Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes

One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.     

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.     

Triple transduction with adeno-associated virus vectors expressing tyrosine hydroxylase, aromatic-L-amino-acid decarboxylase, and GTP cyclohydrolase I for gene therapy of Parkinson's disease

Parkinson's disease (PD), a neurological disease suited to gene therapy, is biochemically characterized by a severe decrease in the dopamine content of the striatum. One current strategy for gene therapy of PD involves local production of dopamine in the striatum achieved by inducing the expression of enzymes involved in the biosynthetic pathway for dopamine. We previously showed that the coexpression of tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), using two separate adeno-associated virus (AAV) vectors, resulted in more effective dopamine production and more remarkable behavioral recovery in 6-hydroxydopamine-lesioned parkinsonian rats, compared with the expression of TH alone. Not only levels of TH and AADC but also levels of tetrahydrobiopterin (BH4), a cofactor of TH, and GTP cyclohydrolase I (GCH), a rate-limiting enzymes for BH4 biosynthesis, are reduced in parkinsonian striatum. In the present study, we investigated whether transduction with separate AAV vectors expressing TH, AADC, and GCH was effective for gene therapy of PD. In vitro experiments showed that triple transduction with AAV-TH, AAV-AADC, and AAV-GCH resulted in greater dopamine production than double transduction with AAV-TH and AAV-AADC in 293 cells. Furthermore, triple transduction enhanced BH4 and dopamine production in denervated striatum of parkinsonian rats and improved the rotational behavior of the rats more efficiently than did double transduction. Behavioral recovery persisted for at least 12 months after stereotaxic intrastriatal injection. These results suggest that GCH, in addition to TH and AADC, is important for effective gene therapy of PD.     

Striatal pleiotrophin overexpression provides functional and morphological neuroprotection in the 6-hydroxydopamine model

Neurotrophic factors are integrally involved in the development of the nigrostriatal system and in combination with gene therapy, possess great therapeutic potential for Parkinson's disease (PD). Pleiotrophin (PTN) is involved in the development, maintenance, and repair of the nigrostriatal dopamine (DA) system. The present study examined the ability of striatal PTN overexpression, delivered via psueudotyped recombinant adeno-associated virus type 2/1 (rAAV2/1), to provide neuroprotection and functional restoration from 6-hydroxydopamine (6-OHDA). Striatal PTN overexpression led to significant neuroprotection of tyrosine hydroxylase immunoreactive (THir) neurons in the substantia nigra pars compacta (SNpc) and THir neurite density in the striatum, with long-term PTN overexpression producing recovery from 6-OHDA-induced deficits in contralateral forelimb use. Transduced striatal PTN levels were increased threefold compared to adult striatal PTN expression and approximated peak endogenous developmental levels (P1). rAAV2/1 vector exclusively transduced neurons within the striatum and SNpc with approximately half the total striatal volume routinely transduced using our injection parameters. Our results indicate that striatal PTN overexpression can provide neuroprotection for the 6-OHDA lesioned nigrostriatal system based upon morphological and functional measures and that striatal PTN levels similar in magnitude to those expressed in the striatum during development are sufficient to provide neuroprotection from Parkinsonian insult.     

Effect of (R)-salsolinol and N-methyl-(R)-salsolinol on the balance impairment between dopamine and acetylcholine in rat brain: involvement in pathogenesis of Parkinson disease

BACKGROUND: Parkinson disease (PD), a progressive neurodegenerative disease, affects at least 1% of population above the age of 65. Although the specific etiology of PD remains unclear, recently the endogenous neurotoxins such as (R)-salsolinol [(R)-Sal] and N-methyl-(R)-salsolinol [(R)-NMSal] have been thought to play a major role in PD. Much interest is focused on the degeneration of dopamine neurons induced by these neurotoxins. However, little literature is available on the impact of endogenous neurotoxins on the balance between dopamine (DA) and acetylcholine (ACh). METHODS: After injection of (R)-Sal or (R)-NMSal into the rat brain striatum, the concentrations of DA and its metabolites were detected by HPLC with electrochemical detection. We assessed the influence of neurotoxins on acetylcholinesterase (AChE) activity and developed a microdialysis-electrochemical device to measure ACh concentrations with enzyme-modified electrodes. RESULTS: (R)-Sal and (R)-NMSal led to concentration-dependent decreases in the activity of AChE. ACh concentrations in striatum treated with (R)-Sal or (R)-NMSal were increased to 131.7% and 239.8% of control, respectively. As to the dopaminergic system, (R)-NMSal caused a significant decrease in DA concentrations and (R)-Sal reduced the concentrations of DA metabolites in the striatum. CONCLUSIONS: (R)-Sal and (R)-NMSal exerted a considerable effect on the balance between DA and ACh by impairing the cholinergic system as well as the dopaminergic system. It is likely that the disruption of balance between DA and ACh plays a critical role in the pathogenesis of neurotoxin-induced PD.     

Recovery From Experimental Parkinsonism by Semaphorin-guided Axonal Growth of Grafted Dopamine Neurons

Cell therapy in animal models of Parkinson''s disease (PD) is effective after intrastriatal grafting of dopamine (DA) neurons, whereas intranigral transplantation of dopaminergic cells does not cause consistent behavioral recovery. One strategy to promote axonal growth of dopaminergic neurons from the substantia nigra (SN) to the striatum is degradation of inhibitory components such as chondroitin sulphate proteoglycans (CSPG). An alternative is the guidance of DA axons by chemotropic agents. Semaphorins 3A and 3C enhance axonal growth of embryonic stem (ES) cell–derived dopaminergic neurons in vitro, while Semaphorin 3C also attracts them. We asked whether intranigral transplantation of DA neurons, combined with either degradation of CSPG or with grafts of Semaphorin 3–expressing cells, towards the striatum, is effective in establishing a new nigrostriatal dopaminergic pathway in rats with unilateral depletion of DA neurons. We found depolarization-induced DA release in dorsal striatum, DA axonal projections from SN to striatum, and concomitant behavioral improvement in Semaphorin 3–treated animals. These effects were absent in animals that received intranigral transplants combined with Chondroitinase ABC treatment, although partial degradation of CSPG was observed. These results are evidence that Semaphorin 3–directed long-distance axonal growth of dopaminergic neurons, resulting in behavioral improvement, is possible in adult diseased brains.     

Inflammatory process in Parkinson disease: neuroprotection by neuropeptide Y

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the nigro‐striatal pathway. Interestingly, it has already been shown that an intracerebral administration of neuropeptide Y (NPY) decreases the neurodegeneration induced by 6‐hydroxydopamine (6‐OHDA) in rodents and prevents loss of dopamine (DA) and DA transporter density. The etiology of idiopathic PD now suggest that chronic production of inflammatory mediators by activated microglial cells mediates the majority of DA‐neuronal tissue destruction. In an animal experimental model of PD, the present study shows that NPY inhibited the activation of microglia evaluated by the binding of the translocator protein (TSPO) ligand [3H]PK11195 in striatum and substantia nigra of 6‐OHDA rats. These results suggest a potential role for inflammation in the pathophysiology of the disease and a potential treatment by NPY in PD.     

Gene therapy for Parkinson's disease using recombinant adeno-associated viral vectors

Existing strategies for gene therapy in the treatment of Parkinson's disease include the delivery of genes encoding dopamine (DA)-synthesising enzymes, leading to localised production of DA in the striatum; genes encoding factors that protect nigral neurons against ongoing degeneration, such as glial cell line-derived neurotrophic factor; and genes encoding proteins that produce the inhibitory transmitter gamma-aminobutylic acid (GABA) in the subthalamic nucleus (STN), thus suppressing the hyperactive STN. Recombinant adeno-associated viral (rAAV) vectors, which are derived from non-pathogenic viruses, have been shown to be suitable for clinical trials. These rAAVs have been found to transduce substantial numbers of neurons efficiently and to express transgenes in mammalian brains for long periods of time, with minimum inflammatory and immunological responses. In vivo imaging using positron emission tomography is useful for monitoring transgene expression and for assessing the functional effects of gene delivery. Vector systems that regulate transgene expression are necessary to increase safety in clinical applications, and the development of such systems is in progress.     

6-羟基多巴胺诱发大鼠帕金森病的实验研究

目的 :研究自由基、神经营养因子及细胞凋亡在帕金森病发病中的变化。方法 :通过脑立体定向注射 6 羟基多巴胺的方法建立大鼠PD模型 ,采用TUNEL法 ,免疫组化技术 ,生化方法 ,观察大鼠黑质细胞凋亡数量 ,纹状体多巴胺含量 ,脑胶质源性神经营养因子 (GDNF)表达及自由基和抗自由基酶的变化。结果 :PD大鼠黑质存在明显的细胞凋亡 ,自由基反应增强 ,抗自由基酶和纹状体多巴胺含量及GDNF减少 ,与对照组存在显著性差异 ( P <0 0 5 )。结论 :自由基反应增强 ,营养因子缺乏及细胞凋亡参与了PD的发病 ,可能是其发病机制之一     

Pharmacological characterization of PD 118717, a putative piperazinyl benzopyranone dopamine autoreceptor agonist.

PD 118717 (7-[3-[4-(2-pyrimidinyl)-1-piperazinyl]-propoxy]-2H-1- benzopyran-2-one sulfate) proved to be a dopamine (DA) D-2 autoreceptor agonist in biochemical and electrophysiological studies in rats and to exhibit an antipsychotic-like profile in behavioral tests in rodents and monkeys. In vitro binding studies indicated that PD 118717 bound selectively to DA D-2 vs. D-1 receptors and exhibited agonist binding properties (biphasic inhibitory curves and GTP shift) similar to DA. It also had significant affinity for serotonin-(5-HT)1A but not 5-HT1B and 5-HT2 receptors. PD 118717 was active in antagonizing the tau-butyrolactone-induced accumulation of dopa in rat striatum and mesolimbic regions. PD 118717 also depressed the firing of DA neurons in substantia nigra pars compacta of rats. In both of the latter tests the effects of PD 118717 were reversed by haloperidol. PD 118717 decreased brain DA metabolism, decreased DA utilization, decreased accumulation of dopa after inhibition of L-aromatic amino acid decarboxylase, stimulated serum corticosterone and inhibited stimulated serum prolactin levels. PD 118717 did not alter striatal acetylcholine levels; nor did it induce locomotor stimulation or stereotypy in normal animals, suggesting a lack of postsynaptic DA stimulation of normosensitive DA receptors. In tests designed to reveal even weak postsynaptic DA agonist effects, PD 118717 stimulated locomotor activity in 6-hydroxydopamine-lesioned animals and relatively higher doses induced a low degree of stereotyped behavior when combined with the DA D-1 agonist SKF 38393. PD 118717 decreased the accumulation of 5-hydroxytryptophan in brain, an effect probably due to an agonist action at 5-HT1A receptors. PD 118717 decreased spontaneous locomotor activity in rodents, antagonized amphetamine-stimulated hyperactivity in mice and inhibited Sidman avoidance in monkeys, effects seen with antipsychotic agents. Unlike DA antagonist antipsychotics, PD 118717 did not induce extrapyramidal dysfunction in monkeys. PD 118717 displayed behavioral activity after p.o. dosing and its effects did not show tolerance on repeated dosing. In conclusion, PD 118717 has the profile of a DA autoreceptor agonist in neurochemical and neurophysiological tests and produces effects suggestive of antipsychotic efficacy without neurological side effect liability in preclinical behavioral tests.     

Selective impairment of prediction error signaling in human dorsolateral but not ventral striatum in Parkinson's disease patients: evidence from a model-based fMRI study

Animal studies have found that the phasic activity of dopamine neurons during reward-related learning resembles a “prediction error” (PE) signal derived from a class of computational models called reinforcement learning (RL). An apparently similar signal can be measured using fMRI in the human striatum, a primary dopaminergic target. However, the fMRI signal does not measure dopamine per se, and therefore further evidence is needed to determine if these signals are related to each other. Parkinson's disease (PD) involves the neurodegeneration of the dopamine system and is accompanied by deficits in reward-related decision-making tasks. In the current study we used a computational RL model to assess striatal error signals in PD patients performing an RL task during fMRI scanning. Results show that error signals were preserved in ventral striatum of PD patients, but impaired in dorsolateral striatum, relative to healthy controls, a pattern reflecting the known selective anatomical degeneration of dopamine nuclei in PD. These findings support the notion that PE signals measured in the human striatum by the BOLD signal may reflect phasic DA activity. These results also provide evidence for a deficiency in PE signaling in the dorsolateral striatum of PD patients that may offer an explanation for their deficits observed in other reward learning tasks.     

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.     

Design of a Single AAV Vector for Coexpression of TH and GCH1 to Establish Continuous DOPA Synthesis in a Rat Model of Parkinson's Disease

Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson''s disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.     

Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged Parkinsonian rat

Injection of an adenoviral (Ad) vector encoding human glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic (DA) neurons in the substantia nigra (SN) of young rats. As Parkinson's disease occurs primarily in aged populations, we examined whether chronic biosynthesis of GDNF, achieved by adenovirus-mediated delivery of a GDNF gene (AdGDNF), can protect DA neurons and improve DA-dependent behavioral function in aged (20 months) rats with progressive 6-OHDA lesions of the nigrostriatal projection. Furthermore, the differential effects of injecting AdGDNF either near DA cell bodies in the SN or at DA terminals in the striatum were compared. AdGDNF or control vector was injected unilaterally into either the striatum or SN. One week later, rats received a unilateral intrastriatal injection of 6-OHDA on the same side as the vector injection. AdGDNF injection into either the striatum or SN significantly reduced the loss of FG labelled DA neurons 5 weeks after lesion (P 相似文献   

高效液相色谱法快速检测单胺类神经递质及其代谢产物

背景:单胺类神经递质及其代谢产物对实验研究帕金森病和实验诊断嗜铬细胞瘤具有重要意义,是当今的研究热点。目的:建立高效液相色谱法同时快速测定肾上腺素、去甲肾上腺素、多巴胺、5-羟色胺、5-羟吲哚乙酸、高香草酸及多巴柯7种单胺类神经递质及其代谢产物。设计:病例-对照观察和随机对照动物实验。单位:青岛大学医学院附属医院检验科及青岛大学医学院附属医院海阳分院。对象:实验于2003-10/12在青岛大学医学院生理教研室和青岛大学医学院附属医院检验科完成。①2例嗜铬细胞瘤患者,均经CT,MRI或病理切片和临床证实为该病。12例正常对照均为在校大学生,且经查排除高血压、内分泌系统疾病和其他疾病。②选用健康雌性Wistar大白鼠12只,体质量150～180g。随机数字表法分为2组,正常对照组和帕金森病组,每组大鼠各6只。方法:制备7种物质样品(肾上腺素、去甲肾上腺素、多巴胺、5-羟色胺、5-羟吲哚乙酸、高香草酸及多巴柯)的标准色谱图,同时进行样品精密度和回收率的检测。采用高效液相色谱法检测12例正常对照和2例嗜铬细胞瘤患者24h尿中的儿茶酚胺类物质。同时以6-羟基多巴胺损毁帕金森病组大鼠大脑一侧前脑束,以监测正常和帕金森病大鼠健侧及损毁侧尾壳核区纹状体内单胺类神经递质及其代谢产物的释放特点。主要观察指标:①样品精密度和回收率检测结果。②12例正常对照和2例嗜铬细胞瘤患者24h尿中儿茶酚胺类物质的测定结果。③各组大鼠纹状体内单胺类神经递质及其代谢产物的释放特点。结果:12例正常对照和2例嗜铬细胞瘤患者均获得24h尿中儿茶酚胺类物质检测结果,并进入结果分析。实验动物在实验中无死亡。①高效液相色谱法对肾上腺素、去甲肾上腺素、多巴胺、5-羟色胺、5-羟吲哚乙酸、高香草酸及多巴柯7种单胺类物质的线性范围为2～1000μg/L,最低检测限为2～30μg/L,批内精密度为3.0%～8.6%,批间精密度为3.8%～9.7%。多巴胺的平均回收率为107.3%、去甲肾上腺素为89.4%、肾上腺素为80.1%,其余4种物质为89.2%～95.7%。②2例嗜铬细胞瘤患者24h尿中儿茶酚胺类物质的含量显著高于正常人,其中1例以肾上腺素明显增高为主,另1例以去甲肾上腺素明显增高为主,均高于正常人10倍左右。③帕金森病大鼠健侧尾壳核内多巴胺及其代谢产物多巴柯、高香草酸含量均在正常范围内,损毁侧尾壳核内多巴胺、5-羟色胺、多巴柯及高香草酸均显著降低,差异有显著性意义(P<0.01);5-羟色胺及其代谢产物5-羟吲哚乙酸含量无明显改变。④帕金森病大鼠健侧尾壳核内多巴胺更新率(多巴柯/多巴胺和高香草酸/多巴胺的比值)升高;损毁侧尾壳核内多巴胺更新速度也加快,与正常对照组比较差异均有显著性意义(P<0.05)。结论:高效液相色谱法能满足对大白鼠帕金森病模型的研究和对嗜铬细胞瘤患者实验诊断的需要。     

Embryonic stem cells as a cell source for treating Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease characterised by a loss of midbrain dopaminergic (DA) neurons. Transplantation of DA neurons represents a promising treatment for PD, and embryonic stem (ES) cells are a good candidate source for DA neurons. However, although recent reports have demonstrated that DA neurons can be efficiently induced from ES cells and function therapeutically in an animal model of PD, many problems remain to be solved in order for ES cells to be used for clinical applications. This review will describe the current status of this field and the obstacles yet to be overcome, and will outline future research approaches from the clinical perspective.     

Efficient gene therapy for Parkinson's disease using astrocytes as hosts for localized neurotrophic factor delivery

Current gene therapy approaches for Parkinson's disease (PD) deliver neurotrophic factors like glial cell line-derived neurotrophic factor (GDNF) or neurturin via neuronal transgene expression. Since these potent signaling-inducing neurotrophic factors can be distributed through long-distance neuronal projections to unaffected brain sites, this mode of delivery may eventually cause side effects. To explore a localized and thus potentially safer alternative for gene therapy of PD, we expressed GDNF exclusively in astrocytes and evaluated the efficacy of this approach in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rat 6-hydroxy-dopamine (6-OHDA) models of PD. In terms of protection of dopaminergic cell bodies and projections, dopamine (DA) synthesis and behaviour, astrocyte-derived GDNF demonstrated the same efficacy as neuron-derived GDNF. In terms of safety, unilateral striatal GDNF expression in astrocytes did not result in delivery of bio-active GDNF to the contralateral hemispheres (potential off-target sites) as happened when GDNF was expressed in neurons. Thus, astrocytic GDNF expression represents a localized but efficient alternative to current gene therapeutic strategies for the treatment of PD, especially if viral vectors with enhanced tissue penetration are considered. Astrocytic neurotrophic factor expression may open new venues for neurotrophic factor-based gene therapy targeting severe diseases of the brain.     

Heterogeneous dopamine neurochemistry in the striatum: the fountain-drain matrix

In contrast to the relatively high attention paid to the structural heterogeneity of striatal dopamine (DA) innervation, little attention has been focused on the possible striatal heterogeneity for release and uptake of DA. By using amperometric methods, we found striatal regions showing a DA decrease during the medial forebrain bundle stimulation (drain areas) near to other zones that showed an increase in DA concentration (fountain areas). Both areas were intermixed to form a tridimensional matrix to regulate DA concentration throughout the striatum (fountain-drain matrix). The response to electrical stimuli of different amplitudes and durations and to different drugs (alpha-methyl-l-tyrosine, cocaine, gamma-butyrolactone, and haloperidol) suggests that regional differences for both DA release/DA uptake and DA cell firing autoregulation are behind the striatal fountain-drain matrix. The high diversity of DA activity observed in the striatum is a new framework for analyzing experimental and clinical phenomena.     

Correction of a rat model of Parkinson's disease by coexpression of tyrosine hydroxylase and aromatic amino acid decarboxylase from a helper virus-free herpes simplex virus type 1 vector

We previously reported long-term biochemical and behavioral correction of the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease (PD) by expression of tyrosine hydroxylase (TH) in the partially denervated striatum, using a herpes simplex virus type 1 (HSV-1) vector. This study had a number of limitations, including the use of a helper virus packaging system, limited long-term expression, and expression of only TH. To address these issues, we developed a helper virus-free packaging system, a modified neurofilament gene promoter that supports long-term expression in forebrain neurons, and a vector that coexpresses TH and aromatic amino acid decarboxylase (AADC). Coexpression of TH and AADC supported high-level (80%), behavioral correction of the 6-OHDA rat model of PD for 5 weeks. Biochemical correction included increases in extracellular dopamine and DOPAC concentrations between 2 and 4 months after gene transfer. Histologic analyses demonstrated neuronal-specific coexpression of TH and AADC at 4 days to 7 months after gene transfer, and cell counts revealed 1000 to 10,000 TH positive cells per rat at 2 months after gene transfer. This improved system efficiently corrects the rat model of PD.