Dopaminergic Neurons In Primary Cultures Research Articles

The dopamine receptor agonist apomorphine stabilizes neurotoxic α-synuclein oligomers.

The misfolding and aggregation of the protein α-synuclein (aSyn) into potentially neurotoxic oligomers is believed to play a pivotal role in the neuropathogenesis of Parkinson's disease (PD). Herein, we explore how apomorphine (Apo), a nonselective dopamine D1 and D2 receptor agonist utilized in the therapy for PD, affects the aggregation and toxicity of aSyn in vitro. Our data indicated that Apo inhibits aSyn fibrillation leading to the formation of large oligomeric species (Apo-aSyn-O), which exhibit remarkable toxicity in mesencephalic dopaminergic neurons in primary cultures. Interestingly, purified Apo-aSyn-O, even at very low concentrations, seems to be capable of converting unmodified aSyn monomer into neurotoxic species. Collectively, our findings warn for a possible dangerous effect of Apo on aSyn misfolding/aggregation pathway.

GDNF to the rescue: GDNF delivery effects on motor neurons and nerves, and muscle re-innervation after peripheral nerve injuries.

Peripheral nerve injuries commonly occur due to trauma, like a traffic accident. Peripheral nerves get severed, causing motor neuron death and potential muscle atrophy. The current golden standard to treat peripheral nerve lesions, especially lesions with large (≥ 3 cm) nerve gaps, is the use of a nerve autograft or reimplantation in cases where nerve root avulsions occur. If not tended early, degeneration of motor neurons and loss of axon regeneration can occur, leading to loss of function. Although surgical procedures exist, patients often do not fully recover, and quality of life deteriorates. Peripheral nerves have limited regeneration, and it is usually mediated by Schwann cells and neurotrophic factors, like glial cell line-derived neurotrophic factor, as seen in Wallerian degeneration. Glial cell line-derived neurotrophic factor is a neurotrophic factor known to promote motor neuron survival and neurite outgrowth. Glial cell line-derived neurotrophic factor is upregulated in different forms of nerve injuries like axotomy, sciatic nerve crush, and compression, thus creating great interest to explore this protein as a potential treatment for peripheral nerve injuries. Exogenous glial cell line-derived neurotrophic factor has shown positive effects in regeneration and functional recovery when applied in experimental models of peripheral nerve injuries. In this review, we discuss the mechanism of repair provided by Schwann cells and upregulation of glial cell line-derived neurotrophic factor, the latest findings on the effects of glial cell line-derived neurotrophic factor in different types of peripheral nerve injuries, delivery systems, and complementary treatments (electrical muscle stimulation and exercise). Understanding and overcoming the challenges of proper timing and glial cell line-derived neurotrophic factor delivery is paramount to creating novel treatments to tend to peripheral nerve injuries to improve patients’ quality of life.

LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD.

Quinacrine and Niclosamide Promote Neurite Growth in Midbrain Dopaminergic Neurons Through the Canonical BMP-Smad Pathway and Protect Against Neurotoxin and α-Synuclein-Induced Neurodegeneration.

Parkinson's disease is a neurodegenerative disorder characterised by nigrostriatal dopaminergic degeneration, and intracellular α-synuclein aggregation. Current pharmacological treatments are solely symptomatic so there is a need to identify agents that can slow or stop dopaminergic degeneration. One proposed class of therapeutics are neurotrophic factors which promote the survival of nigrostriatal dopaminergic neurons. However, neurotrophic factors need to be delivered directly to the brain. An alternative approach may be to identify pharmacological agents which can reach the brain to stimulate neurotrophic factor expression and/or their signalling pathways in dopaminergic neurons. BMP2 is a neurotrophic factor that is expressed in the human substantia nigra; exogenous BMP2 administration protects against dopaminergic degeneration in in vitro models of PD. In this study, we investigated the neurotrophic potential of two FDA-approved drugs, quinacrine and niclosamide, that are modulators of BMP2 signalling. We report that quinacrine and niclosamide, like BMP2, significantly increased neurite length, as a readout of neurotrophic action, in SH-SY5Y cells and dopaminergic neurons in primary cultures of rat ventral mesencephalon. We also show that these effects of quinacrine and niclosamide require the activation of BMP-Smad signalling. Finally, we demonstrate that quinacrine and niclosamide are neuroprotective against degeneration induced by the neurotoxins, MPP+ and 6-OHDA, and by viral-mediated overexpression of α-synuclein in vitro. Collectively, this study identifies two drugs, that are safe for use in patients' to 'are approved for human use, that exert neurotrophic effects on dopaminergic neurons through modulation of BMP-Smad signalling. This rationalises the further study of drugs that target the BMP-Smad pathway as potential neuroprotective pharmacotherapy for Parkinson's disease.

In Vitro Protective Action of Monomeric and Fibrillar α-Synuclein on Neuronal Cells Exposed to the Dopaminergic Toxins Salsolinol and DOPAL

The aggregation of α-synuclein (aSyn) is believed to be mechanistically linked to the degeneration of dopamine (DA)-producing neurons in Parkinson's disease (PD). In this respect, one crucial question that yet remains unsolved is whether aSyn aggregation is associated with either a gain- or loss-of-function of the protein in neuronal cells. Herein, we investigated the effect of monomeric versus fibrillar aSyn on mesencephalic dopaminergic neurons in primary cultures challenged with the neurotoxic catechols: salsolinol (SALSO; 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) and 3,4-dihydroxyphenylacetaldehyde (DOPAL). aSyn monomer protected cells against either SALSO- or DOPAL-induced toxicity via inhibition of caspase-3-mediated apoptosis. While fibrillar aSyn failed in attenuating SALSO neurotoxicity, it increased the viability of DOPAL-treated cells, which was apparently not associated with the inhibition of caspase-3 cleavage. The fact that DOPAL-derived aSyn adducts exhibit lower toxicity compared with DOPAL itself raises the question of whether the generation of these adducts could be part of or a collateral effect of aSyn-mediated protection in neurons exposed to DOPAL. Overall, our work provides important evidence on the impact of the fibrillation of aSyn on its protective role in neuronal cells exposed to the toxic catechols SALSO and DOPAL.

Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures

Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure–affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [3H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5μM (THCA) to 14,711μM (CBDV), whereas Ki values to CB2 range from 8.5μM (THCA) to 574.2μM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10μM, whereas CBC and THCA also displayed slightly positive activities. These findings are neither linked to structural characteristics nor to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Is Selectively Toxic to Primary Dopaminergic Neurons In Vitro

Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and nondopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pretreatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.

Gypenosides protects dopaminergic neurons in primary culture against MPP +-induced oxidative injury

Oxidative injury has been implicated in the etiology of Parkinson's disease (PD). Gypenosides (GPs), the saponins extract derived from the Gynostemma pentaphyllum, has various bioactivities. In this study, GPs was investigated for its neuroprotective effects on the 1-methyl-4-phenylpyridinium ion (MPP +)-induced oxidative injury of dopaminergic neurons in primary nigral culture. It was found that GPs pretreatment, cotreatment or posttreatment significantly and dose-dependently attenuated MPP +-induced oxidative damage, reduction of dopamine uptake, loss of tyrosine hydrolase (TH)-immunopositive neurons and degeneration of TH-immunopositive neurites. However, the preventive effect of GPs was more potential than its therapeutical effect. Most importantly, the neuroprotective effect of GPs may be attributed to GPs-induced strengthened antioxidation as manifested by significantly increased glutathione content and enhanced activity of glutathione peroxidase, catalyze and superoxide dismutase in nigral culture. The neuroprotective effects of GPs are specific for dopaminergic neurons and it may have therapeutic potential in the treatment of PD.

Chemicals Possessing a Neurotrophin-Like Activity on Dopaminergic Neurons in Primary Culture

BackgroundNeurotrophic factors have been shown to possess strong neuroprotective and neurorestaurative properties in Parkinson's disease patients. However the issues to control their delivery into the interest areas of the brain and their surgical administration linked to their unability to cross the blood brain barrier are many drawbacks responsible of undesirable side effects limiting their clinical use. A strategy implying the use of neurotrophic small molecules could provide an interesting alternative avoiding neurotrophin administration and side effects. In an attempt to develop drugs mimicking neurotrophic factors, we have designed and synthesized low molecular weight molecules that exhibit neuroprotective and neuritogenic potential for dopaminergic neurons.Principal FindingsA cell-based screening of an in-house quinoline-derived compound collection led to the characterization of compounds exhibiting both activities in the nanomolar range on mesencephalic dopaminergic neurons in spontaneous or 1-methyl-4-phenylpyridinium (MPP+)-induced neurodegeneration. This study provides evidence that rescued neurons possess a functional dopamine transporter and underlines the involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in these processes.ConclusionCell-based screening led to the discovery of a potent neurotrophic compound possessing expected physico-chemical properties for blood brain barrier penetration as a serious candidate for therapeutic use in Parkinson disease.

Disruption of dopamine homeostasis underlies selective neurodegeneration mediated by α‐synuclein

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.

Activation of c-Jun N-Terminal Protein Kinase Is a Common Mechanism Underlying Paraquat- and Rotenone-Induced Dopaminergic Cell Apoptosis

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the substantia nigra of the brain. Although the underlying causes are not well characterized, epidemiological studies suggest an elevated risk of PD with occupational pesticide exposure. Here, we utilized pheochromocytoma (PC) 12 and SH-SY5Y cells as well as rat primary cultured dopaminergic neurons to investigate mechanisms for dopaminergic cell death induced by paraquat and rotenone, pesticides that are used to model PD in rodents. Both paraquat and rotenone induce selective loss of dopaminergic neurons in primary cultures. We discovered that paraquat induces apoptosis in PC12 cells but not in SH-SY5Y cells, while rotenone exposure causes apoptosis in SH-SY5Y cells but not in PC12 cells. The selective ability of paraquat and rotenone to induce apoptosis in different cell lines correlates with their ability to activate c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinases. Furthermore, JNK and p38 are required for rotenone-induced apoptosis in SH-SY5Y cells (K. Newhouse et al., 2004, Toxicol. Sci. 79, 137-146) as well as primary neurons, and for paraquat-induced apoptosis in PC12 cells. However, JNK but not p38 plays a role in paraquat-induced loss of primary cultured dopaminergic neurons. Our data identify JNK activation as a common mechanism underlying dopaminergic cell death induced by both paraquat and rotenone in model cell lines and primary cultures.

Microarray Analyses Support a Role for Nurr1 in Resistance to Oxidative Stress and Neuronal Differentiation in Neural Stem Cells

Nurr1 is an orphan nuclear receptor required for the development of midbrain dopaminergic neurons. To better understand the molecular consequences of Nurr1 expression, we compared the transcriptomes of two independent control and Nurr1-expressing NSC lines using Affymetrix cDNA microarrays. These data reveal the regulation of genes involved in promoting cell survival (trophic/growth factors and stress response genes) and in preventing cell death (decreased caspase-3 and caspase-11 expression). We found that conditioned medium from Nurr1-expressing NSC lines enhanced the survival of midbrain dopaminergic neurons in primary cultures and that Nurr1-expressing NSC lines themselves were more resistant to oxidative stress. These findings are accompanied by a dynamic pattern of gene regulation that is consistent with a role for Nurr1 in promoting both the acquisition of brain-region-specific identity (Engrailed-1) and neuronal differentiation (tubulin beta III). Interestingly, our gene expression profiles suggested that tenascin-C was regulated by Nurr1 in developing dopaminergic neurons. This was further confirmed in vitro and in Nurr1 knockout mice where low levels of tenascin-C mRNA were observed. Analysis of tenascin-C-null mice revealed an increase in the number of Nurr1(+) cells that become tyrosine hydroxylase-positive (TH(+)) dopaminergic neurons at embryonic day 11.5, suggesting that tenascin-C normally delays the acquisition of TH by Nurr1(+) precursors. Thus, our results confirm the presence of both secreted and cell-intrinsic survival signals modulated by Nurr1 and suggest that Nurr1 is a key regulator of both survival and dopaminergic differentiation.

Synergistic effect of α-dihydroergocryptine and L-dopa or dopamine on dopaminergic neurons in primary culture

There is an ongoing controversy about potential toxicity of L-3,4-dihydroxyphenylalanine (L-dopa) to dopaminergic neurons in Parkinson's disease (PD). Neuroimaging data suggest that L-dopa accelerates the loss of dopamine nerve terminals, especially at higher doses. The disputed aspect of toxicity and the frequently observed motor complications accompanying L-dopa therapy have led to an increased use of dopamine agonists during the past two decades. Reports describing their neuroprotective potential to dopaminergic neurons have attracted much attention. Here, we describe the novel finding that the combination of a dopamine (DA) agonist, alpha-dihydroergocryptine (DHEC), with L-dopa or DA exerts a synergistic stimulatory effect on dopaminergic neurons in primary culture, while each substance alone had no or less effect. DA receptor stimulation plays a decisive role. The synergistic effect suggests that a combinatory therapy can be beneficial to slow the degeneration of dopaminergic neurons.

Pergolide protects dopaminergic neurons in primary culture under stress conditions.

Dopamine agonists are an important therapeutic strategy in the treatment of Parkinson's disease. They postpone the necessity for and reduce the required dose of L-3,4-dihydroxyphenylalanine (L-DOPA) medication thus protecting against the development of motor complications and potential oxidative stress due to L-DOPA metabolism. In primary cultures from mouse mesencephalon we show that pergolide, a preferential D(2) agonist enhanced the survival of healthy dopaminergic neurons at low concentrations of 0.001 microM. About 100 fold higher concentrations (0.1 microM) were necessary to partially reverse the toxic effects of 10 microM 1-methyl-4-phenylpyridinium (MPP(+)). Pergolide was equally effective in preventing the reduction of dopamine uptake induced by 200 microM L-DOPA. Furthermore, between 0.001-0.1 microM it also reduced lactate production thus promoting aerobic metabolism. The present findings suggest that pergolide protects dopaminergic neurons under conditions of elevated oxidative stress.

Protection of dopaminergic neurons in primary culture by lisuride.

Dopamine agonists play an important role in the treatment of Parkinson's disease by reducing the administration of L-3,4-dihydroxyphenylalanine (L-DOPA). The enzymatic and non-enzymatic conversion of L-DOPA is suspected to increase oxidative stress, which leads to the degeneration of dopaminergic neurons in Parkinson's disease. In primary mouse mesencephalic cultures we show that the dopamine D1/D2 receptor agonist lisuride, in a concentration range of 0.001-1 microM, enhances the survival of dopaminergic neurons, protects against toxicity induced by L-DOPA or 1-methyl-4-phenylpyridinium ion (MPP+) and stimulates 3H-dopamine uptake. Lisuride also reduces anaerobic metabolism during incubation with L-DOPA. The present findings suggest that lisuride may have trophic/survival-promoting properties and potentially reduces oxidative stress.

Decelerated rate of dendrite outgrowth from dopaminergic neurons in primary cultures from brains of hypoxanthine phosphoribosyltransferase-deficient knockout mice

Lesch–Nyhan syndrome (LNS), caused by the complete deficiency of hypoxanthine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is still unclear. Evidence has accumulated indicating that it reflects dopamine deficiency associated with defective arborization of dopaminergic dendrites. We monitored the differentiation in vitro of dopaminergic neurons, cultured from HPRT-deficient knockout mice. The HPRT-deficient dopaminergic neurons exhibited a decelerated rate of outgrowth of dendrites in comparison to that of control neurons resulting, after 8 days in culture, in 32% smaller average total length of dendrites per neuron ( P<0.025). The results suggest that the abnormal dendrite outgrowth in LNS reflects a defective developmental process.

Modulation of glutamate neurotoxicity on mesencephalic dopaminergic neurons in primary cultures by the presence of striatal target cells

Glutamate toxicity was compared in substantia nigra (SN)/striatum (STR) and SN/cerebellum (CRB) co-cultures on both the entire neuronal population (neuron specific enolase (NSE) immunopositive cells) and dopaminergic neurons (tyrosine hydroxylase (TH) immunopositive cells). In SN/CRB co-cultures NSE- and TH-positive cells were more sensitive to glutamate-induced toxicity than in SN/STR co-cultures. Moreover, in SN/STR co-cultures as compared to SN/CRB and SN cultures, glutamate toxicity was prevented to a larger extent by TCP, a non-competitive NMDA antagonist. These results suggest that target cells induce a differential expression of the different glutamate receptor subtypes in mesencephalic dopaminergic cells. Alternatively, the presence of target cells may induce the selective development of a subpopulation of dopaminergic neurons expressing predominantly NMDA receptors.

Membranes from pituitary intermediate lobe cells enhance differentiation of fetal hypothalamic dopaminergic neurons in primary culture

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.

Brain-derived and glial cell line-derived neurotrophic factors protect a catecholaminergic cell line from dopamine-induced cell death

Brain-derived neurotrophic factor (BDNF) promotes the survival of dopaminergic neurons in primary cultures and protects these neurons from the neurotoxic effects of 6-hydroxydopamine. The protective mechanism of BDNF on neurotoxicity was evaluated using CATH.a cells, a clonal catecholaminergic cell line derived from the central nervous system. Dopamine produced a dose-dependent cell death in CATH.a cells. Treatment of CATH.a cells with BDNF or glia cell line-derived neurotrophic factor (GDNF) reduced dopamine-induced cell death by approximately 60–70%. Nerve growth factor, basic fibroblast growth factor, neurotrophin-4/5 and insulin had no protective effect on dopamine-induced cell death. Dopamine decreased the activity of superoxide dismutase and the levels of glutathione in the CATH.a cells and these decreases were reversed by BDNF. In addition, BDNF treatment alone increased superoxide dismutase activity by 108%. These results suggest that BDNF may safeguard CATH.a cells from dopamine-induced cell death by maintaining or enhancing components of the cell, which protect from oxidative stress.

Standardized methods to bioassay neurotrophic factors for dopaminergic neurons

The search for specific neurotrophic factors that will eventually be used to reduce or arrest the rate of degeneration of dopaminergic neurons in Parkinson's disease is being pursued by first testing the ability of putative compounds to increase the survival of dopaminergic neurons in primary cultures of the fetal, ventral mesencephalon. This research has intensified in recent years. The experimental procedures used by different laboratories in these studies differ widely, and meaningful comparisons of the results obtained are accordingly difficult to make. Some important experimental variables include the age of the fetal tissue used; the dissection technique used to isolate the ventral mesencephalon; the percentage of dopaminrgic neurons present in the culture initially; handling of the tissue during dissection; the technique used to disperse the cells; the use of serum; the technique of plating the cells; the attachment factors used; detachment and loss of cells during the staining procedure; the age of the cultures at the time of analysis; the uneven distribution of cells at the time of analysis and the use of imaging techniques in the analysis. We show that when the E14 rat embryo is used, it is possible to consistently obtain a culture with 20% of tyrosine hydroxylase-positive neurons. Neither the plating density in the range of 7.8 × 10 3 to 1.25 × 10 5 cells/cm 2, nor the percentage of serum in the growth medium affected the percentage of cells that expressed TH initially, at 4 or 12 h after plating. When the cells were plated as 25 μl droplets, called microislands (area ≈ 12.5 mm 2), and allowed to attach before additional growth medium was added, cell density remained uniform at the center of the microisland for the duration of the culture. Restriction of the analysis of cell survival to the center of the microisland therefore helped to decrease the variability in counting that could occur when cells are dispersed over a larger area. In contrast, in an 8-well chamber slide or 35 mm petri dish, in which the whole area is plated, cell density was consistently higher at the edge (edge effect), versus the centre, by a factor of about three. The use of microisland cultures also has the additional benefit of increasing by a factor of about five the number of individual cultures that can be set up per litter, and a proportionate reduction in the number of animals used per experiment. When the percentage of serum in the growth medium was 0% always, or 10% for the first 12 h, and 0% thereafter, or 10% always, the number of TH-pos neurons per field (using a × 20 objective, column factor 1.25; area 320 μm 2) after 5 days in culture (DIV5) was < 1, 3–8 and 14–22, respectively. Under the same experimental conditions, the number of neurons (MAP2-positive) per field was 5–8, 18–30 and 45–65 ( N = 10 in all cases), respectively. Serum deprivation therefore has a highly deleterious effect on neuronal survival in culture. We suggest that cultures that were exposed to serum at any stage of the experiment, should not be referred to as ‘serum-free’, since even a brief exposure to serum exerts a protective effect on neurons, and especially on dopaminergic neurons. Instead, the percentage and kind of serum used, the exact usage, and the duration of exposure of the cells to serum should be stated. Finally, it is suggested that where possible, an imaging system with manual count and journaling capabilities be used in the analysis. The methods described are illustrated by dose-response curves of the neurotrophic effects of BDNF, NGF-β and IL-6 versus percentage survival on dopaminergic neurons, when grown in serum-free medium throughout.