AAV vector-mediated gene transfer and its application to the nervous system]

AAV vectors are considered to be promising gene-delivery vehicles for gene therapy, because they are derived from non-pathogenic virus, efficiently transduce non-dividing cells, and cause long-term gene expression. Appropriate AAV serotypes are utilized depending on the type of target cells; e.g., neurons are efficiently transduced with AAV2 and AAV5 vectors, and an AAV1 vector is most suitable for muscles. Among various neurological disorders, Parkinson's disease (PD) is one of the most appropriate candidates of gene therapy. PD is a progressive neurodegenerative disorder that predominantly affects dopaminergic neurons in the substantia nigra. There are two major approaches to gene therapy of PD; i.e., 1) intrastriatal expression of dopamine (DA)-synthesizing enzyme genes, and 2) neuroprotection using the glial cell line-derived neurotrophic factor (GDNF) gene to prevent the disease progression. As for the initial step of clinical application, AADC (aromatic L-amino acid decarboxylase; the enzyme converting L-DOPA to DA) gene transfer in combination with oral administration of L-DOPA would be appropriate, since DA production can be regulated by the dose of L-DOPA. Preclinical studies are being conducted in MPTP-parkinsonian monkeys. AAV vector-mediated gene therapy would be feasible as a novel treatment of PD in the near future.     

Gene therapy for Parkinson's disease: studies in animal models]

Recent developments in viral vectors capable of providing high levels of long-term transgene expression in the brain have led to the pursuit of two strategies in gene therapy for the treatment of Parkinson's disease (PD). One is the local production of dopamine in the striatum achieved by inducing the expression of dopamine-synthesizing enzymes. Three enzymes are necessary for efficient dopamine synthesis: tyrosine hydroxylase (TH) converts tyrosine to L-DOPA, aromatic L-amino acid decarboxylase (AADC) then converts L-DOPA to dopamine, and guanosine triphosphate cyclohydrolase I (GCH) is the rate-limiting enzyme for the synthesis of TH co-factor tetrahydrobiopterine. We have previously demonstrated that transduction with separate adeno-associated virus (AAV) vectors expressing TH, AADC, and GCH is effective in reducing motor abnormalities in 6-hydroxydopamine-lesioned rats and in MPTP-treated monkeys. Behavioral recovery persisted for at least 18 months after intrastriatal injection in parkinsonian rats. In MPTP monkeys, the amelioration of motor abnormalities was remarkable on the contralateral side, accompanied by robust transgene expression and elevated dopamine synthesis in the AAV-injected putamen. The second strategy entails the expression of neurotrophic factors or brain vesicular monoamine transporter in the striatum or the substantia nigra to slow the degeneration of dopamine neurons. Gene therapy using viral vectors offers a promising approach in the treatment of PD patients.     

Towards a neuroprotective gene therapy for Parkinson's disease: use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model

During the last few years, recombinant viral vectors derived from adenovirus (Ad), adeno-associated virus (AAV) or lentivirus (LV) have been developed into highly effective vehicles for gene transfer to the adult central nervous system. In recent experiments, in the rat model of Parkinson's disease, all three vector systems have been shown to be effective for long-term delivery of glial cell line-derived neurotrophic factor (GDNF) at biologically relevant levels in the nigrostriatal system. Injection of the GDNF encoding vectors into either striatum or substantia nigra thus makes it possible to obtain a regionally restricted over-expression of GDNF within the nigrostriatal system that is sufficient to block the toxin-induced degeneration of the nigral dopamine neurons. Injection of GDNF vectors in the striatum, in particular, is effective not only in rescuing the cell bodies in the substantia nigra, but also in preserving the nigrostriatal projection and a functional striatal dopamine innervation in the rat Parkinson model. Long-term experiments using AAV-GDNF and LV-GDNF vectors show, moreover, that sustained GDNF delivery over 3-6 months can promote regeneration and significant functional recovery in both 6-OHDA-lesioned rats and MPTP-lesioned monkeys. The impressive efficacy of the novel AAV and LV vectors in rodent and primate Parkinson models suggests that the time may now be ripe to explore these vector systems as tools for neuroprotective treatments in patients with Parkinson's disease.     

Targeting Parkinson's - tyrosine hydroxylase and oxidative stress as points of interventions

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons leading to decrease in striatal dopamine (DA) levels. In the present review, our focus was on recent advances in the treatment procedures of PD to achieve an increase in deficient tyrosine hydroxylase (TH) activity and/or expression. Stimulation of residual TH activity by the cofactors, 6R-L-erythro-tetrahydrobiopterin (BPH4) or NADH, or by brain transplant of natural TH-containing cells (fetal substantia nigra) or genetically engineered TH-containing cells, has been tried experimentally and clinically lately. As a promising approach to the gene therapy, intrastriatal expression of DAsynthesizing enzymes through transduction with separate adeno-associated virus (AAV) vectors/ marrow stromal cells (MSCs) or nonviral intravenous administration of rat transferrin receptor monoclonal antibody (TfRmAb)-targeted PEGylated immunoliposomes (PILs) has been found to be effective in animal models. Oxidative stress has been identified as one of the intermediary risk factors that could initiate and/or promote degeneration of DA neurons. TH itself is a prime target of oxidative/nitrosative injury. Certain superoxide dismutase and catalase mimetic prevented nitration of TH in cultured dopaminergic neurons. Therefore, development of therapeutic agents that can prevent formation of or specifically remove nitrating agents without interfering with normal neuronal function may protect protein from inactivation and provide means of limiting neuronal injury in PD. Non-pharmacological approaches such as diet therapy or use of active constituents of plants and phytomedicines have also emerged as a new - area of high interest. New treatment strategies for TH dysfunction rectification, a provision for neuroprotection in PD, seem to be on the horizon with many therapies under investigation.     

HSV vector-delivery of GDNF in a rat model of PD: partial efficacy obscured by vector toxicity

Herpes simplex virus (HSV)-derived vectors have been suggested for potential use in gene therapy for Parkinson's disease (PD). HSV naturally infects adult neuronal cells and possesses a large genome for the insertion of transgenes. In the present study, we have used two different HSV constructs to deliver glial cell line-derived neurotrophic factor (GDNF) to the striatum, and to assess the neuroprotective effects of the GDNF product in an intrastriatal 6-hydroxydopamine lesion model. One construct is blocked for IE gene expression whereas the other is deleted in the thymidine kinase gene. Both constructs induced a significant protection of the dopaminergic neurons in the substantia nigra from the lesions, whereas only one induced a transient behavioural recovery in amphetamine-induced rotation. Unexpectedly, the more deleted virus caused the greater toxicity. This was found to be due to the way the vector was purified. The issue of toxicity, which might account for the variable functional effects, needs resolving prior to therapeutic application of these vectors.     

帕金森病大鼠三重目的基因共表达的长期行为改善

目的　探讨多巴胺合成酶基因的三重共转导对帕金森病基因治疗的效果。方法　分别构建编码酪氨酸羟化酶 (TH)、芳香族氨基酸脱羧酶 (AADC)和三磷酸鸟苷环水解酶I(GCH)基因的腺伴随病毒 (AAV)载体 ,用复合感染的方式将AAV TH、AAV AADC及AAV GCH通过立体定向法注射入帕金森病大鼠损毁侧纹状体。采用免疫组化方法确定TH、AADC和GCH的表达 ;并连续观测基因转导后大鼠行为改善的情况长达 1年。结果　组织学依据显示TH、AADC和GCH基因均可在纹状体内长期有效而稳定的进行表达 ;三重基因转导可引起大鼠较TH和AADC共转导更为显著的行为改善(P <0 0 1)。结论　AAV载体介导的TH、AADC和GCH三重基因纹状体内共表达对于帕金森病大鼠的基因治疗更为有效。     

An adeno-associated virus vector-mediated multiple gene transfer for dopamine synthetic enzymes

Objective: To explore a multiple gene transfer approach with separate adeno-associated virus vectors. Methods: The genes of dopamine synthetic enzymes, tyrosine hydroxylasc (TH), GTP cyclohydrolase I (GCH, an enzyme critical for tetrahydrobioptcrin synthesis), and aromatic L-amino acid decarboxylase (AADC), were cotransduced into 293 cells with separate AAV vectors. Expressions of TH, GCH, and AADC were detected by Western blot analysis. L-dopa and dopamine levels in the ceils were assayed by HPLC. Results: TH, GCH, and AADC proteins were effectively cocxpressed in the transduced cells with three separate AAV vectors, AAV-TH, AAV-GCH, and AAV-AADC. Furthermore, the coexpression of these three proteins resulted in an effectively spontaneous dopainc production in the cotransduced cells. Conclusion: The triple transduction of TH, GCH, and AADC genes with separate AAV vectors is effective, which might be important to gene therapy for Parkinson's disease.     

Delayed gene therapy of glial cell line-derived neurotrophic factor is efficacious in a rat model of Parkinson's disease

Gene transfer of glial cell line-derived neurotrophic factor (GDNF) in rodent models of Parkinson's disease (PD) has been shown to protect against neurodegeneration either prior to or immediately after neurotoxin-induced lesions; however, the nigrostriatal pathway was largely intact when gene delivery was completed in these models, which may not accurately reflect the clinical situation encountered with Parkinson's patients. In this study, replication-incompetent adenoviral vectors encoding the rat GDNF gene were administered into the striatum 4 weeks following 6-hydroxydopamine (6-OHDA) injection in the unilateral striatum, more closely resembling fully developed PD. Apomorphine-induced rotational behavior testing was performed every week following 6-OHDA injection. At the 10th week after gene transfer, the striatal dopamine concentrations were measured by HPLC with an electrochemical detector and the number of tyrosine hydroxylase (TH)-positive dopamine neurons in the substantia nigra (SN) was determined by immunohistochemistry. Injection of 6-OHDA into the striatum produced stable increases in rotation, which reached a plateau between 4 and 5 weeks post-injection. The number of TH-positive neuron in the SN and dopamine levels in the striatum was significantly lower in the 6-OHDA group compared to the normal group. Gene transfer of GDNF, but not beta-galactosidase, significantly increased the number of TH-positive neurons and dopamine levels, with a subsequent behavioral recovery between 5 and 10 weeks following GDNF transduction. These findings demonstrate that adenovirus-mediated gene transfer of GDNF is efficacious even in the late stages of 6-OHDA-induced PD rats. They also provide further evidence on the effectiveness of GDNF-based gene therapy for experimental Parkinson's disease.     

AAV2-mediated delivery of human neurturin to the rat nigrostriatal system: long-term efficacy and tolerability of CERE-120 for Parkinson's disease

Neurturin (NTN) is a neurotrophic factor with known potential to protect and restore the function of dopaminergic substantia nigra neurons whose degeneration has been most closely linked to the major motor deficits in Parkinson's disease (PD). CERE-120, an adeno-associated virus serotype 2 (AAV2)-based gene delivery vector encoding human NTN, is being developed as a potential therapeutic for PD. In a series of preclinical studies reported herein, CERE-120 delivery to the striatum produced a dose-related neuroprotection of nigrostriatal neurons in the rat 6-hydroxydopamine (6-OHDA) lesion model. Long-lasting efficacy of CERE-120 was evidenced by substantia nigra cell protection, preserved fiber innervation of the striatum, and behavioral recovery for at least 6 months. In addition, striatal infusion of CERE-120 was found to have a safety and tolerability profile devoid of side effects or toxicological responses, for at least 12 months post-treatment, even at dose multiples 125 times that of the lowest efficacious dose tested. These results support the ongoing CERE-120 clinical program in PD patients.     

Bioactivity of AAV2‐neurturin gene therapy (CERE‐120): Differences between Parkinson's disease and nonhuman primate brains

Background: AAV2‐neurturin (CERE‐120) is designed to deliver the neurotrophic‐factor, neurturin, to the striatum to restore and protect degenerating nigrostriatal neurons in Parkinson's disease (PD). A common hypothesis is that following expression in the striatum, neurotrophic‐factors like neurturin (NRTN) will be transported from degenerating terminals to their cell bodies in the substantia nigra pars compacta (SNc). Methods: We tested this concept using immunohistochemistry, comparing the bioactivity of AAV2‐neurturin in brains of PD patients versus those of nonhuman primates similarly treated. Results: NRTN‐immunostaining in the targeted striatum was seen in all PD cases (mean putaminal coverage: ～15% by volume); comparable expression was observed in young, aged, and parkinsonian monkeys. In the SNc cell bodies, however, only rare evidence of neurturin was seen in PD, while ample evidence of intense nigral‐NRTN was observed in all monkeys. NRTN‐expression was associated with occasional, sparse TH‐induction in the striatum of PD, but nothing apparent in the SNc. In primates, NRTN produced robust TH‐induction throughout the nigrostriatal neurons. Discussion: These data provide the first evidence that gene therapy can increase expression of a neurotrophic‐factor deep in the PD brain and that clear but modest enhancement of degenerating neurons can be induced. They also provide important insight regarding deficiencies in the status of nigrostriatal neurons in advanced PD, suggesting that serious axon‐transport deficits reduced the bioactivity of AAV2‐NRTN by limiting the protein exposed to the cell body. Thus, future efforts using neurotrophic‐factors to treat neurodegenerative diseases will need to target both the terminal fields and the cell bodies of degenerating neurons to assure maximal benefit is achieved. © 2010 Movement Disorder Society     

Intrastriatal glial cell line-derived neurotrophic factors for protecting dopaminergic neurons in the substantia nigra of mice with Parkinson disease

BACKGROUND: Substantia nigra is deep in position and limited in range, the glial cell line-derived neurotrophic factor (GDNF) injection directly into substantia nigra has relatively greater damages with higher difficulty. GDNF injection into striatum, the target area of dopaminergic neuron, may protect the dopaminergic neurons in the compact part of substantia nigra through retrograde transport. OBJECTIVE: To investigate the protective effect of intrastriatal GDNF on dopaminergic neurons in the substantia nigra of mice with Parkinson disease (PD), and analyze the action pathway. DESIGN: A controlled observation. SETTING: Neurobiological Laboratory of Xuzhou Medical College. MATERIALS: Twenty-four male Kunming mice of 7–8 weeks old were used. GDNF, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were purchased from Sigma Company (USA); LEICAQWin image processing and analytical system. METHODS: The experiments were carried out in the Neurobiological Laboratory of Xuzhou Medical College from September 2005 to October 2006. The PD models were established in adult KunMing mice by intraperitoneal injection of MPTP. The model mice were were randomly divided into four groups with 6 mice in each group: GDNF 4-day group, phosphate buffer solution (PSB) 4-day group, GDNF 6-day group and PSB 6-day group. Mice in the GDNF 4 and 6-day groups were administrated with 1 μL GDNF solution (20 μg/L, dispensed with 0.01 mol/L PBS) injected into right striatum at 4 and 6 days after model establishment. Mice in the PSB 4 and 6-day groups were administrated with 0.01 mol/L PBS of the same volume to the same injection at corresponding time points. ② On the 12th day after model establishment, the midbrain tissue section of each mice was divided into 3 areas from rostral to caudal sides. The positive neurons of tyroxine hydroxylase (TH) and calcium binding protein (CB) with obvious nucleolus and clear outline were randomly selected for the measurement, and the number of positive neurons in unit area was counted. MAIN OUTCOME MEASURES: Number of positive neurons of TH and CB in midbrain substantia nigra of mice in each group. RESULTS: All the 24 mice were involved in the analysis of results. The numbers of TH+ and CB+ neurons in the GDNF 4-day group (54.33±6.92, 46.33±5.54) were obviously more than those in the PBS 4-day group (27.67±5.01, 21.50±5.96, P < 0.01). The numbers of TH+ and CB+ neurons in the GDNF 6-day group (75.67±5.39, 69.67±8.69) were obviously more than those in the PBS 6-day group (27.17±4.50, 21.33±5.72, P < 0.01) and those in the GDNF 4-day group (P < 0.01). CONCLUSION: Intrastriatal GDNF can protect dopaminergic neurons in substantia nigra of PD mice, and it may be related to the increase of CB expression.     

Neuroprotection in a rat Parkinson model by GDNF gene therapy using EIAV vector

Vectors based on lentiviruses are opening up new approaches for the treatment of neurodegenerative diseases. Currently, the equine infectious anaemia virus (EIAV) vector is one of the most attractive gene delivery systems with respect to neuronal tropism. The aim was to validate EIAV-lentiviral vectors as a gene delivery system for neurotrophic factor genes in an animal model of Parkinson's disease. EIAV carrying the glial cell line-derived neurotrophic factor (GDNF) gene was unilaterally injected into rat striatum and above the substantia nigra (SN). One week later, the rats received a 6-OHDA lesion into the ipsilateral striatum. GDNF delivery led to extensive expression of GDNF protein within the striatum. In addition, near complete protection against dopaminergic cell death was observed in the GDNF-treated group.     

Effect of 6-hydroxydopamine on striatal GDNF and nigral GFRalpha1 and RET mRNAs in the adult rat

Exogenous GDNF as well as vectors containing the gene for this trophic factor has been shown to be neuroprotective in animal models of Parkinson's disease. We therefore investigated whether changes in striatal GDNF protein and nigral mRNA levels of its co-receptors GFRalpha1 and RET occur in response to lesions of dopamine (DA) neurons and examined the temporal profile of these changes as they relate to the loss of dopaminergic markers. Rats were lesioned with 6-hydroxydopamine and sacrificed 3 h to 60 days post-infusion. DA tissue levels in the striatum and tyrosine hydroxylase immunoreactivity in the substantia nigra (SN) and ventral tegmental area (VTA) were used to determine the size of the lesions. GDNF protein was measured in the striatum using radioimmunocytochemistry. In situ hybridization was used to determine alterations in the mRNAs of RET and GFRalpha1 in the SN and VTA. We observed no persistent changes in GDNF protein in the striatum in response to 6-hydroxydopamine over the 60-day observation period, suggesting that compensatory changes in this trophic factor do not occur in response to injury. Dramatic decreases in RET and GFRalpha1 were observed in both SN and VTA that were generally correlated with the loss of TH protein and striatal DA content, strongly suggesting that these receptors are located on DA neurons and that the protective effect of GDNF reflects a direct action of the trophic factor on these neurons.     

Inducing agent tamoxifen of CreERT2 to reduce the side effects of gene therapy for Parkinson's disease

BACKGROUND:Gene therapy for Parkinson's disease is being explored as an effective strategy to restore and protect the function of neuronal cells in the substantia nigra. Regulation of gene expression is necessary for gene therapy to avoid adverse effects due to excessive synthesis of transgene products.OBJECTIVE:Here we developed recombinant adeno-associated virus (AAV) as a viral vector-mediated gene regulation system based on Cre recombinase fused to the mutated ligand-binding domain of the estrogen receptor (CreERT2) + inducing agent tamoxifen. Inducible Cre recombinase was used to reduce tyrosine hydroxylase gene expression and to prevent the excessive increase in dopamine.DESIGN, TIME AND SETTING:A genetic engineering in vitro comparative study and randomized controlled animal experiment. This study was conducted at the Gene Therapy Center, Jichi Medical School, Japan from June 2002 to June 2004.METHODS:To construct a recombinant AAV vector carrying a dopamine synthase gene. The tyrosine hydroxylase gene was inserted using a IoxP fragment that could be regulated by Cre recombinase. The recombinant AAV vector carrying the CreERT2 gene was co-transduced with HEK293 cells and the corpus striatum in a rat model of Parkinson's disease, with inducing agent tamoxifen to regulate gene expression.MAIN OUTCOME MEASURES:The levels of dopamine and aromatic L-amino acid decarboxylase (AADC) activity were detected in HEK293 cell medium and in the corpus striatum in a rat model of Parkinson's disease using high-performance liquid chromatography. Immunofluorescence double staining was used to observe tyrosine hydroxylase and Cre or AADC co-expression in HEK293 cell medium. Immunohistochemical staining was employed to observe tyrosine hydroxylase and AADC expression and behavioral changes were measured in Parkinson's rats.RESULTS:Transfected AAV-CreERT2 and AAV expressing dopamine synthesis enzymes could increase the synthesis of dopamine in HEK293 medium and Parkinson's rat striatum (P < 0.01) and improve the rotational behavior of Parkinson's rats. While tamoxifen markedly reduced overproduction of dopamine caused by cotransfection of viral vectors (P < 0.01), but did not affect the expression and activity of AADC.CONCLUSION:The application of AAV vector-encoded tyrosine hydroxylase gene under the gene regulation system of Cre-ERT2>, after tamoxifen treatment, can effectively control the generation of genetically modified products to reduce the production of excessive dopamine in vivo and in vitro. Therefore, this method can increase the safety of gene therapy.     

Gene therapy and cell transplantation for Parkinson's disease]

Increasing enthusiasm in the field of stem cell research is raising the hope of novel cell replacement therapies for Parkinson's disease (PD), but it also raises both scientific and ethical concerns. In most cases, dopaminergic cells are transplanted ectopically into the striatum instead of the substantia nigra. If the main mechanism underlying any observed functional recovery with these cell replacement therapies is restoration of dopaminergic neurotransmission, then viral vector-mediated gene delivery of dopamine-synthesizing enzymes is a more straight forward approach. The development of a recombinant adeno-associated viral (AAV) vector is making gene therapy for PD a feasible therapeutic option in the clinical arena. Efficient and long-term expression of genes for dopamine-synthesizing enzymes in the striatum restored local dopamine production and allowed behavioral recovery in animal models of PD. A clinical trial to evaluate the safety and efficacy of AAV vector-mediated gene transfer of aromatic L-amino acid decarboxylase, an enzyme that converts L-dopa to dopamine, is underway. With this strategy patients would still need to take L-dopa to control their PD symptoms, however, dopamine production could be regulated by altering the dose of L-dopa. Another AAV vector-based clinical trial is also ongoing in which the subthalamic nucleus is transduced to produce inhibitory transmitters.     

Downregulation of tyrosine hydroxylase phenotype after AAV injection above substantia nigra: Caution in experimental models of Parkinson’s disease

Adeno-associated virus (AAV) vector-mediated delivery of human α-synuclein (α-syn) gene in rat substantia nigra (SN) results in increased expression of α-syn protein in the SN and striatum which can progressively degenerate dopaminergic neurons. Therefore, this model is thought to recapitulate the neurodegeneration in Parkinson's disease. Here, using AAV to deliver α-syn above the SN in male and female rats resulted in clear expression of human α-syn in the SN and striatum. The protein was associated with moderate behavioral deficits and some loss of tyrosine hydroxylase (TH) in the nigrostriatal areas. However, the immunohistochemistry results were highly variable and showed little to no correlation with behavior and the amount of α-syn present. Expression of green fluorescent protein (GFP) was used as a control to monitor gene delivery and expression efficacy. AAV-GFP resulted in a similar or greater TH loss compared to AAV-α-syn and therefore an additional vector that does not express a protein was tested. Vectors with double-floxed inverse open reading frame (DIO ORF) encoding fluorescent proteins that generate RNA that is not translated also resulted in TH downregulation in the SN but showed no significant behavioral deficits. These results demonstrate that although expression of wild-type human α-syn can cause neurodegeneration, the variability and lack of correlation with outcome measures are drawbacks with the model. Furthermore, design and control selection should be considered carefully because of conflicting conclusions due to AAV downregulation of TH, and we recommend caution with having highly regulated TH as the only marker for the dopamine system.     

Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture

Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes.     

Neuronal GDNF Expression in the Adult Rat Nervous System Identified By In Situ Hybridization

Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor which has been purified on the basis of its ability to promote the survival of dopaminergic neurons in vitro. GDNF has subsequently been cloned and its sequence shown to be distantly related to transforming growth factor-β (TGF-β). To identify GDNF expressing cells in the adult rat brain, in situ hybridization using a digoxygenin (DIG)-labelled riboprobe has been performed. Our results show that GDNF mRNA is mainly expressed in neurons and that its synthesis is not restricted to dopaminergic areas. It is widely expressed in the cortex, the hippocampus, the striatum, the substantia nigra, the thalamus, the cerebellum and the spinal cord. Neuronal GDNF expression varies among brain regions as determined by the intensity of the in situ signal. Double labelling of the substantia nigra using tyrosine hydroxylase immunohistochemistry, associated with GDNF in situ hybridization, show that the majority of dopaminergic neurons express GDNF. The widespread expression of GDNF throughout the adult brain suggests that its administration in Parkinson's disease should be restricted to the altered structures, in order to avoid possible deleterious side effects.     

尼古丁对帕金森病大鼠纹状体GDNF和多巴胺含量的影响

目的 研究尼古丁对帕金森病（PD）大鼠纹状体脑胶质细胞源性神经营养因子(GDNF)和多巴胺（DA）含量的影响。方法 将6－羟多巴胺（6－OHDA）立体定向注射到大鼠右侧中脑腹侧背盖部（VTA）和黑质致密部（SNpc)，建立PD大鼠模型。采用生化、免疫组织化学方法观察不同剂量尼古丁对PD大鼠的作用，检测纹状体GDNF表达及DA含量的变化。结果 造模前及造模后皮下注射尼古丁的PD大鼠，纹状体GDNF表达及DA含量较PD组有明显改善（P＜0．05）。结论 尼古丁可减轻6－OHDA对黑质DA能神经元的损伤，对PD大鼠具有保护作用。     

Marked dopaminergic cell loss subsequent to developmental, intranigral expression of glial cell line-derived neurotrophic factor

Glial cell line-derived neurotrophic factor (GDNF) shows potent neuroprotective as well as neurorestorative actions on the adult neurons impacted in animal models of Parkinson's disease (PD). Long-term pharmaco-physiological effects of GDNF on developing dopaminergic (DA) neurons have not yet been explored because of technical difficulties in producing prolonged cell type-specific delivery of this neurotrophic factor in mammalian embryonic brain. The current studies used our previously characterized 9.0-kb tyrosine hydroxylase promoter to produce transgenic mice with neuronal cell type-specific expression of GDNF in substantia nigra pars compacta (SNc) and locus coeruleus (LC). These mice were used to test the parsimonious hypothesis that increased developmental expression of GDNF in SNc and LC would significantly enhance the number of postmitotic adult neurons. To our surprise, adult transgenic mice carrying the TH9.0kb-GDNF hybrid gene showed dramatic reductions in both the numbers and the volumes of SNc-DA and LC-noradrenergic (NA) neurons by quantitative morphometric analysis. The decrease in the number of DA neurons was apparent as early as postnatal day 2, the period before the major naturally occurring apoptotic cell death in midbrain. Aged transgenic mice exhibited no further significant deficits in motor behaviors. These data suggest that continuous, early developmental GDNF expression exerts physiological effects on newly differentiated, immature dopamine neurons that differ from those observed on more mature and adult DA neurons. Further elucidation of the mechanisms underlying differential GDNF actions will greatly improve the pharmacological efficacy of GDNF in fetal neural transplantation as well as adult neuronal gene therapy in PD patients.