Barry J. Hoffer

GLIAL CELL LINE- DERIVED NEUROTROPHIC FACTOR REVERSES TOXIN-INDUCED INJURY TO MIDBRAIN DOPAMINERGIC NEURONS IN VIVO

Fischer 344 rats were unilaterally injected into the medial forebrain bundle with 6-hydroxydopamine (6-OHDA). Apomorphine-induced rotational behavior was used to select animals whose rotation exceeded 300 turns/h, corresponding to greater than 95% dopamine (DA) depletion in the ipsilateral striatum. Four weeks later, glial cell line-derived neurotrophic factor (GDNF) or vehicle was injected intranigrally ipsilateral to the lesion (0.1-100 micrograms). The highest dose of GDNF tested produced a marked decrease in rotational behavior. This dose also produced levels of DA in the ipsilateral substantia nigra (SN) which were not statistically different from the contralateral side. Vehicle-treated animals showed a marked DA depletion in the ipsilateral SN. These results demonstrate neurochemical and behavioral improvements in unilaterally DA-lesioned rats following intranigral administration of GDNF, suggesting that GDNF may develop into a useful therapy for Parkinsons disease.

Glial cell line-derived neurotrophic factor is expressed in the developing but not adult striatum and stimulates developing dopamine neurons in vivo.

The potential role of glial cell line-derived neurotrophic factor (GDNF) as a trophic molecule for midbrain dopamine neurons was examined using two different approaches: in situ hybridization and intraocular transplantation. The presence of mRNA for GDNF was noted in striatal and ventral limbic dopaminergic target areas in the developing (E20-P7) rat, but not the adult rat. Signals were also found in nondopaminergic areas during maturation, such as the cerebellar anlage, spinal cord, and thalamus. Lesions of the nigrostriatal pathway in neonatal or adult rats, using 6-hydroxydopamine injected into the medial forebrain bundle, did not elicit upregulation of mRNA for GDNF. Grafts of fetal ventral mesencephalon in the anterior eye chamber were exposed to repeated injections of GDNF, which elicited a marked and dose-dependent increase in transplant volume. A low (0.1 microgram/eye) and high (1 microgram/eye) dose of GDNF both led to a somewhat larger mean area of dopamine fiber outgrowth into host irides. In the transplants, cell counts of tyrosine hydroxylase (TH)-immunoreactive neurons revealed a doubling of cell numbers in the low-dose group and about four times as many cells in the high-GDNF-dose group compared to controls. Moreover, the density of TH-immunoreactive nerve fibers was markedly and significantly higher in transplants treated with the high GDNF dose. Since the volumes of these transplants were also larger, the total amount of both TH-positive cells and TH-positive nerve fibers was many-fold greater in the high-GDNF group than that in the controls. Taken together, these data support the concept that GDNF functions as a dopaminotrophic factor in vivo.

Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons

Mitochondrial dysfunction is implicated in the pathophysiology of Parkinson′s disease (PD), a common age-associated neurodegenerative disease characterized by intraneuronal inclusions (Lewy bodies) and progressive degeneration of the nigrostriatal dopamine (DA) system. It has recently been demonstrated that midbrain DA neurons of PD patients and elderly humans contain high levels of somatic mtDNA mutations, which may impair respiratory chain function. However, clinical studies have not established whether the respiratory chain deficiency is a primary abnormality leading to inclusion formation and DA neuron death, or whether generalized metabolic abnormalities within the degenerating DA neurons cause secondary damage to mitochondria. We have used a reverse genetic approach to investigate this question and created conditional knockout mice (termed MitoPark mice), with disruption of the gene for mitochondrial transcription factor A (Tfam) in DA neurons. The knockout mice have reduced mtDNA expression and respiratory chain deficiency in midbrain DA neurons, which, in turn, leads to a parkinsonism phenotype with adult onset of slowly progressive impairment of motor function accompanied by formation of intraneuronal inclusions and dopamine nerve cell death. Confocal and electron microscopy show that the inclusions contain both mitochondrial protein and membrane components. These experiments demonstrate that respiratory chain dysfunction in DA neurons may be of pathophysiological importance in PD.

Correlation of apomorphine- and amphetamine-induced turning with nigrostriatal dopamine content in unilateral 6-hydroxydopamine lesioned rats

In the unilateral 6-hydroxydopamine (6-OHDA)-lesioned rat model of Parkinsons disease, controversy exists concerning the use of apomorphine- or D-amphetamine-induced rotations as reliable indicators of nigrostriatal dopamine depletion. Our objective was to evaluate which, if either, drug-induced behavior is more predictive of the extent of nigrostriatal dopamine depletion. Fischer 344 and Sprague-Dawley rats were unilaterally injected with 9 micrograms/4 microliters/4 min 6-hydroxydopamine into the medial forebrain bundle. The animals were behaviorally tested with apomorphine (0.05 mg/kg, s.c.) and D-amphetamine (5.0 mg/kg, s.c.). Following testing, the brains were removed and the right and left striata, substantia nigra and ventral tegmental area were dissected free and quickly frozen at -70 degrees C for analysis of catecholamine content by high performance liquid chromatography coupled with electrochemical detection. Our results indicate that an animal which has greater than a 90% depletion of dopamine in the striatum might not rotate substantially on apomorphine, without a concomitant depletion of > 50% of the DA content in the corresponding substantia nigra. No correlations were seen involving depletions of the ventral tegmental area and the extent of the lesions to the striatum. Submaximally lesioned (75-90% depleted) rats were found to rotate on D-amphetamine but not on apomorphine. In addition, control rats that did not receive lesions were often seen to rotate extensively on D-amphetamine. We therefore conclude that maximal lesions of the striatum and substantia nigra are required to generate rotations demonstrable with low dose apomorphine but not with D-amphetamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. II. Sensitivity of Purkinje cells to norepinephrine and related substances administered by microiontophoresis.

Abstract The sensitivity of rat cerebellar Purkinje cells to norepinephrine and to adrenergic antagonists and protagonists was studied by microiontophoresis. (1) Virtually all Purkinje cells respond to microiontophoresis of norepinephrine with a reduction in mean spontaneous discharge rate. (2) The effects of norepinephrine are reproduced by epinephrine, isoproterenol, amphetamine, and tyramine. (3) Responses to norepinephrine are blocked by the beta receptor antagonist, MJ-1999. (4) Desmethylimipramine, which blocks re-uptake of norepinephrine, markedly potentiates the response to this agent. (5) Norepinephrine, tyramine, amphetamine and MJ-1999 are effective in animals treated with 6-hydroxydopamine, indicating that their effects are not exerted on adrenergic presynaptic terminals. (6) These data support the hypothesis that norepinephrine is a Purkinje cell neurotransmitter.

Studies on norepinephrine-containing afferents to Purkinje cells of rat cerebellum. I. Localization of the fibers and their synapses

A process is described for the purification and recovery of cyanuric acid from a hot slurry of cyanuric acid in an organic solvent. In the process, the hot slurry is quenched in a quench liquid which is at a temperature for cooling the hot slurry to form a cooled slurry of cyanuric acid particles in a mixture of the solvent and the quench liquid. Cyanuric acid particles are separated from the mixture of solvent and quench liquid and recovered. The process effectively cools and separates cyanuric acid from the solvent and any color bodies present to produce a pure white crystalline product.

GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet β-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine–induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinsons disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD.

Glial Cell Line-Derived Neurotrophic Factor Protects against Ischemia-Induced Injury in the Cerebral Cortex

Glial cell line-derived neurotrophic factor (GDNF), a recently described and cloned member of the transforming growth factor (TGF)-β superfamily, has been shown to have marked trophic activity on several populations of central neurons. Survival-promoting and injury protectant activity in vitro and in vivo, using several paradigms, has been demonstrated for ventral mesencephalic dopaminergic neurons and spinal cord motoneurons. In view of a proposed commonality of mechanisms, involving intracellular free radical generation, depolarization-induced Ca2+ influx, and mitochondrial respiratory enzyme injury, between such GDNF-responsive paradigms and those of ischemia-induced injury, we tested the effects of GDNF on the extent of neural degeneration induced by transient middle cerebral artery (MCA) occlusion. We now report that intracerebroventricular and intraparenchymal administration of GDNF potently protects the cerebral hemispheres from damage induced by MCA occlusion. In addition, the increase in nitric oxide that accompanies MCA occlusion and subsequent reperfusion is blocked almost completely by GDNF. Thus, this protein may play an important role in the treatment of cerebrovascular occlusive disease.

Neurturin and glial cell line-derived neurotrophic factor receptor-beta (GDNFR-beta), novel proteins related to GDNF and GDNFR-alpha with specific cellular patterns of expression suggesting roles in the developing and adult nervous system and in peripheral organs.

Cloning strategies were used to identify a gene termed glial cell line-derived neurotrophic factor receptor-β (GDNFR-β) related to GDNFR-α. In situ hybridization was then used to map cellular expression of the GDNF-related trophic factor neurturin (NTN) and GDNFR-β mRNA in developing and adult mice, and comparisons with GDNFR-α and RET were made. Neurturin is expressed in postnatal cerebral cortex, striatum, several brainstem areas, and the pineal gland. GDNFR-β mRNA was more widely expressed in the developing and adult CNS, including cerebral cortex, cerebellum, thalamus, zona incerta, hypothalamus, brainstem, and spinal cord, and in subpopulations of sensory neurons and developing peripheral nerves. NTN colocalized with RET and GDNFR-α in ureteric buds of the developing kidney. The circular muscle layer of the developing intestines, smooth muscle of the urether, and developing bronchiolae also expressed NTN. GDNFR-β was found in myenteric but not submucosal intestinal plexuses. In developing salivary glands NTN had an epithelial expression, whereas GDNFR-β was expressed in surrounding tissue. Neurturin and GDNFR-β were present in developing sensory organs. In the gonads, NTN appeared to be expressed in Sertoli cells and in the epithelium of the oviduct, whereas GDNFR-β was expressed by the germ cell line. Our findings suggest multiple roles for NTN and GDNFR-β in the developing and adult organism. Although NTN and GDNFR-β expression patterns are sometimes complementary, this is not always the case, suggesting multiple modi operandi of GDNF and NTN in relation to RET and the two binding proteins, GDNFR-α and GDNFR-β.

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.