism rats. The parkinsonian striatum showed a 3.4-fold increase of dopamine turnover relative to the control striatum, as often observed in the brain of PD patients. A 7-day course of levodopa therapy markedly increased dopamine turnover in the parkinsonian striatum (53-fold of control level) than in the control striatum (5-fold of the control level). However, bromocriptine specifically and markedly suppressed the levodopa-induced abnormal activation of dopamine turnover in the parkinsonian striatum. Our findings explain the pharmacological basis for the introduction of bromocriptine during long-term levodopa therapy.     

Sonic hedgehog maintains cellular and neurochemical homeostasis in the adult nigrostriatal circuit

Non cell-autonomous processes are thought to play critical roles in the cellular maintenance of the healthy and diseased brain but mechanistic details remain unclear. We report that the interruption of a non cell-autonomous mode of sonic hedgehog (Shh) signaling originating from dopaminergic neurons causes progressive, adult-onset degeneration of dopaminergic, cholinergic, and fast spiking GABAergic neurons of the mesostriatal circuit, imbalance of cholinergic and dopaminergic neurotransmission, and motor deficits reminiscent of Parkinson's disease. Variable Shh signaling results in graded inhibition of muscarinic autoreceptor- and glial cell line-derived neurotrophic factor (GDNF)-expression in the striatum. Reciprocally, graded signals that emanate from striatal cholinergic neurons and engage the canonical GDNF receptor Ret inhibit Shh expression in dopaminergic neurons. Thus, we discovered a mechanism for neuronal subtype specific and reciprocal communication that is essential for neurochemical and structural homeostasis in the nigrostriatal circuit. These results provide integrative insights into non cell-autonomous processes likely at play in neurodegenerative conditions such as Parkinson's disease.     

Selenite benefits embryonic stem cells therapy in Parkinson's disease

Embryonic stem cells (ESC) transplantation is a potential therapeutic approach for Parkinson's disease (PD). However, one of the main challenges to this therapy is the post-transplantation survival of dopaminergic (DA) neurons. In this study, mouse ESC were differentiated into DA neurons by a modified serum free protocol. These ESC-derived neurons were then transplanted into striatum of 6-OHDA lesioned rat. The viability of grafted DA neurons was decreased, accompanied by activated microglia and high levels of proinflammatory factors, such as TNF-α and iNOS, in the graft niche. This suggested that the local neuroinflammation might be involved in the reduced cells viability. Selenite, the source of essential micronutrient selenium, could inhibit NF-κB p65 nuclear translocation and subsequently reduce iNOS, COX-2 and TNF-α expression in LPS-treated BV2 cells in a dose dependant manner. Before the transplantation of ESC-derived DA neurons, 6-OHDA lesioned rats were intraperitoneally injected with selenite. The expression levels of TNF-α and iNOS were decreased by 30% and 50%, respectively, in selenite treated group. The survival of implanted DA neurons and the rotational behavior of transplanted rats were also remarkably improved by selenite treatment. To sum up, selenite might benefit ESCs transplantation therapy in PD through anti-inflammation effects.