Carolyn Hyman

Brain-derived neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-methyl-4-phenylpyridinium ion toxicity: involvement of the glutathione system.

Brain‐derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopamine neurons in cultures derived from the embryonic rat mesencephalon. We now extend this study by demonstrating that, in addition to the effect of sustaining survival of dopaminergic neurons, BDNF also confers protection against the neurotoxic effects of 6‐hydroxydopamine (6‐OHDA) and N‐methyl‐4‐phenylpyridinium ion (MPP+). Exposure of mesencephalic cultures to either 6‐OHDA or MPP+ resulted in a loss of 70–80% of dopaminergic neurons, as determined by tyrosine hydroxylase (TH) immunocytochemistry. In BDNF‐treated cultures, loss of TH‐positive cells after exposure to either toxin was reduced to only 30%. To facilitate biochemical measurements, we studied SH‐SY5Y dopaminergic neuroblastoma cells. BDNF was found to protect these cells from the dopaminergic neurotoxins, 6‐OHDA and MPP+. Indicative of oxidative stress, treatment of SH‐SY5Y cells with 10 μM 6‐OHDA for 24 h caused a fivefold increase in the levels of oxidized glutathione (GSSG). Pre‐treatment with BDNF for 24 h completely prevented the rise in GSSG. Further examination revealed that BDNF increased the activity of the protective enzyme, glutathione reductase, by 100%. In contrast, BDNF had no effect on the activity of catalase. These results add further impetus to exploring the therapeutic potential of BDNF in animal models of Parkinsons disease.

Efficacy of brain-derived neurotrophic factor and neurotrophin-3 on neurochemical and behavioral deficits associated with partial nigrostriatal dopamine lesions.

Abstract: Brain‐derived neurotrophic factor (BDNF) promotes the survival of dopamine (DA) neurons, enhances expression of DA neuron characteristics, and protects these cells from 6‐hydroxydopamine (6‐OHDA) toxicity in vitro. We tested the ability of BDNF or neurotrophin‐3 (NT‐3) to exert similar protective effects in vivo during chronic delivery of 6‐OHDA to the rat neostriatum. Chronic infusions of BDNF or NT‐3 (12 µg/day) above the substantia nigra were started 6 days before and continued during an 8‐day chronic intrastriatal infusion of 6‐OHDA. In control and neurotrophin‐treated animals, 6‐OHDA treatment selectively depleted 50–60% of nigrostriatal DA nerve terminals but produced little if any loss of pars compacta DA cell bodies. This partial DA lesion resulted in three rotations per minute toward the lesioned hemisphere after treatment with the DA release‐inducing drug d‐amphetamine. Compared with supranigral infusions of vehicle, BDNF and NT‐3 decreased the number of these ipsiversive rotations by 70 and 48% and increased by 20‐ and 10‐fold, respectively, the number of contraversive rotations observed after amphetamine injection. When challenged with the DA receptor agonist apomorphine, BDNF‐ and NT‐3‐treated animals also exhibited a seven‐ and 3.5‐fold increase in the number of contraversive rotations relative to the vehicle group, respectively. Compared with vehicle, BDNF increased striatal levels of homovanillic acid (HVA; 86%), 3,4‐dihydroxyphenylacetic acid (DOPAC; 42%), and 5‐hydroxyindoleacetic acid (5‐HIAA; 32%) and the HVA/DA (43%) and 5‐HIAA/serotonin (34%) ratios in the DA‐denervated striatum. NT‐3 augmented only striatal 5‐HIAA levels (24%). Neither factor altered the 6‐OHDA‐induced decrease in striatal DA levels or high‐affinity DA uptake and thus did not protect against the destruction of DA terminals and did not alter striatal D1 or D2 ligand binding. Choline, GABA, and glutamate uptake in the striatum were not altered by the lesion or neurotrophin treatment. Thus, BDNF and to a lesser extent NT‐3 reverse rotational behavioral deficits and augment striatal DA and 5‐HT metabolism in a partial DA lesion model.

The Neurotrophins NT-4/5 and BDNF Augment Serotonin, Dopamine, and GABAergic Systems during Behaviorally Effective Infusions to the Substantia Nigra

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) have both been identified as ligands for the TrkB receptor, yet differences have emerged in terms of their in vitro potencies for neuronal survival and differentiation. This has prompted the in vivo study of their effects on behavior and neuro-chemical parameters associated with dopamine, serotonin, and GABAergic neurons in the basal ganglia. Two-week supranigral infusions of NT-4/5 and BDNF were similar in their ability to augment levels of the dopamine metabolite homovanilic acid (HVA) (63 and 78%, respectively) and the ratios of dihydroxphenylacetic acid/dopamine (DOPAC/DA) (39, 48%) and HVA/DA (85, 77%) in the caudate-putamen of the hemisphere ipsilateral to the nigral infusion. Striatal concentrations of DOPAC were elevated 45% by BDNF but not by NT-4/5. The 3-MT/dopamine ratio, an indicator of dopamine release, was elevated by 38 and 32% in the striata of BDNF- and NT-4/5-infused rats, respectively. Striatal indoleamine metabolism, determined by the ratio of 5-hydroxyindoleacetic acid (5HIAA)/serotonin was also elevated by NT-4/5 and BDNF in the caudate-putamen (29, 32%), and the 5HIAA content of the substantia nigra was elevated by both factors (43, 40%). The activity of GAD within the superior colliculus was elevated 21 and 41% by BDNF and NT-4/5, respectively. A contraversive rotational bias was induced in BDNF and NT-4/5-treated rats challenged with d-amphetamine, and these responses were blocked by pretreatment with selective D1 or D2 receptor antagonists but not by opiate receptor antagonism. Thus, NT-4/5 and BDNF can elevate the turnover of dopamine through both metabolic and release pools and augment the behavioral response to d-amphetamine. The role for dopamine in this behavioral response is indicated by the requirement of unoccupied D1 and D2 receptors, but may also involve changes in serotonergic, GABAergic, or other pathways. The TrkB receptor-specific actions of BDNF and NT-4/5 may have implications for understanding the etiology or treatment of basal ganglia disorders.

Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon

The neurotrophin brain-derived neurotrophic factor (BDNF) was tested for its ability to promote the survival and regulation of expression of phenotypic markers of dopaminergic, serotonergic, and GABAergic neurons in free-floating roller tube cultures of human fetal ventral mesencephalon. This culture system contains neurons of the anlage of the substantia nigra as well as that of the rostral raphe nucleus. Dopaminergic neuron number and tyrosine hydroxylase (TH) fiber density was monitored by TH immunocytochemistry. Measurement of dopamine (DA) content, TH enzymatic activity, serotonin (5-HT) content, and glutamic acid decarboxylase (GAD) activity were used as indices of their respective neurotransmitter function. The presence of GABAergic and serotonergic neurons in this culture system was confirmed by GABA and 5-HT immunocytochemistry. In cultures maintained in the presence of BDNF (10 ng/ml), the density of TH-positive cells was increased by 2.5-fold (P F 0.05), and the TH-positive fiber density was increased by 3.5-fold (P F 0.01), relative to control cultures. Similarly, the relative increases in DA content and TH activity were 2.6- and 2.3-fold, respectively, in the BDNF-treated cultures (P F 0.01 and P F 0.01). On a per neuron basis, DA content and TH activity were not markedly changed by BDNF treatment, suggesting that the increases in DA content and TH activity are due to more DA neurons surviving. Relative elevations were also observed in serotonin content (2.0-fold, P F 0.01) and GAD enzymatic activity (1.4-fold, P F 0.01). Future studies will need to determine whether these changes result from the direct action of BDNF on these neurons or through some indirect mechanism. The results demonstrate that BDNF has beneficial effects on cultured human fetal tissue, which may be relevant in optimizing neuronal transplantation techniques, and that multiple systems are simultaneously influenced by BDNF.

Comparison of the Effects of the Neurotrophins on the Morphological Structure of Dopaminergic Neurons in Cultures of Rat Substantia Nigra

The effect of the various neurotrophin family members on the morphological structure of dopaminergic neurons was compared in dissociated cultures of embryonic rat ventral mesencephalon. Cultures were maintained in vitro in the presence of brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin‐4/5 (NT‐4/5), nerve growth factor (NGF) or no added growth factors. Three‐dimensional reconstructions of 48 neurons were made in each of the experimental groups following immunocytochemical staining for tyrosine hydroxylase to detect dopaminergic neurons. In addition [3H]mazindol binding analyses were carried out in replicate cultures in order to quantify the effects of the neurotrophins on the number of dopamine uptake sites. Among the neurotrophins tested, NT‐4/5 influenced the proximal morphological parameters most, as determined by a 36% increase in the soma profile area and 35% in the number of stem neurites. Analysis of neuritic size and complexity in these cultures revealed that combined neuritic length and number of segments/cell were increased by 45 and 40% respectively. A change in neurite complexity in the NT‐4/5 treated cultures was further confirmed using Scholls concentric sphere analysis. In addition, relative to the control, NT‐4/5 increased the neuronal differentiation as evidenced by increases in varicosity density and [3H]mazindol binding by 114 and 101% respectively. BDNF and, to a lesser extent, NT‐3 also increased both proximal parameters and parameters of differentiation, but were without effect on parameters of neuritic size and Complexity. No effects on neuronal structure were observed in NGF treated cultures. These findings demonstrate that BDNF, NT‐3 and NT‐4/5 influence the morphological differentiation of dopaminergic neurons in vitro, suggesting they may play a role in the structural development and plasticity of these neurons in the mesencephalon.

Effects of Donor Age and Brain-Derived Neurotrophic Factor on the Survival of Dopaminergic Neurons and Axonal Growth in Postnatal Rat Nigrostriatal Cocultures

Early postnatal rat brain tissue can be grown for several weeks as organotypic slice cultures by the roller-tube method. We have here used this method to study the effects of donor age and brain-derived neurotrophic factor (BDNF) on the survival and growth of tyrosine hydroxylase immunoreactive (TH-i), dopaminergic (DA) neurons during the postnatal period when their nerve fibers normally innervate the striatal target. Tissue slices of ventral mesencephalon (VM) and striatum were prepared from newborn and 7-day-old rats and cocultured for 3--3 1/2 weeks with different combinations of the two donor ages. After immunocytochemical staining the number of TH-i, ventral mesencephalic neurons were counted, and the growth of TH-i fibers into the striatal part of the cocultures was evaluated. Co-cultures, with both VM and striatal slices prepared from newborn rats, contained a significantly higher number of TH-i neurons and displayed a significantly increased innervation of the striatal slices compared with other combinations of donor ages. Addition of BDNF resulted in both an increased survival of TH-i neurons and an increased growth of TH-i fibers into the cocultured striatal slices. Significant neurotrophic effect of BDNF did, however, require young donor age of both VM and striatal slices. It is suggested that BDNF induces more cells, possibly progenitor cells, to express TH immunoreactivity. Alternatively BDNF may suppress apoptotic cell death documented by others to occur in the postnatal rat substantia nigra pars compacta. Irrespective of the mechanisms, survival of more TH-i neurons was related to an increased innervation of the striatal slices by TH-i nerve fibers. The observed effects of BDNF on both survival and fiber growth of TH-i neurons indicate a potential role of BDNF for treatment of Parkinsons disease or grafts of immature DA neurons transplanted to patients with Parkinsons disease. A significant trophic effect of BDNF did, however, seem to depend on young developmental age of both striatum and VM. Parallel treatment with striatal neurotrophic factors may therefore be a necessary prerequisite to a trophic effect of BDNF under clinical conditions.

The neurotrophin family of NGF-related neurotrophic factors

The recent molecular cloning of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) has established the existence of an NGF-related family of neurotrophic factors - the neurotrophins. Purification and recombinant production of BDNF and NT-3 has allowed the initiation or extension of in vitro studies of the neuronal specificity of each of these factors. We have found that NT-3, like NGF and BDNF, promotes survival and neurite outgrowth from certain populations of sensory neurons. There appear to be both distinct and overlapping specificities of the 3 neurotrophins towards peripheral neurons - sympathetic neurons and subpopulations of neural crest and neural placode-derived sensory neurons. Using cultures of central nervous system neurons, we have recently established that BDNF: (i) promotes the survival and phenotypic differentiation of rat septal cholinergic neurons, a property consistent with the discovery of high levels of BDNF mRNA expression within the hippocampus; (ii) promotes the survival of rat nigral dopaminergic neurons and furthermore protects these neurons from two dopaminergic neurotoxins, 6-hydroxydopamine (6-OHDA) and MPTP. Thus the neurotrophic effects of these factors towards peripheral neurons and neuronal populations known to degenerate in two of the major human neurodegenerative diseases - Alzheimers and Parkinsons disease - provokes the question of whether neurotrophic factors may have therapeutic potential in halting the progression and ameliorating the symptoms of devastating neurological disorders of the CNS or PNS, or improving regeneration of neurons of CNS or PNS after traumatic injury.

A continuous striatal infusion of 6-hydroxydopamine produces a terminal axotomy and delayed behavioral effects

Rat models of Parkinsons disease typically employ a rapid nigral injection of 6-hydroxydopamine (6-OHDA) to produce a near-complete loss of nigrostriatal dopamine neurons, and thus, model end stage disease. The present report describes the use of a continuous, low dose infusion of 6-OHDA into the striatum which produces a terminal axotomy of nigrostriatal dopamine neurons and protracted behavioral response. A solution of 6-OHDA in 0.4% ascorbate, delivered at 37 degrees C from osmotic minipumps, was stable for 8 days as determined by its retained toxicity to a dopaminergic neuroblastoma cell line. The continuous infusion of 0.2 mu g 6-OHDA per h did not affect the striatal uptake of [3H]%GABA, [3H]choline, or [3H]glutamate but reduced [3H]dopamine uptake by 55% within 1.5 days after the start of the infusion. The striatal infusion of 6-OHDA produced a dose-dependent reduction of striatal dopamine and DOPAC levels but did not alter HVA, 5-HT, or 5-HIAA. An increase in amphetamine-induced ipsiversive rotations occurred within 1.5 days after the acute striatal injection of 20 mu g or 30 mu g of 6-OHDA but required 4 days to develop with the continuous 6-OHDA infusion. The topography of the lesion mapped by [3H]mazindol binding showed that, beginning by 1.5 days, a diffuse depletion of terminals encompassed much of the striatum in the 30 mu g acute injection group, whereas in the continuously infused rats, the lesion was apparent only by 4 days and was restricted to a smaller and more completely lesioned area. Unlike acutely lesioned animals, continuously infused rats revealed no obvious loss of dopamine neurons in the pars compacta by 5 weeks after 6-OHDA. The continuous striatal infusion of 6-OHDA can produce a topographically limited terminal axotomy of dopamine neurons and a protracted behavioral impairment.

Potential Therapeutic Use of BDNF or NT-4/5 in Parkinson’s and Alzheimer’s Diseases

A growing body of evidence has revealed that the neuropathologies of Parkinson’s and Alzheimer’s diseases reflect numerous system atrophies, rather than singular deficits of ldopaminergic or cholinergic neurons, respectively (see Agid et al, 1987 and Reisberg, 1983 for reviews). Both diseases can eventually result in losses of catecholaminergic, indoleaminergic, cholinergic, and peptidergic neurons but vary in the degree to which these cell groups are affected, particularly at early stages. Research efforts have focused on the identification of common mechanisms contributing to these conditions, as well as determinations of growth factor responsiveness of the affected neuronal populations. The present report summarizes in vitro and in vivo evidence that two members of the neurotrophin family of growth factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5), support the survival or maturation of phenotypic markers in rat neuronal populations that correspond to those severely affected in Parkinson’s and Alzheimer’s diseases. BDNF also promotes the expression of at least several of the neuropeptides that are also decreased in Alzheimer’s disease (Nawa et al, 1993; Croll et al, 1993). The present review, however, will survey the more extensive information currently available for the in vivo and in vitro actions of BDNF or NT-4/5 on monoaminergic, cholinergic, and GABAergic neurotransmitters.