Survival Of Mesencephalic Dopaminergic Neurons Research Articles

Human Amniotic Epithelial Cells Alleviate a Mouse Model of Parkinson's Disease Mainly by Neuroprotective, Anti-Oxidative and Anti-Inflammatory Factors.

Human amniotic epithelial cells (hAECs) have been reported to have neuroprotective roles in Parkinson's disease (PD) animal models. However, the molecular mechanism is not fully understood. The present study was designed to explore the possible mechanism by which hAECs ameliorate PD symptoms and the important paracrine factors produced by hAECs that attribute to the recovery of dopaminergic neurons. Thus, we performed in vivo and in vitro experiments with hAECs in PD models or lesioned dopaminergic neurons, respectively. First, hAECs were transplanted into the striatum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice and motor deficits were significantly attenuated. Second, the grafts prevented the loss of nigral dopaminergic neurons and promoted the outgrowth of neurites and striatal axon fibers in PD mice. In addition, decreased microglial activation, inflammatory factor levels and MPTP-induced excessive reactive oxygen species (ROS) levels were also observed in hAEC-treated PD mice. In vitro, we found that the conditioned medium (CM) from hAECs promoted the survival of mesencephalic dopaminergic neurons stimulated with 1-methyl-4-phenylpyridine (MPP+) and induced neurite outgrowth. Next, analysis of hAEC-CM with an antibody array of 507 soluble target proteins revealed that the levels of many neurotrophic factors, growth factors, neuronal cell adhesion molecule (NrCAM) and anti-inflammatory factors were evidently high. In addition, antibody neutralization experiments showed that many of these factors contributed to the survival and growth of dopaminergic neurons and neurite outgrowth. More importantly, we found that the anti-inflammatory factor interleukin-1 receptor antagonist (IL-1ra) also augmented the survival of dopaminergic neurons, demonstrating for the first time an anti-oxidative and anti-inflammatory role of hAECs in PD mice, which represents a novel molecular mechanism of hAECs in the treatment of PD. The molecular mechanism of hAECs recovering lesioned dopaminergic neurons and attenuating PD symptoms. First, hAECs secret manyneurotrophic factors, growth factors, and neuronal cell adhesion molecule (NrCAM) which promote the growth of the damaged dopaminergic neurons andtheir neurites. Second, hAECs produce many anti-inflammatory factors and other factors contributing to reducing the activation of microglia andsuppressing the neuroinflammation. Third, hAECs reduce the excessive ROS levels by upregulating some anti-oxidative signals.

Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease

Current research on Parkinson's disease (PD) pathogenesis requires relevant animal models that mimic the gradual and progressive development of neuronal dysfunction and degeneration that characterizes the disease. Polymorphisms in engrailed 1 (En1), a homeobox transcription factor that is crucial for both the development and survival of mesencephalic dopaminergic neurons, are associated with sporadic PD. This suggests that En1 mutant mice might be a promising candidate PD model. Indeed, a mouse that lacks one En1 allele exhibits decreased mitochondrial complex I activity and progressive midbrain dopamine neuron degeneration in adulthood, both features associated with PD. We aimed to further characterize the disease-like phenotype of these En1+/− mice with a focus on early neurodegenerative changes that can be utilized to score efficacy of future disease modifying studies. We observed early terminal defects in the dopaminergic nigrostriatal pathway in En1+/− mice. Several weeks before a significant loss of dopaminergic neurons in the substantia nigra could be detected, we found that striatal terminals expressing high levels of dopaminergic neuron markers TH, VMAT2, and DAT were dystrophic and swollen. Using transmission electron microscopy, we identified electron dense bodies consistent with abnormal autophagic vacuoles in these terminal swellings. In line with these findings, we detected an up-regulation of the mTOR pathway, concurrent with a downregulation of the autophagic marker LC3B, in ventral midbrain and nigral dopaminergic neurons of the En1+/− mice. This supports the notion that autophagic protein degradation is reduced in the absence of one En1 allele. We imaged the nigrostriatal pathway using the CLARITY technique and observed many fragmented axons in the medial forebrain bundle of the En1+/− mice, consistent with axonal maintenance failure. Using in vivo electrochemistry, we found that nigrostriatal terminals in the dorsal striatum were severely deficient in dopamine release and reuptake. Our findings support a progressive retrograde degeneration of En1+/− nigrostriatal neurons, akin to what is suggested to occur in PD. We suggest that using the En1+/− mice as a model will provide further key insights into PD pathogenesis, and propose that axon terminal integrity and function can be utilized to estimate dopaminergic neuron health and efficacy of experimental PD therapies.

Characterization of the Engrailed mutant mice as experimental models for Parkinson's disease

Engrailed genes are homeodomain-containing transcription factors necessary for the development and maintenance of mesencephalic dopaminergic neurons. Deletion in the Engrailed genes has been shown to affect the survival of mesencephalic dopaminergic neurons both during development and in the adult. Here we describe for the first time a significant reduction in striatal dopamine levels in En1+/−;En2+/− mice compared with their En2+/− littermates, accompanied by a modest reduction in the number of nigral DA neurons. Our results strengthen previous evidence indicating Engrailed genes as survival factors for mature dopaminergic neurons. Furthermore, our data suggest a role for these transcription factors in the maintenance of synaptic dopaminergic neurotransmission in adult neurons.

Pitx3 promoter directs Cre-recombinase specifically in a human neuroblastoma cell line

The Pitx3 gene is a homeobox transcription factor. This gene is expressed only in midbrain dopaminergic-neurons in the central nervous system, where its expression is important for development and survival of mesencephalic-dopaminergic neurons. The promoter region of the Pitx3 gene is not yet completely delimited. We used the Cre-loxP system to demonstrate the efficiency and specificity of a 4.2-kbp sequence in the 5'-flanking region of the Pitx3-gene promoter inserted in the 5'-terminus of the Cre-recombinase gene. A Cre-recombinase-reporter assay indicated that this 5'-flanking region possesses promoter activity. The cell-specific gene regulation of the Pitx3 promoter in neurons was demonstrated by a reverse-transcription polymerase chain reaction (RT-PCR) and Western blot detection of Cre-recombinase in SH-SY5Y cells but not in MCF7 cells. Functionality of the Cre-recombinase was confirmed in vitro. The Pitx3-promoter-Cre cassette here described can be used to develop transgenic mice with the specific expression of Cre-recombinase in midbrain-dopaminergic neurons to elucidate the gene function in which the conventional knockout leads to an early lethal phenotype. Such specific expression of the Pitx3 promoter may be used for gene therapy studies of Parkinson's disease.

Slow progressive degeneration of nigral dopaminergic neurons in postnatal Engrailed mutant mice

The homeobox transcription factors Engrailed-1 and Engrailed-2 are required for the survival of mesencephalic dopaminergic neurons in a cell-autonomous and gene-dose-dependent manner. Because of this requirement, the cells die by apoptosis when all four alleles of the Engrailed genes are genetically ablated (En1-/-;En2-/-). In the present study, we show that viable and fertile mice, heterozygous null for Engrailed-1 and homozygous null for Engrailed-2 (En1+/-;En2-/-), have an adult phenotype that resembles key pathological features of Parkinson's disease. Specifically, postnatal mutant mice exhibit a progressive degeneration of dopaminergic neurons in the substantia nigra during the first 3 mo of their lives, leading to diminished storage and release of dopamine in the caudate putamen, motor deficits similar to akinesia and bradykinesia, and a lower body weight. This genetic model may provide access to the molecular etiology for Parkinson's disease and could assist in the development of novel treatments for this neurodegenerative disorder.

A look at life from the homeodomain

​ThisThis workshop brought together developmental biologists and oncologists for three intense days of discussions about the recent progress that has been made on the roles of homeodomain proteins in development, haematopoiesis and leukaemogenesis. The homeo-domain is a 61-amino-acid DNA-binding motif with specific sequence and structural characteristics (Gehring & Hiromi, 1986). Homeodomain proteins were initially identified in Drosophila as being responsible, when mutated, for homeotic transformations—that is, the conversion of a part of the body into the likeness of another. Since their initial discovery, the number of homeodomain proteins has increased enormously, and they comprise several different families of transcription factors. The main stars of the meeting were members of the Hox family and of the three amino-acid loop extension (TALE) families, with some other homeodomain proteins making guest appearances. TALE proteins include the PBC family (Exd in Drosophila and Pbx proteins in vertebrates) and the Hth/Meis superfamily (Hth in Drosophila, and Meis and Prep proteins in vertebrates). The link between Hox and TALE proteins is their functional interactions, which were initially revealed in flies by genetic analyses and later supported by biochemical data (Mann & Affolter, 1998; Moens & Selleri, 2006). PBC proteins have been shown to act as Hox cofactors that mainly determine Hox DNA-binding specificity and selectivity. Hth/Meis proteins interact with PBC proteins, participating in ternary complexes with Hox proteins, or modulating PBC protein stability and subcellular localization. This European Molecular Biology Organization/European School of Molecular Medicine workshop on Homeodomain Proteins, Hematopoietic Development and Leukemias took place in Riva del Garda, Italy, between 23 and 25 March 2006, and was organized by F. Blasi, ... Functional specificities and redundancies A large part of the meeting was devoted to the presentation and discussion of the consequences of the complete or partial inactivation of different TALE products in several vertebrates. These studies arrived at two main conclusions: first, TALE proteins are essential for the development of most areas of the organism; and second, they have both individual functions and a large degree of redundancy. This was clearly shown in the Pbx gene family, for which an advanced phenotypic analysis of single and compound mutants was presented. In mice, the individual contribution of each member of the family seemed to be different because, whereas the Pbx1 single mutant had a wide range of malformations, only minor or no phenotypes were obvious in Pbx2 and Pbx3 mutants. However, when a reduced Pbx2 and/or Pbx3 dosage was added to the Pbx1−/− background, strong exacerbations of the malformations were detected, as reported by L. Selleri (New York, NY, USA) and M. Cleary (Stanford, CA, USA). In particular, the cardiovascular system, the axial skeleton, the limbs and the craniofacial area were shown to be strongly affected. A.J. Waskiewicz (Edmonton, Canada) also described redundancy among Pbx genes in zebrafish, in which the functions of the Pbx2 and Pbx4 gene products seem to overlap in a range of tissues, including the hindbrain and tectum, and during early haematopoiesis. Hox gene products are known to be largely redundant, especially within paralogous groups. In the case of the Meis and Prep gene families, pleiotropic functions in patterning and differentiation processes were also described (see below); however, genetic analyses designed to evaluate the possible extent of redundancy among the different members of these gene families are still at an early stage. At the meeting, several tissues were reported to require the activity of homeodomain proteins for proper development; we highlight some of these data below.

Substance P, Neurokinins A and B, and Synthetic Tachykinin Peptides Protect Mesencephalic Dopaminergic Neurons in Culture via an Activity-Dependent Mechanism

We evaluated the neuroprotective potential of tachykinin peptides using a model system in which mesencephalic dopaminergic (DA) neurons die spontaneously and selectively as they mature. The three native tachykinins, substance P (SP), neurokinin (NK) A, and NKB afforded substantial protection against DA cell demise. The selective NK1 receptor antagonist [D-Pro9,[spiro-gamma-lactam] Leu10,Trp11]substance P (GR71251) was sufficient in itself to suppress the effect of SP, whereas a cotreatment with GR71251 and the NK3 receptor antagonist (R)-N-[alpha-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (SB218795) was required to prevent the effects of both NKA and NKB. Consistent with these results, D-Ala-[L-Pro9,Me-Leu8]substance P(7-11) (GR73632), a selective agonist of NK1 receptors and [pro7]-NKB, a selective agonist of NK3 receptors, conferred protection to DA neurons, whereas (Lys3, Gly8-R-gamma-lactam-Leu9)neurokinin A(3-10) (GR64349), which activates specifically NK2 receptors, did not. DA neurons rescued by tachykinins accumulated [3H]DA efficiently, which suggests that they were also totally functional. Neuroprotection by tachykinins was highly selective for DA neurons, rapidly reversed upon treatment withdrawal, and reproduced by but independent of glial cell line-derived neurotrophic factor. Survival promotion by tachykinins was abolished by blocking voltage-gated Na+ channels with tetrodotoxin or N-type voltage-gated Ca2+ channels with omega-conotoxin-MVIIA, which indicates that an increase in neuronal excitability was crucially involved in this effect. Together, these data further support the notion that the survival of mesencephalic DA neurons during development depends largely on excitatory inputs, which may be provided in part by tachykinins.

Factors promoting survival of mesencephalic dopaminergic neurons.

Growth factors promoting survival of mesencephalic dopaminergic neurons are discussed in the context of their requirement during development and adulthood. The expression of growth factors should be detectable in the nigrostriatal system during critical periods of development, i.e., during the period of ontogenetic cell death and synaptogenesis and during neurite extension and neurotransmitter synthesis. Growth factors discussed include members of the family of glial-cell-line-derived neurotrophic factors (GDNF), neurotrophins, transforming growth factors beta, and low molecular compounds mimicking growth factor activities. To date, the available data support the notion that GDNF is a highly promising candidate, although GDNF-null mice lack a dopaminergic phenotype. There remains a possibility that endogenous dopaminotrophic factors remain to be discovered.

TGF-β promotes survival on mesencephalic dopaminergic neurons in cooperation with Shh and FGF-8

Impaired neuronal survival is a key event in the development of degenerative diseases, such as Parkinson's disease (PD). Here we show that transforming growth factor beta (TGF-β) acts directly on rat E14 midbrain dopaminergic neurons in vitro, its survival-promoting effect being not mediated by BDNF, NT-3, or GDNF. Treatment with TGF-β, sonic hedgehog (Shh), or fibroblast growth factor-8 (FGF8) significantly increased number of tyrosine hydroxylase (TH)-immunoreactive neurons after 7 days, whereas application of these factors added together further increased number of TH-positive neurons, compared to single-factor treatments. Neutralization of endogenous TGF-β, Shh, or FGF8 significantly reduced number of dopaminergic neurons. TGF-β treatment decreased number of apoptotic cells, having no effect on cell proliferation. Neutralization of TGF-β in vivo during chick E6-10 resulted in reduced number of midbrain dopaminergic neurons. The results suggest that TGF-β is required for survival of mesencephalic dopaminergic neurons acting in cooperation with Shh and FGF8.

Identification and Characterization of TMEFF2, a Novel Survival Factor for Hippocampal and Mesencephalic Neurons

We have identified a novel mammalian gene, TMEFF2, that encodes a putative transmembrane protein containing two follistatin-like domains and one epidermal growth factor (EGF)-like domain. The TMEFF2 gene is predominantly expressed in the brain. In situ hybridization analysis revealed that TMEFF2 is widely expressed in the brain, including hippocampal cornu ammonis, dentate gyrus, and substantia nigra pars compacta. We evaluated the survival effect of TMEFF2 using primary cultured neurons from several regions of fetal rat brain following treatment with a recombinant TMEFF2 protein fragment consisting of the putative extracellular domain. TMEFF2 increased survival of neurons from the hippocampus and midbrain, but not from the cerebral cortex, indicating that the survival effects of TMEFF2 are specific to certain cell types. Recombinant TMEFF2 also promoted survival of mesencephalic dopaminergic neurons. Together, these findings suggest that TMEFF2 may be a novel survival factor for hippocampal and mesencephalic, but not for cortical, neurons.

Neurotrophins prevent death and differentially affect tyrosine hydroxylase of adult rat nigrostriatal neurons in vivo.

Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) promote survival of mesencephalic dopaminergic neurons in vitro and affect normal and damaged ones in vivo. Here, these neurotrophins had markedly different potencies to prevent the death of axotomized nigrostriatal dopaminergic neurons when infused close to the rostral end of the nigral nucleus of adult rats (NT-4 > BDNF > NT-3; nerve growth factor or NGF without effect). With a high dose of BDNF (30 micrograms/day) complete protection was achieved in the rostral but not caudal nigral regions, consistent with its poor diffusion characteristics in brain tissue. Measurements of tyrosine hydroxylase immunoreactivity suggest that BDNF and NT-4 (presumably through their TrkB receptor) reduce the synthesis of this rate-limiting enzyme for dopamine synthesis in rescued as well as in normal neurons. In sharp contrast, survival-promoting doses of NT-3 (presumably through its TrkC receptor) maintained normal levels of tyrosine hydroxylase immunoreactivity in the rescued nigrostriatal neurons. These results suggest that for these adult central nervous system neurons, some neurotrophic factors are predominantly involved in facilitating cell survival, whereas others are more involved in regulating neurotransmitter function.

Glial cell line-derived neurotrophic factor prevents death, but not reductions in tyrosine hydroxylase, of injured nigrostriatal neurons in adult rats

Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mesencephalic dopaminergic neurons in vitro and when injected locally into the brains of lesioned adult animals. Here, we show that GDNF (3 micrograms per day and higher) can promote the survival of all (retrogradely labeled) axotomized nigrostriatal dopaminergic neurons of adult rats when continuously infused for 2 weeks close to the substantia nigra, compared to only approximately 30% survival with control infusions. Based on our previous observations, GDNF was as potent as ciliary neurotrophic factor and neurotrophin-4 and approximately five to ten times more potent than brain-derived neurotrophic factor and was most effective in promoting survival. GDNF prevented neuronal death induced by 6-hydroxydopamine to a lesser extent than after axotomy. GDNF treatments begun 1 week after axotomy could maintain those neurons that had not yet died. When a 2 week GDNF treatment was interrupted, most of the GDNF-rescued neurons died over the following 2 weeks. This suggests that longer trophic factor treatments or nigrostriatal connections are needed to achieve permanent survival. Measurements of tyrosine hydroxylase (TH) immunoreactivity of the rescued neuronal cell bodies suggest that GDNF cannot prevent the lesion-induced loss of this rate-limiting enzyme for dopamine synthesis. In fact, GDNF induced a decrease in TH in normal animals, suggesting an active down-regulation of TH synthesis. Levels of TH immunoreactivity were recovered between 7 and 14 days after withdrawal of a 2 week GDNF infusion, in the neurons that survived axotomy. These results may have implications for developing new treatment strategies for Parkinson's disease.

Protein from chromaffin granules promotes survival of mesencephalic dopaminergic neurons by an EGF-receptor ligand-mediated mechanism

Chromaffin cells grafted to the brain of animals with experimental parkinsonism and patients with Parkinson's disease can restore nigrostriatal functions. Mechanisms underlying these beneficial effects are unknown, but may include growth factors rather than the minute amounts of dopamine (DA) liberated from chromaffin cells. We now report that protein from chromaffin granules, which release their contents by exocytosis, promotes survival and uptake of 3H-DA of mesencephalic DAergic neurons in vitro and protect against N-methylpyridinium ion toxicity. This neurotrophic effect is accompanied by cell proliferation and mediated by astroglial cells induced in these cultures. Inhibition of cell proliferation and concomitant astrogliosis by 5-fluorodeoxyuridine and alpha-aminoadipic acid abolishes the trophic effect. Two highly specific inhibitors of the epidermal growth factor receptor (EGFR) signal transduction pathway, 4,5-dianilinophthalimide (10 microM) and tyrphostin B56 (10 microM), selectively block the neurotrophic capacity of chromaffin granule protein. As expected, they also block the mitogenic effects of EGF and TGF-alpha. However, these two mitogens do not mimic the pronounced mitogenic and trophic actions of chromaffin granule protein. Culture medium conditioned by mesencephalic cells pretreated with chromaffin granule protein promotes survival of DAergic neurons without increasing numbers of astroglial cells. The effective molecule is unlikely to be glial cell line-derived neurotrophic factor, whose mRNA is not detectable in cultures treated with chromaffin granule protein. We conclude that chromaffin granules contain a putatively novel growth factor, which signals through the EGFR and may be responsible for the known protective and restorative actions of chromaffin cell grafts to the lesioned nigrostriatal system.

Protein from chromaffin granules promotes survival of mesencephalic dopaminergic neurons by an EGF‐receptor ligand‐mediated mechanism

Protein from chromaffin granules promotes survival of mesencephalic dopaminergic neurons by an EGF‐receptor ligand‐mediated mechanism

Chronic activation of the cyclic AMP signaling pathway promotes development and long-term survival of mesencephalic dopaminergic neurons.

Dibutyryl cyclic AMP (dbcAMP), a permeant analogue of cyclic AMP (cAMP), prevented, for at least 3 weeks, the death of tyrosine hydroxylase (TH)-immunopositive dopaminergic neurons, which occurred spontaneously by apoptosis in mesencephalic cultures. Treatment with the cyclic nucleotide analogue also led to a significant increase in the uptake of [3H] dopamine, attesting that the rescued TH+ neurons were fully functional and differentiated. dbcAMP was most effective when added immediately after plating, but delayed treatment could still arrest the ongoing degenerative process. Trophic/survival effects were long-lasting, declining only progressively after withdrawal of dbcAMP from the culture medium. They were independent of cell density and still detectable in the absence of serum proteins. The effects of dbcAMP were mimicked by depolarizing concentrations of potassium and by agents that increase endogenous production of cAMP, such as forskolin or 3-isobutyl-1-methylxanthine, but not by native cAMP, which cannot cross cell membranes. Elimination of glial cells by arabinoside-C did not reduce the activity of dbcAMP. GABAergic neurons, also present in these cultures, were much less dependent on the cyclic nucleotide analogue for their survival, and serotoninergic cells were not dependent at all. Therefore, cAMP-dependent signaling may be particularly crucial for the maturation and long-term survival of mesencephalic dopaminergic neurons.

Cyclic AMP, but not basic FGF, increases the in vitro survival of mesencephalic dopaminergic neurons and protects them from MPP(+)-induced degeneration.

We studied how stimulation of protein kinase C and cAMP-dependent protein kinases affect the development of mesencephalic dopaminergic neurons in primary cell cultures derived from fetal rats at embryonic day E14. The effects of compounds which activate these second messenger systems were compared to those of basic fibroblast growth factor (bFGF) and insulin-like growth factor I (IGF-I). In mesencephalic cultures, there was a continuous loss of dopaminergic neurons. Despite this decline in cell number, neurotransmitter uptake per neuron increased with time, indicating that the surviving dopaminergic neurons continued their biochemical differentiation while others degenerated. IGF-I and bFGF did not affect the number of dopaminergic neurons. However, dopamine uptake per neuron was significantly higher in bFGF and IGF-I treated cultures, suggesting that these factors stimulated differentiation. Protein kinase C and cAMP-dependent protein kinases were not involved in mediating the effects of bFGF and IGF-I. Treatment of cultures with phorbol esters did not affect dopamine uptake, whereas elevated levels of intracellular cAMP resulted in an increase in dopamine uptake which was additive to that elicited by bFGF or IGF-I. Further analysis revealed that exposure of mesencephalic cultures to dibutyryl cAMP (dbcAMP) during the first 3 days after plating increased the survival of dopaminergic neurons, whereas prolonged treatment attenuated the development of the dopamine uptake system. Moreover, cyclic AMP, but not bFGF, was able to prevent the degeneration of dopaminergic neurons induced by 1-methyl-4-phenyl-pyridinium ion (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results suggest that increased intracellular levels of cAMP protect dopaminergic neurons in situations of stress like the process of dissociation and plating or the exposure to neurotoxic compounds. Our results reveal novel possibilities for the treatment of Parkinson's disease.