Neuroprotective effects of human umbilical cord mesenchymal stromal cells in an immunocompetent animal model of Parkinson's disease

Microglial activation in the substantia nigra (SN) is a ubiquitous feature in PD which could mediate toxic effects. Human mesenchymal stromal cells (hMSCs) possess immunomodulatory properties. We evaluated whether the transplantation of hMSCs obtained from umbilical cord had a neuroprotective effect in a not-immunosuppressed rat Parkinson's disease (PD) model. Rats receiving hMSCs in the SN displayed significant preservation in the number of dopaminergic neurons in the SN at 21 days after lesion and an improved performance in behavioral tests compared to control rats. However, no differences in any inflammatory parameter tested were found. These results suggest that grafted hMSCs exert neuroprotection but not neuromodulatory effects on degenerating dopaminergic neurons.     

Transient elevation of adult hippocampal neurogenesis after dopamine depletion

Degeneration of the midbrain dopaminergic neurons during Parkinson's disease (PD) may affect remote regions of the brain that are innervated by the projections of these neurons. The dentate gyrus (DG), a site of continuous production of new neurons in the adult hippocampus, receives dopaminergic inputs from the neurons of the substantia nigra (SN). Thus, depletion of the SN neurons during disease or in experimental settings may directly affect adult hippocampal neurogenesis. We show that experimental ablation of dopaminergic neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydopyridine (MPTP) mouse model of PD results in a transient increase in cell division in the subgranular zone (SGZ) of the DG. This increase is evident for the amplifying neural progenitors and for their postmitotic progeny; our results also indicate that MPTP treatment affects division of the normally quiescent stem cells in the SGZ. We also show that l-DOPA, used in the clinical treatment of PD, while attenuating the MPTP-induced death of dopaminergic neurons, does not alter the effect of MPTP on cell division in the DG. Our results suggest that a decrease in dopaminergic signaling in the hippocampus leads to a transient activation of stem and progenitor cells in the DG.     

Oxidative stress and microglial activation in substantia nigra following striatal MPP+

The present study aims to study sequential alterations occurring in both dopaminergic neurons and microglia in substantia nigra (SN) following intrastriatal injection of 1-methyl-4-phenylpridium ion (MPP+) in rats. Heme oxygenase-1 (HO-1), a marker of oxidative stress, first appeared in dopaminergic neurons in SN at 1 day post-lesion. Subsequently, microglia in SN exhibited morphological changes indicative of activation. At 7 days post-lesion, those findings increased severity and 7a significant reduction in the number of dopaminergic neurons was observed. The present finding suggests that extensive oxidative stress and secondary-induced neuroinflammation play a relevant role in MPP(+)-induced retrograde dopaminergic neuron degeneration. We hope that this model will be useful in developing a disease modifying therapy of Parkinson's disease.     

Trisialoganglioside GT1b induces in vivo degeneration of nigral dopaminergic neurons: role of microglia

We recently showed that trisialoganglioside (GT1b) induces cell death of dopaminergic neurons in rat mesencephalic cultures (Chung et al., Neuroreport 12:611-614, 2001). The present study examines the in vivo neurotoxic effects of GT1b on dopaminergic neurons in the substantia nigra (SN) of Sprague-Dawley rats. Seven days after GT1b injection into the SN, immunocytochemical staining of SN tissue revealed death of nigral neurons, including dopaminergic neurons. Additional immunostaining using OX-42 and OX-6 antibodies showed that GT1b-activated microglia were present in the SN where degeneration of nigral neurons was found. Western blot analysis and double-labeled immunohistochemistry showed that inducible nitric oxide synthase (iNOS) was expressed in the SN, where its levels were maximal at 8 h post-GT1b injection, and that iNOS was localized exclusively within microglia. GT1b-induced loss of dopaminergic neurons in the SN was partially inhibited by N(G)-nitro-L-arginine methyl ester hydrochloride, an NOS inhibitor. Our results indicate that in vivo neurotoxicity of GT1b against nigral dopaminergic neurons is at least in part mediated by nitric oxide released from activated microglia. Because GT1b exists abundantly in central nervous system neuronal membranes, our data support the hypothesis that immune-mediated events triggered by endogenous compounds such as GT1b could contribute to the initiation and/or the progression of dopaminergic neuronal cell death that occurs in Parkinson's disease.     

Protection from 1-methyl-4-phenylpyridinium (MPP) toxicity and stimulation of regrowth of MPP-damaged dopaminergic fibers by treatment of mesencephalic cultures with EGF and basic FGF

Several peptide growth factors can maintain survival or promote recovery of injured central neurons. In the present study, the effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the toxicity produced by the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), were investigated in rat mesencephalic dopaminergic neurons in culture. High affinity [3H]DA uptake and morphometric analyses of tyrosine hydroxylase immunostained neurons were used to assess the extent of MPP+ toxicity, dopaminergic neuronal survival and growth of neurites. Consistent with previous reports, EGF and bFGF treatments stimulated neuritic outgrowth in dopaminergic neurons, increased DA uptake and enhanced their long-term survival in vitro. These growth factors also stimulated proliferation of astrocytes. The time course of EGF and bFGF effects on dopaminergic neurons coincided with the increase in glial cell density, suggesting that proliferation of glia mediates their trophic effects. Several findings from our study support this possibility. When MPP+ was applied to cultures at 4 days in vitro, before glial cells had proliferated, the damage to dopaminergic neurons was not affected by EGF or bFGF pretreatments. However, when cultures maintained in the presence of the growth factors for 10 days were exposed to MPP+, after they had become confluent with dividing glial cells, the MPP(+)-induced decreases in DA uptake and cell survival were significantly attenuated. Furthermore, when glial cell proliferation was inhibited by 5-fluoro-2'-deoxyuridine, the protective effects of EGF and bFGF against MPP+ toxicity were abolished. Continuous treatment of MPP(+)-exposed cultures with EGF or bFGF resulted in the stimulation of process regrowth of damaged dopaminergic neurons with concomitant recovery of DA uptake, suggesting that the injured neurons are able to respond to the trophic effects of EGF and bFGF. In summary, our study shows that the trophic effects of EGF and bFGF on mesencephalic dopaminergic neurons include protection from the toxicity produced by MPP+ and promotion of recovery of MPP(+)-damaged neurons. Stimulation of glial cell proliferation is necessary for these effects.     

Delayed increase of tyrosine hydroxylase expression in rat nigrostriatal system after traumatic brain injury

Tyrosine hydroxylase (TH) is the key enzyme for synthesizing dopamine (DA) in dopaminergic neurons and its terminals. Emerging experimental and clinical evidence support the hypothesis of a disturbance in dopamine neurotransmission following traumatic brain injury (TBI). However, the effect of controlled cortical impact (CCI) injury on TH in the nigrostriatal system is currently unknown. To determine if there is an alteration in TH after CCI injury, we examined TH levels at 1 day, 7 days, and 28 days post-injury by utilizing a commercially available antibody specific to TH. Rats were anesthetized and surgically prepared for CCI injury (4 m/s, 3.2 mm) or sham surgery. Injured (N=6) and sham animals (N=6) were sacrificed and coronally sectioned (35 microm thick) through the striatum and substantia nigra (SN) for immunohistochemistry. Additionally, semiquantitative measurements of TH protein in striatal and SN homogenates from injured (N=6) and sham (N=6) rats sacrificed at the appropriate time post-surgery were assessed using Western blot analysis. TH protein is bilaterally increased at 28 days post-injury in nigrostriatal system revealed by immunohistochemistry in injured rats compared to sham controls. Western blot analysis confirms the findings of immunohistochemistry in both striatum and SN. We speculate that the increase in TH in the nigrostriatal system may reflect a compensatory response of dopaminergic neurons to upregulate their synthesizing capacity and a delayed increase in the efficiency of DA neurotransmission after TBI.     

EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture.

Epidermal growth factor (EGF) immunoreactive material has been demonstrated to be present in the basal ganglia. In this study, we investigated the effect of EGF on cells cultured from 16-day embryonic rat mesencephalon, which included dopamine neurons that project to the striatum in vivo. EGF receptors were detected in untreated cultures by [125I]-EGF binding. Treatment of the cultures with EGF resulted in up to 50-fold increases in neuronal high-affinity dopamine uptake. Scatchard analysis of uptake kinetics and counting of tyrosine hydroxylase-immunoreactive cells suggest that the effect of EGF on uptake is due to increased survival and maturation of dopaminergic neurons. By contrast, the high-affinity uptake for serotonin was increased only threefold over its controls. There was no significant effect on high-affinity gamma-aminobutyric acid (GABA) uptake. These results suggest that EGF is acting as a neurotrophic agent preferential for dopaminergic neurons in E16 mesencephalic cultures. Immunocytochemistry for glial fibrillary acidic protein demonstrated an increase in astroglia with EGF treatment. Fluorodeoxyuridine, an agent that is toxic to proliferating cells was able to eliminate the effect of EGF on dopamine uptake, suggesting that EGF may be increasing dopaminergic cell survival largely through a population of dividing cells.     

Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson's disease

We examined in Sprague-Dawley rats whether intranigral administration of complex-I inhibitor, rotenone, produces biochemical lesions in the striatum similar to those observed in Parkinson's disease (PD). Unilateral stereotaxic infusion of rotenone (2-12 mug in 1 mul) into substantia nigra (SN) pars compacta caused significant inhibition of complex-I activity and increased production of hydroxyl radicals in vivo as measured employing spectrophotometric and HPLC-electrochemical procedures, respectively. It also caused a significant time- and dose-dependent reduction of dopamine level, but not serotonin, in the ipsilateral striatum when assayed using an HPLC electrochemical method. This effect was found to be progressive for 90 days. A dose-dependent decrease in nigral glutathione level, as measured fluorimetrically, was also observed to be progressive till 90th day. A significant decrease in tyrosine hydroxylase immunoreactivity in the striatum (73 +/- 8.4% as assessed by densitometric studies) or in SN ipsilateral to the side of infusion suggested nigrostriatal neuronal degeneration. A dose of rotenone (6 microg in 1 microl) that caused 55% striatal dopamine depletion when infused into the SN failed to affect serotonin levels in the terminal regions when infused into the nucleus raphe dorsalis, indicating rotenone's specificity of action towards dopaminergic neurons. Our findings suggest that unilateral infusion of rotenone reproduces neurochemical and neuropathological features of hemiparkinsonism in rats and indicate an active involvement of oxidative stress in rotenone-induced nigrostriatal neurodegeneration. The present study also demonstrates more sensitivity of dopaminergic neurons towards rotenone and establishes mitochondrial complex-I damage as one of the major contributory components of neurodegeneration in PD. The progressive nature of pathology in this model closely mimics idiopathic PD, and absence of mortality warrants the use of this model in drug discovery programs.     

Gene expression profiling in the midbrain of striatal 6-hydroxydopamine-injected mice

In order to clarify mechanisms underlying dopaminergic neuronal death in Parkinson's disease (PD), a gene expression profiling study was performed in a rodent model of PD. In this model, mice are intrastriatally injected with 6-hydroxydopamine (6-OHDA) and dopaminergic neurons in the substantia nigra (SN) gradually die by retrograde degeneration. The SN were removed 2 h, 24 h, or 14 days after 6-OHDA administration. Levels of mRNAs related to cell death or survival were quantified using adaptor-tagged competitive PCR (ATAC-PCR). The cyclin D1 gene showed an immediate increase in mRNA expression. After 24 h, when dopaminergic neurons were under intense degeneration, levels of caspase 8 mRNA and p53 apoptosis effecter related to pmp 22 (PERP) mRNA increased and, conversely, FAS mRNA decreased. After 14 days, when the degeneration was attenuated, levels of PERP mRNA and serum- and glucocorticoid-regulated kinase (SGK) mRNA still increased. SGK has a similarity to AKT, which is an important molecule involved in nerve growth factor signal transduction. AKT mRNA levels are low in dopaminergic neurons. These results suggest that an increase in cyclin D1 mRNA triggers dopaminergic neurons to enter an abnormal cell cycle, which leads to neuronal degeneration and cell death, possibly induced by PERP and caspase 8. In addition to cell death-related genes, several survival-related genes are activated. SGK might function as a key enzyme for the survival of dopaminergic neurons.     

Long-term high-frequency electro-acupuncture stimulation prevents neuronal degeneration and up-regulates BDNF mRNA in the substantia nigra and ventral tegmental area following medial forebrain bundle axotomy

Electroacupuncture (EA) has been used in China for many years to treat Parkinson's disease (PD) with reportedly effective results. However, the physiological and biological mechanism behind its effectiveness is still unknown. In the present study, different frequencies of chronic EA stimulation (0, 2, 100 Hz) were tested in a partially lesioned rat model of PD which was induced by transection of the medial forebrain bundle (MFB). After 24 sessions of EA stimulation (28 days after MFB transection), dopaminergic neurons in the ventral midbrain were examined by immunohistochemical staining, and brain-derived neurotrophic factor (BDNF) mRNA levels in ventral midbrain were measured by in situ hybridization. The results show a marked decrease of dopaminergic neurons on the lesioned side of the substantia nigra (SN) comparing with the unlesioned side. Zero Hz and 2 Hz EA stimulation had no effect on the disappearance of dopaminergic neurons. However, after 100 Hz EA, about 60% of the tyrosine hydroxylase (TH)-positive neurons remained on the lesioned side of the SN. In addition, levels of BDNF mRNA in the SN and ventral tegmental area (VTA) of the lesioned side were significantly increased in the 100 Hz EA group, but unchanged in the 0 and 2 Hz groups. Our results suggest that long-term high-frequency EA is effective in halting the degeneration of dopaminergic neurons in the SN and up-regulating the levels of BDNF mRNA in the subfields of the ventral midbrain. Activation of endogenous neurotrophins by EA may be involved in the regeneration of the injured dopaminergic neurons, which may underlie the effectiveness of EA in the treatment of PD.     

Delivery of a GDNF gene into the substantia nigra after a progressive 6-OHDA lesion maintains functional nigrostriatal connections

The effects of delivering GDNF via an adenoviral vector (AdGDNF) 1 week after lesioning dopaminergic neurons in the rat substantia nigra (SN) with 6-hydroxydopamine (6-OHDA) were examined. Rats were unilaterally lesioned by injection of 6-OHDA into the striatum, resulting in progressive degeneration of dopaminergic neurons in the SN. One week later, when substantial damage had already occurred, AdGDNF or a control vector harboring beta-galactosidase (AdLacZ) was injected into either the striatum or SN (3.2 x 10(7) PFU/microl in 2 microl). Rats were examined behaviorally with the amphetamine-induced rotation test and for forelimb use for weight-bearing movements. On day 30 postlesion, the extent of nigrostriatal tract degeneration was determined by injecting a retrograde tracer (FluoroGold) bilaterally into the lesioned striatum. Five days later, rats were sacrificed within 2 h of amphetamine injection to examine amphetamine-induced Fos expression in the striatum, a measure of dopaminergic-dependent function in target neurons. AdGDNF injection in the SN rescued dopaminergic neurons in the SN and increased the number of dopaminergic neurons that maintained a connection to the striatum, compared to rats injected with AdLacZ. Further support that these spared SN cells maintained functional connections to the striatum was evidenced by increased Fos expression in striatal target neurons and a decrease in amphetamine-induced rotation. In contrast to the effects observed in rats injected with AdGDNF in the SN, rats injected with AdGDNF in the striatum did not exhibit significant ameliorative effects. This study demonstrates that experimentally increasing levels of GDNF biosynthesis near the dopaminergic neuronal soma is effective in protecting the survival of these neurons and their function even when therapy is begun after 6-OHDA-induced degeneration has commenced. Thus, GDNF gene therapy may ameliorate the consequences of Parkinson's disease through rescuing compromised dopaminergic neurons.     

Dopaminergic lesion enhances growth factor-induced striatal neuroblast migration

Adult neurogenesis persists in the subventricular zone and is decreased in Parkinson disease (PD). The therapeutic potential of neurogenesis in PD requires understanding of mechanisms of 1) neural stem cell generation; 2) their guidance to the lesion site; and 3) the environment that enables neuronal differentiation, survival, and functional integration. We examined the combined intraventricular infusion of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) in a 6-hydroxydopamine-induced rodent model of PD. Epidermal growth factor and FGF-2 induced a massive increase in cell proliferation and in numbers of doublecortin-expressing neuroblasts in the subventricular zone. These growth factors also increased dopaminergic neurogenesis in the olfactory bulb and promoted the migration of newly generated neuroblasts from the subventricular zone into the adjacent striatum. The effects of EGF and FGF-2 were present in unlesioned animals but were dramatically enhanced in 6-hydroxydopamine-lesioned animals.These findings suggest that newly generated neuroblasts may be redirected to the region of dopaminergic deficit, and that EGF and FGF-2 can enhance dopaminergic neurogenesis in the olfactory bulb but not in the striatum. Similar mechanisms may be involved in the increased numbers of dopaminergic neurons observed in the olfactory bulbs of PD patients and their functional olfactory deficits.     

Organotypic slice cultures of dopaminergic neurons of substantia nigra

Morphological methods were used to study the plasticity of target-deprived mammalian dopaminergic (DA) neurons. Slices of substantia nigra (SN) were taken from the midbrain of rats aged one to twelve days, and cultured for one to two weeks. Localization of tyrosine hydroxylase (TH) was used to examine the distribution and shapes of DA neurons. Histochemical staining for acetylcholinesterase (AChE) was carried out to estimate both survival and biosynthesis of SN neurons. We found that some DA neurons can survive in vitro without their usual target neurons. This was demonstrated by injecting rhodamine-conjugated microspheres (RD) into the caudate putamen, a SN target area, at 6 to 8 days prior to culturing. RD-labeled cells survived in SN cultures and some of them were doubly labeled with AChE. TH neurons had different shapes and their axon terminals formed close contacts with adjacent nondopaminergic neurons. These findings suggest that a subset of DA neurons may switch targets, but the majority of them require target interactions with the caudate putamen for survival in vitro.     

Inhibition of the serotonin transporter induces microglial activation and downregulation of dopaminergic neurons in the substantia nigra

Drugs that selectively inhibit the serotonin transporter (SERT) are widely used in the treatment of depression and anxiety disorders. These agents are associated with a range of extrapyramidal syndromes such as akathisia, dystonia, dyskinesia and parkinsonism, suggesting an effect on dopaminergic transmission. We studied the time course of changes in dopaminergic neurons in the substantia nigra (SN) after initiation of two different SERT inhibitors, citalopram and fluoxetine. In the first experiment, groups of Sprague-Dawley rats received daily meals of rice pudding either alone (N = 9) or mixed with citalopram 5 mg/kg/day (N = 27). Rats were sacrificed after 24 h, 7 days or 28 days of treatment. Sections of SN were processed for tyrosine hydroxylase (TH) immunohistochemistry. Citalopram induced a significant decrease in TH-positive cell counts at 24 h (44%), 7 days (38%) and 28 days (33%). No significant differences among the citalopram treatment groups were observed in the SN. To determine whether these changes would occur with other SERT inhibitors, we conducted a second experiment, this time with a 28 day course of fluoxetine. As was observed with citalopram, fluoxetine induced a significant 21% reduction of TH cell counts in the SN. Immunoblot analysis showed that fluoxetine also induced a 45% reduction of striatal TH. To investigate a possible role for the innate immune system in mediating these changes, we also studied the microglial marker OX42 after administration of fluoxetine and noted a significant 63% increase in the SN of fluoxtine-treated animals. These results indicate that SERT inhibition can activate microglia and alter the regulation of TH, the rate limiting enzyme for dopamine biosynthesis. These changes may play a role in mediating the extrapyramidal side effects associated with SERT inhibitors.     

Role of the subthalamic nucleus in the regulation of nigral dopamine neuron activity.

The influence of subthalamic nucleus (STN) afferents on dopaminergic (DA) neurons of the rat substantia nigra (SN) was investigated. Hemisections of the brain placed between the STN and the SN or located anterior to the STN caused an increase in the firing rate of DA cells without producing significant changes in their firing pattern. In contrast, electrolytic and ibotenic acid lesions of the STN resulted in 93% and 49% reductions, respectively, in the level of burst firing without affecting the firing rate of DA cells recorded in the lateral SN. Furthermore, procedures which interrupted the STN input to the SN produced rapid pacemaker-like firing in 18% of the lateral SN DA neurons recorded. Activation of the STN using single pulses of electrical stimulation caused: 1) a 20-50 msec inhibition of DA cell firing followed by an excitation, which in 35% of DA cells was accompanied by spikes occurring in a burst-like pattern, and 2) a short-latency inhibition lasting 5-25 msec in 75% of non-DA SN zona reticulata (ZR) neurons. On the other hand, stimulation of the STN for 1 minute at 20 Hz resulted in an initial decrease in DA cell burst firing followed by elevated firing rates and increased burst firing by 30-60 minutes after the stimulation. Pharmacological activation of the STN by infusion of bicuculline caused a rapid inhibition of DA cells followed by a two-fold increase in burst firing 6-14 minutes later, whereas SN ZR cells responded with an elevation in firing rate which dissipated in 6-14 minutes. Muscimol-induced STN inhibition produced complimentary biphasic changes in SN neuron firing: 1) an initial increase followed by a decrease in burst firing and firing rate of DA neurons and 2) a rapid inhibition followed by an excitation of ZR cells over a similar time course. Thus, the STN appears to exert a dual action on SN DA cells: 1) initial inhibition possibly mediated through STN excitation of the inhibitory SN ZR projections to DA cells, and 2) a facilitation of burst firing which may be a direct effect of excitatory STN afferents.     

Dietary restriction and 2-deoxyglucose administration improve behavioral outcome and reduce degeneration of dopaminergic neurons in models of Parkinson's disease.

Parkinson's disease (PD) is an age-related disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and corresponding motor deficits. Oxidative stress and mitochondrial dysfunction are implicated in the neurodegenerative process in PD. Although dietary restriction (DR) extends lifespan and reduces levels of cellular oxidative stress in several different organ systems, the impact of DR on age-related neurodegenerative disorders is unknown. We report that DR in adult mice results in resistance of dopaminergic neurons in the SN to the toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP-induced loss of dopaminergic neurons and deficits in motor function were ameliorated in DR rats. To mimic the beneficial effect of DR on dopaminergic neurons, we administered 2-deoxy-D-glucose (2-DG; a nonmetabolizable analogue of glucose) to mice fed ad libitum. Mice receiving 2-DG exhibited reduced damage to dopaminergic neurons in the SN and improved behavioral outcome following MPTP treatment. The 2-DG treatment suppressed oxidative stress, preserved mitochondrial function, and attenuated cell death in cultured dopaminergic cells exposed to the complex I inhibitor rotenone or Fe2+. 2-DG and DR induced expression of the stress proteins heat-shock protein 70 and glucose-regulated protein 78 in dopaminergic cells, suggesting involvement of these cytoprotective proteins in the neuroprotective actions of 2-DG and DR. The striking beneficial effects of DR and 2-DG in models of PD, when considered in light of recent epidemiological data, suggest that DR may prove beneficial in reducing the incidence of PD in humans.     

The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system

We have injected lipopolysaccharide (LPS) into the nigrostriatal pathway of rats in order to address the role of inflammation in Parkinson's disease (PD). LPS induced a strong macrophage/microglial reaction in Substantia nigra (SN), with a characteristic clustering of macrophage cells around blood-vessels. The SN was far more sensitive than the striatum to the inflammatory stimulus. Moreover, only the dopaminergic neurons of the SN were affected, with no detectable damage to either the GABAergic or the serotoninergic neurons. The damage to the DA neurons in the SN was permanent, as observed 1 year postinjection. Unlike the direct death of dopaminergic neurons caused by agents as MPP(+) or 6-OHDA, LPS seems to cause indirect death due to inflammatory reaction. Therefore, we suggest that the injection of a single dose of LPS within the SN is an interesting model for studying the selective effects of inflammatory reaction on dopaminergic system and also potentially useful for studying PD.     

1-methyl-4-phenylpyridinium neurotoxicity is attenuated by adenoviral gene transfer of human Cu/Zn superoxide dismutase

Oxidative stress has been suggested to be an important mediator of dopaminergic cell death in Parkinson's disease (PD). We investigated the neuroprotective potential of Cu/Zn superoxide dismutase (SOD1) overexpression in the rat substantia nigra (SN) following adenovirus-mediated gene transfer. Human dopaminergic SK-N-SH cells were transduced with adenoviral vectors expressing either human SOD1 (Ad-SOD1) or beta-galactosidase (Ad-betagal) before exposure to 1 mM of the 1-methyl-4-phenylpyridinium ion (MPP+). A strong neuroprotective effect of SOD1 gene transfer was observed in the SK-N-SH cells exposed to MPP+ compared with controls. Adult rats were then given unilateral injections of either Ad-SOD1 or Ad-betagal into the striatum, and MPP+ was administered 8 days later at the same location. Strong transgene expression was detected in the SN dopaminergic neurons, a consequence of retrograde axonal transport of the adenoviral particles. The amphetamine-induced rotational behavior of the rats was markedly lower in Ad-SOD1-injected rats than in control animals. Also, behavioral recovery significantly correlated with the number of tyrosine hydrolase-expressing neurons in the SN of the treated rats. These results are consistent with oxidative stress contributing to the MPP+ -induced neurodegenerative process. They also indicate that SOD1 gene transfer into the nigrostriatal system may be a potential neuroprotective strategy for treating PD.     

HIV-1 gp120 neurotoxicity proximally and at a distance from the point of exposure: Protection by rSV40 delivery of antioxidant enzymes

Toxicity of HIV-1 envelope glycoprotein (gp120) for substantia nigra (SN) neurons may contribute to the Parkinsonian manifestations often seen in HIV-1-associated dementia (HAD). We studied the neurotoxicity of gp120 for dopaminergic neurons and potential neuroprotection by antioxidant gene delivery. Rats were injected stereotaxically into their caudate-putamen (CP); CP and (substantia nigra) SN neuron loss was quantified. The area of neuron loss extended several millimeters from the injection site, approximately 35% of the CP area. SN neurons, outside of this area of direct neurotoxicity, were also severely affected. Dopaminergic SN neurons (expressing tyrosine hydroxylase, TH, in the SN and dopamine transporter, DAT, in the CP) were mostly affected: intra-CP gp120 caused approximately 50% DAT+ SN neuron loss. Prior intra-CP gene delivery of Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1) protected SN neurons from intra-CP gp120. Thus, SN dopaminergic neurons are highly sensitive to HIV-1 gp120-induced neurotoxicity, and antioxidant gene delivery, even at a distance, is protective.