Paul A. Lapchak

Glial cell line-derived neurotrophic factor attenuates behavioural deficits and regulates nigrostriatal dopaminergic and peptidergic markers in 6-hydroxydopamine-lesioned adult rats: comparison of intraventricular and intranigral delivery

The effects of intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor were tested on low dose (0.05 mg/kg) apomorphine-induced rotations and tyrosine hydroxylase activity in the substantia nigra and striatum of stable 6-hydroxydopamine-lesioned rats. In addition, we determined if 6-hydroxydopamine lesions in the absence or presence of treatment affected neuropeptide (substance P, met-enkephalin, dynorphin) content in the striatum. Glial cell line-derived neurotrophic factor, when administered intranigrally, prevented apomorphine-induced rotational behaviour for 11 weeks following a single injection. In comparison, intraventricularly-administered glial cell line-derived neurotrophic factor produced a transient reduction in rotational behaviour that lasted for two to three weeks following a single injection. We also show that rotational behaviour is reduced following each subsequent intraventricular injection of glial cell line-derived neurotrophic factor given every six weeks, a time-point when baseline rotation deficits were re-established. Intranigrally- or intraventricularly-administered glial cell line-derived neurotrophic factor significantly reduced weight gain in all 6-hydroxydopamine-lesioned rats in this study. Following behavioural analysis where a confirmed improvement of behaviour was established, tissues were dissected for neurochemical analysis. In lesioned rats with intranigral injections of administered glial cell line-derived neurotrophic factor, significant increases of nigral, but not striatal tyrosine hydroxylase activity were measured. Additionally, 6-hydroxydopamine lesions significantly increased striatal dynorphin (61-139%) and met-enkephalin (81-139%), but not substance P levels. In these rats, intranigrally-administered glial cell line-derived neurotrophic factor injections reversed lesion-induced increases in nigral dynorphin A levels and increased nigral dopamine levels, but did not alter nigral met-enkephalin or substance P levels nor striatal dopamine levels. In lesioned rats with intraventricular injections of glial cell line-derived neurotrophic factor, tyrosine hydroxylase ispilateral to the lesion was increased in the substantia nigra, but not in the striatum. Intraventricularly-administered glial cell line-derived neurotrophic factor did not reverse lesion-induced increases in nigral dynorphin A or met-enkephalin levels nor did glial cell line-derived neurotrophic factor affect substance P levels in the striatum. These results suggest that in an animal model of Parkinsons disease, the neurotrophic factor glial cell line-derived neurotrophic factor reverses behavioural consequences of 6-hydroxydopamine administration, an effect that may involve both dopaminergic and peptidergic neurotransmission.

Adenoviral vector-mediated GDNF gene therapy in a rodent lesion model of late stage Parkinson's disease

A recombinant adenoviral vector encoding the human glial cell line-derived neurotrophic factor (GDNF) gene (Ad-GDNF) was used to express the neurotrophic factor GDNF in the unilaterally 6-hydroxydopamine (6-OHDA) denervated substantia nigra (SN) of adult rats ten weeks following the 6-OHDA injection. 6-OHDA lesions significantly increased apomorphine-induced (contralateral) rotations and reduced striatal and nigral dopamine (DA) levels by 99% and 70%, respectively. Ad-GDNF significantly (P < 0.01) decreased (by 30-40%) apomorphine-induced rotations in lesioned rats for up to two weeks following a single injection. Locomotor activity, assessed 7 days following the Ad-GDNF injection, was also significantly (P < 0.05) increased (by 300-400%). Two weeks after the Ad-GDNF injection, locomotor activity was still significantly increased compared to the Ad-beta-gal-injected 6-OHDA lesioned (control) group. Additionally, in Ad-GDNF-injected rats, there was a significant decrease (10-13%) in weight gain which persisted for approximately two weeks following the injection. Consistent with the behavioral changes, levels of DA and the metabolite dihydroxyphenylacetic acid (DOPAC) were elevated (by 98% and 65%, respectively) in the SN, but not the striatum of Ad-GDNF-injected rats. Overall, a single Ad-GDNF injection had significant effects for 2-3 weeks following administration. These results suggest that virally delivered GDNF promotes the recovery of nigral dopaminergic tone (i.e.: increased DA and DOPAC levels) and improves behavioral performance (i.e.: decreased rotations, increased locomotion) in rodents with extensive nigrostriatal dopaminergic denervation. Moreover, our results suggest that viral delivery of trophic factors may be used eventually to treat neurodegenerative diseases such as Parkinsons disease.

Glial cell line-derived neurotrophic factor: distribution and pharmacology in the rat following a bolus intraventricular injection

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase dopaminergic parameters in vitro and in vivo and can reduce parkinsonian behaviors in animal models of the disease. This study determined the potential of the lateral ventricle as an administration route for GDNF by examining the distribution and neurochemical consequences of a single intraventricular injection. Autoradiographic analysis showed that intraventricularly administered [125I]GDNF was distributed throughout the ventricular system at 1 and 24 h following injection. The cerebral cortex, septum, diagonal band, fimbria, striatum, hippocampus, hypothalamus, substantia nigra/ventral tegmental area, and cerebellum were also labeled. At 7 days, there was still labeling throughout the ventricular system, hypothalamus, substantia nigra, and cerebellum. Twenty-four hours following an intrastriatal injection of [125I]GDNF, label was observed in the substantia nigra/ventral tegmental area, demonstrating retrograde transport. The neurochemical effects of intraventricularly administered GDNF (0.1-100 micrograms) at 7 days post injection were also examined. GDNF significantly increased striatal (approximately 28%) and nigral (up to 40%) dopamine, as well as regulated the dopamine metabolites homovanillic acid and dihydroxyphenylacetic acid. Dopamine levels were unchanged in the frontal cortex. Dopamine content was significantly increased in the hypothalamus (up to 35%), an increase which may contribute to the inhibition of weight gain seen after administration of GDNF. Additionally, dopamine turnover was decreased or unchanged across the brain regions analyzed, which may indicate that in unlesioned rats, intraventricularly administered GDNF stimulates the synthesis and storage of dopamine. This study shows that intraventricularly injected GDNF can access basal ganglia structures, most notably the midbrain dopamine cell body region, and remains present in this area for at least 7 days following a single administration. GDNF differentially increases dopaminergic tone within a variety of brain structures, including the nigrostriatal pathway. These data support the potential effectiveness of intraventricular administered GDNF as a treatment for Parkinsons disease.

GDNF TRIGGERS FIBER OUTGROWTH OF FETAL VENTRAL MESENCEPHALIC GRAFTS FROM NIGRA TO STRIATUM IN 6-OHDA-LESIONED RATS

Abstract.Previous reports have indicated that grafting of fetal ventral mesencephalic tissue to the nigra region of animals unilaterally lesioned with 6-hydroxydopamine (6-OHDA), in conjunction with kainate injection between the nigra and striatum, restores nigrostriatal tyrosine hydroxylase immunoreactivity. Glial-cell-line-derived neurotrophic factor (GDNF), a potent trophic factor for dopaminergic neurons, has been found to be upregulated by kainate. We have investigated the bridging effect of GDNF injection on intra-nigral transplants. Adult Sprague-Dawley rats were anesthetized and unilaterally injected with 6-OHDA into the medial forebrain bundle. The completeness of lesions was tested by measuring methamphetamine-induced rotations. Some 1–2 months after 6-OHDA administration, fetal ventral mesencephalic tissues were grafted into the lesioned nigral area followed by injection of 100 μg GDNF, along a tract from the nigra to striatum. Animals receiving transplantation and GDNF injection showed a significant decrease in rotation 1–3 months after grafting. Immunocytochemical studies indicated that tyrosine-hydroxylase-positive neurons and fibers were present in the nigra and striatum, respectively, after grafting. No effects of similarly injected brain-derived neurotrophic factor were seen. These results indicate that fetal nigral transplantation and GDNF injection restore the nigrostriatal dopaminergic pathway in Parkinsonian animals and support the hypothesis of trophic activity of GDNF on midbrain dopaminergic neurons.

Glial cell line-derived neurotrophic factor induces the dopaminergic and cholinergic phenotype and increases locomotor activity in aged Fischer 344 rats.

Glial cell line-derived neurotrophic factor has been shown to affect dopaminergic and cholinergic neuron markers and functions in young rats. However, it is not known if the response to exogenous glial cell line-derived neurotrophic factor is augmented during normal aging. Thus, the effects of chronic intraventricular infusions of glial cell line-derived neurotrophic factor were determined in young adult (three-months-old) and aged (24-months-old) Fischer 344 (F344) male rats. The effects of glial cell line-derived neurotrophic factor were compared to the effects of the neurotrophin nerve growth factor. Growth factors were administered at a dose of 10 mg/day for 14 days. Locomotor activity and weight changes were also examined in all rats. Aged F344 rats showed significantly reduced (by 75-80%) locomotor activity compared to young rats. In glial cell line-derived neurotrophic factor-treated aged and young rats there was significantly increased (242% and 149%, respectively) locomotor activity measured at seven days. There was also a significant increase in locomotor activity measured 14 days after the start of infusion. Both glial cell line-derived neurotrophic factor and nerve growth factor reduced weight gain by 10% in young and old F344 rats. Two weeks following the start of nerve growth factor or glial cell line-derived neurotrophic factor administration the brains were used for neurochemical analyses. Glial cell line-derived neurotrophic factor significantly increased tyrosine hydroxylase activity in the substantia nigra and striatum of aged rats and in the substantia nigra of young rats. Nerve growth factor treatment did not significantly affect tyrosine hydroxylase activity. However, glial cell line-derived neurotrophic factor and nerve growth factor increased choline acetyltransferase activity in the septum, hippocampus, striatum and cortex of aged rats and in the hippocampus and striatum of young rats to a comparable degree. These findings indicate that specific dopaminergic and cholinergic neuron populations remain responsive to glial cell line-derived neurotrophic factor during the life span of the rat and may be involved in maintaining phenotypic expression within multiple neuronal populations. Additionally, the glial cell line-derived neurotrophic factor-induced up-regulation of brain neurotransmitter systems may be responsible for increased locomotor activity in F344 rats.

Intracerebroventricular glial cell line-derived neurotrophic factor improves motor function and supports nigrostriatal dopamine neurons in bilaterally 6-hydroxydopamine lesioned rats

In order to evaluate the efficacy of glial cell line-derived neurotrophic factor (GDNF) in a model of advanced Parkinsons disease, we studied rats with extensive bilateral lesions of the nigrostriatal pathway. Adult male F344 rats were injected bilaterally into the medial forebrain bundle with the neurotoxin 6-hydroxydopamine. Locomotor ability as measured by total distance traveled in an open field over 20 min, as well as von Frey hair testing of sensorimotor neglect, was monitored weekly. Rats demonstrating severe motor impairment and sensorimotor neglect were used for this study and were sorted to achieve similar average behavioral scores between the two treatment groups. After 2 weeks of pretesting, the rats received 250 microg GDNF or vehicle injected into the right lateral cerebral ventricle. Three weeks later, an additional 500 microg GDNF or vehicle was injected into the contralateral ventricle. The rats were monitored for another 2 weeks prior to sacrifice. Behavioral results indicated that von Frey hair scores were inconsistent between tests for each rat and were unchanged following GDNF treatment. However, GDNF recipients demonstrated significant improvement in locomotor ability compared to vehicle recipients. High-pressure liquid chromatography-electrochemical detection analysis of neurotransmitter levels revealed a significant increase in dopamine content within the substantia nigra and ventral tegmenta, but not the striata, of GDNF-treated rats. Further, immunohistochemical staining of tissues from matched pairs of rats revealed increased numbers of tyrosine hydroxylase-positive ventral mesencephalic neurons in one of the two pairs of rats examined. These results suggest that intracerebroventricular GDNF administration improves motor ability and supports nigrostriatal dopaminergic neurons in a model of severe Parkinsons disease.

Pharmacological characterization of glial cell line-derived neurotrophic factor (GDNF): implications for GDNF as a therapeutic molecule for treating neurodegenerative diseases

Abstract.This review presents a comprehensive survey of the recently described pharmacological activities of glial cell line-derived neurotrophic factor on the central nervous system.

Glial Cell Line-Derived Neurotrophic Factor: A Novel Therapeutic Approach to Treat Motor Dysfunction in Parkinson's Disease

The discovery of the novel neurotrophic factor glial cell-line derived neurotrophic factor (GDNF) in 1993 sparked the interest of basic neuroscientists and clinicians alike. Since that time, many aspects of GDNFs physiology and pharmacology have been studied in great detail. GDNF has been shown to be a potent survival factor for dopaminergic neurons during development. GDNF also has been shown to be a survival factor and neurotrophic factor for nigrostriatal dopaminergic neurons in the adult. The factor also reverses behavioral deficits in a rodent and primate model of Parkinsons disease. The overall goal will be to discuss the pharmacology of GDNF in the context of a potential therapeutic use to treat Parkinsons disease. Thus, the following report presents a comprehensive review of the development of GDNFs pharmacology and evidence which supports the clinical use of GDNF to treat dopaminergic deficits and motor dysfunctions in Parkinsons disease.

Characterization of a Fibrin Glue–GDNF Slow-Release Preparation

One novel method to deliver trophic factor locally in the CNS is to mix it into fibrin glue. In the present studies, [125I]-labeled GDNF-containing fibrin glue balls were used to determine binding and spread of the trophic factor. First, the binding of different concentrations of [125I]-labeled GDNF in fibrin glue was determined in vitro. Within the six concentrations used (from 200 nM to 0.004 nM, 0 M as control), there was a strong linear correlation between the [125I]-GDNF concentration and the recovered radioactivity (r = 0.992). The mean bound radioactivity in 16 samples with 4 nM [125I]-GDNF was 71262 +/- 2710 CPM, and accounted for 89.8% of the mean initial count of free [125I]-GDNF (79369 +/- 3499 CPM). Second, [125I]-GDNF-containing glue balls were implanted into the anterior chamber of adult rats. The implanted fibrin glue balls decreased in size with time, but could still be identified on the irises 2 wk after implantation. Radioactivity was concentrated at the implantation sites in the early stages with a distribution in the surrounding iris tissue, which became separated into focal radioactive spots at the third week. Counts of radioactivity were significantly higher in the [125I]-GDNF glue ball-implanted irises than controls until 14 days after implantation. A study of the [125I] decay over time using least-squares linear regression demonstrated first-order kinetics (r = -0.98, p <0.02) with k = 0.0091 and T 1/2 = 76 h. Finally, [125I]-GDNF-containing glue balls were implanted in the spinal cord of adult rats. Radioactivity was concentrated at the implantation sites in the early stages and was later distributed more widely in the surrounding thoracic cord. The [125I]-GDNF-containing glue degraded over time and became a porous meshwork with decreasing radioactivity at the later time points. Radioactivity in the spinal cords subjected to implantation of [125I]-GDNF-containing glue balls was higher than in controls for 14 days. Study of the [125I] decay by time with least-squares linear regression demonstrated first-order kinetics (r = -0.97, p = 0.001) with T 1/2 = 75.6 h. We conclude that the trophic factor GDNF becomes bound in the fibrin glue matrix from which it is gradually released. Our results suggest that fibrin glue is an effective substrate for keeping a trophic factor localized in situ for a finite period, protected from the circulation, surrounding aqueous humor or CSF.

Glial cell line-derived neurotrophic factor reverses motor impairment in 16–17 month old rats

Aging is accompanied with declines in motoric function which may be the result of deficits in central nervous system dopaminergic function. Glial cell line-derived neurotrophic factor (GDNF) has been shown to have neuroprotective and restorative effects on dopaminergic neurons of the nigrostriatal pathway in young rats. In this study, 10, 40, or 60 micrograms GDNF or vehicle was injected intrastriatally in 16-17 month old Fischer 344 rats. Coordination and muscle strength as determined by performance on an inclined balance beam and a wire grip strength test were monitored for up to 5 weeks post-injection. GDNF elicited dose-dependent improvements in motor coordination without concurrent increases in strength. The highest dose tested produced > 79% improvement in motor coordination, resulting in performance scores approaching those achieved by 3 month old rats tested concurrently. These findings indicate GDNF produces profound improvement in the motoric function of mature rats, which may be related to dopaminergic circuits.