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9-Methyl-beta-carboline up-regulates the appearance of differentiated dopaminergic neurones in primary mesencephalic culture

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Abstract

beta-Carbolines (BCs) derive from tryptophan and its derivatives. They are formed endogenously in humans and mammals and occur inter alia in cooked meat and tobacco smoke. They have been detected in human brain, cerebrospinal fluid, and plasma. Up to now they were predominantly identified as compounds exhibiting neurotoxic actions. Since significantly higher amounts are present in parkinsonian patients, they are regarded as potential pathogenetic factors in Parkinson's disease. We identified for the first time a BC (9-methyl-BC; 9-me-BC) exerting neuroprotective and neuron-differentiating effects. Treatment of primary mesencephalic dopaminergic cultures with 9-me-BC inhibited the basal release of lactate dehydrogenase and reduced the number of cells stained with propidium iodide. Caspase-3 activity was decreased, the total protein content was unchanged and ATP content was increased. Furthermore, the expression of inflammation-related genes was reduced. The number of differentiated dopaminergic neurones was significantly increased and a wide array of neurotrophic/transcription factors (Shh, Wnt1, Wnt5a, En1, En2, Nurr1, Pitx3) and marker genes (Th, Dat, Aldh1a1) decisive for dopaminergic differentiation was stimulated. Consistently, the dopamine content was slightly, although non-significantly, increased and the dopamine uptake capacity was elevated. An anti-proliferative effect was observed in human neuroblastoma SH-SY5Y cells which is consistent with a reduced incorporation of bromodesoxyuridine into the DNA of primary mesencephalic cells. Whether the additional dopaminergic neurones in primary culture derive from dopaminergic precursor cells, previously tyrosine hydroxylase negative dopaminergic neurones or are the result of a transdifferentiation process remains to be established.

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... βCs occur ubiquitously through synthetic mechanisms and are vastly found in medicinal plants across global continents (Hamann et al., 2008;Marwat and ur Rehman, 2011;Wernicke et al., 2007). Harmaline was first isolated from the seed of Peganum harmala (Zygophillaceae, Syrian rue) in 1841 in the middle East (Sarkar and Bhadra, 2014;Wang et al., 2015;Zeng et al., 2010) and since then βC derivatives have been found present in several medicinal plants (Hamid et al., 2017) including Eurycoma spp. ...
... Interaction with imidazoline receptor, antagonism of benzodiazepine receptors, and inhibition of MAO-A. unsubstituted THβC and fully aromatised βC analogues easily cross Blood-Brain Barrier (BBB) (Hamann et al., 2008) and interact with receptors relevant to neurotoxicities such as the imidazoline and GABA A receptors, thereby enhancing the activation of an irreversible opening of mitochondrial pores and then irregular transportation of peptides and ions such as Ca 2+ resulting in cell homeostasis and apoptosis. This pathophysiological property is inclusively induced by β-carbolinium from cyclization of tryptamine, 1-trichloromethyl-1,2,3,4-tetrahydro-β-Carboline (TaClo), a structural analogue of 1-methyl-4-pyridinium (MPP + ) and β-amyloid which promote neurodegeneration and neurotoxicity leading to AD and PD (Bachurin et al., 2003;Seidel and Pfau, 2017;Wiart, 2014). ...
... βCs and some other natural amines similar to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) such as the isoquinolines also act as serotonin-and tryptamine-derived and synthetic toxins (Nagatsu, 2002;Samita et al., 2016;Schott et al., 2006). The mutagenic implications of some derivatives of βCs on eukaryotic and prokaryotic cells at a significantly high amount in the brain, cerebrospinal fluid, and human plasma impart the pathogenesis of addiction, risk of carcinoma and liver cirrhosis especially in alcoholic patients, although, the underlying aetiology is not fully understood yet (Greube and Rommelspacher, 2003;Hamann et al., 2008;Jaqueline et al., 2006;Pfau and Skog, 2004;Wernicke et al., 2007). Antagonism of GABA-A receptor by βC esters reportedly induces seizure and some derivatives like methyl-β-carboline-3-carboxylate, methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) also evoke convulsant and anxiogenic effects at low and high doses respectively in animal models (Chapouthier and Venault, 2001;Harden and Pennell, 2013;Kulick et al., 2014). ...
Article
Neuropsychiatric disorders are diseases of the central nervous system (CNS) which are characterised by complex pathomechanisms that including homeostatic failure, malfunction, atrophy, pathology remodelling and reactivity anomaly of the neuronal system where treatment options remain challenging. β-Carboline (βC) alkaloids are scaffolds of structurally diverse tricyclic pyrido[3,4-b]indole alkaloid with vast occurrence in nature. Their unique structural features which favour interactions with enzymes and protein receptor targets account for their potent neuropharmacological properties. However, our current understanding of their biological mechanisms for these beneficial effects, especially for neuropsychiatric disorders is sparse. Therefore, we present a comprehensive review of the scientific progress in the last two decades on the prospective pharmacology and physiology of the βC alkaloids in the treatment of some neuropsychiatric conditions such as depression, anxiety, Alzheimer’s disease, Parkinson’s disease, brain tumour, essential tremor, epilepsy and seizure, licking behaviour, dystonia, agnosia, spasm, positive ingestive response as demonstrated in non-clinical models. The current evidence supports that βC alkaloids offer potential therapeutic agents against most of these disorders and amenable for further drug design.
... These results were confirmed by confocal microscopy analysis using a live/dead fluorescent test (Figure 2b). In the previous research, Hamman et al. [26] demonstrated that synthetic methylated derivative of norharman (i.e., 9-methyl-β-carboline (9-me-BC)), discovered as a neuroprotective compound, reduced the number of necrotic cells in the primary mesencephalic dopaminergic cell culture derived from embryonic mice as a model with relevance to Parkinson's disease, which corresponds to our results. Moreover, 9-me-BC was identified as an agent capable of increasing the number of dopaminergic neurons in a concentration-dependent manner while maintaining the functionality of these neurons [26,27]. ...
... In the previous research, Hamman et al. [26] demonstrated that synthetic methylated derivative of norharman (i.e., 9-methyl-β-carboline (9-me-BC)), discovered as a neuroprotective compound, reduced the number of necrotic cells in the primary mesencephalic dopaminergic cell culture derived from embryonic mice as a model with relevance to Parkinson's disease, which corresponds to our results. Moreover, 9-me-BC was identified as an agent capable of increasing the number of dopaminergic neurons in a concentration-dependent manner while maintaining the functionality of these neurons [26,27]. On the other hand, dimethyl β-carboline derivatives (2,9-dimethyl-BC) exert highly toxic effects not only on dopaminergic neurons but also on other cell constituents in primary dopaminergic culture [28]. ...
... CASP3 is an effector factor responsible for the proteolytic cascade leading to cell death [39]. A similar effect in a decrease of caspase-3 activity was observed for the primary mesencephalic dopaminergic cell culture derived from embryonic mice after treatment with 9-methyl-β-carboline (a methylated derivative of norharmane) [26]. It can be assumed that the obtained result is a protective effect of β-carbolines. ...
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Some studies have ascribed a protective effect against neurodegenerative diseases to the β-carbolines harman (H) and norharman (NH), which occur mostly in coffee and coffee substitutes. We determined the concentrations of β-carbolines and undesirable compounds (such as acrylamide) in roasted coffee substitute ingredients and found that chicory coffee was optimal. Two in vivo experiments were conducted with seventeen-month-old male Sprague Dawley rats fed a diet with the addition of pure carboline standards in the first stage, and chicory in the second. We observed an increase in the level of H and NH in blood plasma, as well as higher activity of animals in the battery behavioral test, particularly in the second stage. The results of in vitro studies—particularly the level of the expression in brain tissue of genes associated with aging processes and neurodegenerative diseases—clearly show the benefits of a diet rich in β-carbolines.
... Recently, an essential and unexpected role of LRRK2 in the regulation of protein homeostasis during aging was shown in mice implying that LRRK2 mutations cause PD and cell death via impairment of protein degradation pathways, leading to α-synuclein accumulation and aggregation over time (Tong et al. 2010). When the dopamine transporter (DAT), by which 9-me-BC is completely taken up into dopaminergic neurons (Hamann et al. 2008), was blocked, the stimulatory effects on the number of dopaminergic neurons were abolished but a further increase of the neurite outgrowth was observed (Polanski et al. 2010). This discrepancy led to the perception that the effect on neurite outgrowth is independent of the uptake of 9-me-BC into dopaminergic neurons but perhaps contingent on uptake into astrocytes by an organic cation transporter (OCT). ...
... Synthesis of 9-me-BC was performed as described by (Hamann et al. 2008). ...
... Dopaminergic cell cultures were prepared as described earlier (Polanski et al. 2010). Half of the basic medium (composition see (Hamann et al. 2008)) was changed on the first day in vitro (DIV) and on the 3rd DIV, two-thirds of the basic medium were changed. On the 5th DIV, half of the basic medium was replaced with serum-free Dulbecco's modified Eagle's medium (DMEM) containing 2% B-27. ...
Article
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β-Carbolines (BC) are pyridoindoles, which can be found in various exogenous and endogenous sources. Recent studies revealed neurostimulative, neuroprotective, neuroregenerative and anti-inflammatory effects of 9-methyl-BC (9-Me-BC). Additionally, 9-me-BC increased neurite outgrowth of dopaminergic neurons independent of dopamine uptake into these neurons. In this study, the role of astrocytes in neurostimulative, neuroregenerative and neuroprotective properties of 9-me-BC was further explored. 9-Me-BC exerted anti-proliferative effects without toxic properties in dopaminergic midbrain and cortical astrocyte cultures. The organic cation transporter (OCT) but not the dopamine transporter seem to mediate at least part the effect of 9-me-BC on astrocytes. Remarkably, 9-me-BC stimulated the gene expression of several important neurotrophic factors for dopaminergic neurons like Artn, Bdnf, Egln1, Tgfb2 and Ncam1. These factors are well known to stimulate neurite outgrowth and to show neuroprotective and neuroregenerative properties to dopaminergic neurons against various toxins. Further, we show that effect of 9-me-BC is mediated through phosphatidylinositol 3-kinase (PI3K) pathway. Additionally, 9-me-BC showed inhibitory properties to monoamine oxidase (MAO) activity with an IC50 value of 1 µM for MAO-A and of 15.5 µM for MAO-B. The inhibition of MAO by 9-me-BC might contribute to the observed increased dopamine content and anti-apoptotic properties in cell culture after 9-me-BC treatment in recent studies. Thus, 9-me-BC have a plethora of beneficial effects on dopaminergic neurons warranting its exploration as a new multimodal anti-parkinsonian medication.
... For instance, the endogenously formed 2-methyl-b-carbolinum and 2,9-dimethylb-carbolinum ions are suspected to be involved in the pathogenesis of PD (Hamann et al. 2006;Lorenc-Koci et al. 2006). In contrast, an unexpected stimulatory effect of 9methyl-b-carboline (9-me-BC) on dopaminergic neurons was recently discovered in cultures of primary mesencephalic neurons (Hamann et al. 2008;Polanski et al. 2010). This finding has now directed the focus of research on the stimulatory and protective action of 9-me-BC on the nigrostriatal dopaminergic system and its potential use as anti-Parkinson drug (see review by Polanski et al. 2011). ...
... The ethics committee of the State of Thuringia (02-038/08) approved all experimental protocols which were intended to minimize animal suffering and the quantity of experimental animals. 9-me-BC treatment 9-me-BC was synthesized as described by Hamann et al. (2008). At the age of 7 weeks, rats were randomly assigned to one of the following experimental groups: non-injected animals (ni): remained without any pharmacological treatment until used in the experiments; vehicle-treated animals (veh): treated intraperitoneally (i.p.) once daily with 1 mL saline (Merck, Darmstadt, Germany)/100 g body weight (b.w.) for 10 days; animals treated with 9-me-BC for 5 days (9-me-BC-5d): treated i.p. with 2 lmol 9-me-BC/100 g b.w. ...
... Moreover, these findings translate results gained from in vitro approaches to the in vivo system and thereby expand our knowledge of 9-me-BC actions in the intact brain in several directions. Our study revealed direct evidence that 9-me-BC treatment does not only stimulate cultured DA neurons in vitro (Hamann et al. 2008;Polanski et al. 2010Polanski et al. , 2011 but also DA neurons within the intact meso-cortico-limbic pathway in vivo. A recent study in an animal model of PD described that in vivo 9-me-BC is not only able to stimulate dopaminergic cells in drug-naive animals (as shown in the present study), but also in 1-methyl-4-phenyl-pyridinium ion (MPP + )-pre-treated rats and thereby rescuing the PD-like phenotype indicating the neuroprotective properties of 9-me-BC (Wernicke et al. 2010). ...
Article
J. Neurochem. (2012) 121, 924–931. β-Carbolines (BCs) belong to the heterogenous family of carbolines, which have been found exogenously, that is, in various fruits, meats, tobacco smoke, alcohol and coffee, but also endogenously, that is, blood, brain and CSF. These exogenous and endogenous BCs and some of their metabolites can exert neurotoxic effects, however, an unexpected stimulatory effect of 9-methyl-β-carboline (9-me-BC) on dopaminergic neurons in primary mesencephalic cultures was recently discovered. The aim of the present study was to extend our knowledge on the stimulatory effects of 9-me-BC and to test the hypothesis that 9-me-BC may act as a cognitive enhancer. We found that 10 days (but not 5 days) of pharmacological treatment with 9-me-BC (i) improves spatial learning in the radial maze, (ii) elevates dopamine levels in the hippocampal formation, and (iii) results after 10 days of treatment in elongated, more complex dendritic trees and higher spine numbers on granule neurons in the dentate gyrus of 9-me-BC-treated rats. Our results demonstrate that beyond its neuroprotective/neurorestorative and anti-inflammatory effects, 9-me-BC acts as a cognitive enhancer in a hippocampus-dependent task, and that the behavioral effects may be associated with a stimulatory impact on hippocampal dopamine levels and dendritic and synaptic proliferation.
... Recently, exceptional properties of 9-me-BC have been shown, proposing the substance as a potential new candidate for the treatment of PD. 9-Me-BC exerted neurostimulatory, neuroprotective, neuroregenerative and anti-inflammatory effects in primary dopaminergic culture [22,61]. While toxic effects of 9-methylated -carbolines were reported in former in vivo studies with mice [62], recent research has confirmed the neuroprotective properties of 9-me-BC in a rat model [63]. ...
... Stimulation, protection & regeneration of dopaminergic neurons by 9-methyl--carboline 9-methyl--carboline is taken up by the DAT into dopaminergic neurons and reaches maximum uptake kinetics after approximately 3 h [61]. After treatment of primary dopaminergic cell cultures with 9-me-BC for 48 h, an increased number of tyrosine hydroxylase immunoreactive (THir) neurons were observed [22]. ...
... Dopaminergic neurotrophic/morphogenic factors, such as sonic hedgehog (Shh) [71], wingless-type mouse mammary tumor virus (MMTV) integration site family, member 1 (Wnt1) [72] and member 5a (Wnt5a) [73], as well as the dopaminergic transcription factors engrailed homeobox 1 (En1) [74], nuclear receptor related 1 (Nurr1) [75] and paired-like homeodomain transcription factor 3 (Pitx3) [76], and the dopaminergic markers aldehyde dehydrogenase 1a1 (Aldh1), DAT and TH, have also been shown to be upregulated by 9-me-BC [61]. Importantly, recent studies demonstrated that Nurr1 is required not only during the development process of dopaminergic neurons, but is also essential for the maintenance of maturing and adult dopaminergic neurons [77]. ...
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β-carbolines are potential endogenous and exogenous neurotoxins that may contribute to the pathogenesis of Parkinson's disease (PD). 9-methyl-β-carboline exhibits multimodal effects that could be beneficial in the treatment of PD. It shows stimulatory effects to dopaminergic neurons by increasing the expression of tyrosine hydroxylase and its transcription factors in pre-existing dopa decarboxylase immunoreactive neurons. Furthermore, 9-methyl-β-carboline has emerged as a substance with the rare property of a protective and regenerative/restorative potential for dopaminergic neurons by inducing gene expression of several neurotrophic factors and decreasing apoptotic cell signals. It reduces protein levels of α-synuclein and inhibits monoamine oxidase A and B. Finally, 9-methyl-β-carboline acts on multiple targets in the inflammatory cascade by inhibiting the proliferation of microglia, by decreasing chemotactic cytokines and by creating an anti-inflammatory environment in the CNS. This article summarizes our current knowledge of 9-methyl-carboline and discusses its potential role as a new drug for the treatment of PD.
... Recently, 9-methyl-b-carboline (9-me-BC) was identified as a BC with unexpected stimulatory effects on dopaminergic neurons (Hamann et al. 2008). It was transported via the DAT and increased the number of tyrosine hydroxylase (TH) positive cells by approximately 20%, whereas the total cell number remained unchanged. ...
... Consistently, dopamine uptake was increased by 20% as well. 9-Me-BC also exerted protective effects in primary dopaminergic cultures (Hamann et al. 2008), as the rate of necrosis and apoptosis, especially the activity of caspase 3 in the cultures were reduced. Furthermore, 9-me-BC also showed anti-proliferative effects as reflected by a decreased incorporation of BrdU in the culture. ...
... In addition, it stimulated the gene expression of the important dopaminergic neurotrophic factors sonic hedgehog (Shh), wingless-type mouse mammary tumor virus (MMTV) integration site family, member 1 and member 5a of the dopaminergic transcription factors engrailed homeobox 1, nuclear receptor related 1 (Nurr1) and paired-like homeodomain transcription factor 3 (Pitx3), and finally of the dopaminergic markers aldehyde dehydrogenase1a1, dopamine transporter (DAT) and tyrosine hydroxylase (Th). Importantly, 9-me-BC also reduced the expression of inflammatory modulators such as chemokine (C-X-C motif) ligand 9 (Cxcl9), tumor necrosis factor (TNF), Fas ligand and interferon regulatory factor 1 (Hamann et al. 2008). ...
Article
J. Neurochem. (2010) 113, 1659–1675. β-Carbolines (BCs) are potential endogenous and exogenous neurotoxins that may contribute to the pathogenesis of Parkinson’s disease. However, we recently demonstrated protective and stimulatory effects of 9-methyl-BC (9-me-BC) in primary dopaminergic culture. In the present study, treatment with 9-me-BC unmasked a unique tetrad of effects. First, tyrosine hydroxylase (TH) expression was stimulated in pre-existing dopa decarboxylase immunoreactive neurons and several TH-relevant transcription factors (Gata2, Gata3, Creb1, Crebbp) were up-regulated. Neurite outgrowth of TH immunoreactive (THir) neurons was likewise stimulated. The interaction with tyrosine kinases (protein kinase A and C, epidermal growth factor-receptor, fibroblast growth factor-receptor and neural cell adhesion molecule) turned out to be decisive for these observed effects. Second, 9-me-BC protected in acute toxicity models THir neurons against lipopolysaccharide and 2,9-dime-BC+ toxicity. Third, in a chronic toxicity model when cells were treated with 9-me-BC after chronic rotenone administration, a pronounced regeneration of THir neurons was observed. Fourth, 9-me-BC inhibited the proliferation of microglia induced by toxin treatment and installed an anti-inflammatory environment by decreasing the expression of inflammatory cytokines and receptors. Finally, 9-me-BC lowered the content of α-synuclein protein in the cultures. The presented results warrant the exploration of 9-me-BC as a novel potential anti-parkinsonian medication, as 9-me-BC interferes with several known pathogenic factors in Parkinson’s disease as outlined above. Further investigations are currently under way.
... Thus, we rationalized that by modifying substituents, BCs with neuroprotective actions might be detected. We synthesized a large number of BCs and screened them for neuroprotective properties using HEK-293 cells transfected with the human DA transporter (hDAT) [87] and primary dopaminergic cultures of the midbrain from embryonic mice, as models with relevance to PD [22]. We found evidence that 9-methyl-b-carboline (9-me-BC) is a compound with neuroprotective properties. ...
... Quantitative real-time RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons and of several genes involved in neuronal differentiation. In contrast, genes engaged in inflammation and apoptosis were downregulated [22]. These results indicate that 9-me-BC is neuroprotective. ...
... Materials 9-Methyl-b-carboline . HCl was synthesized by Yvonne Schott, Institute of Pharmacy (head: Prof. J. Lehmann), Friedrich-Schiller University, Jena, Germany, as described previously [22]; MPP + was obtained from Sigma Aldrich, Taufkirchen, Germany. The following compounds were obtained as indicated in parentheses: protease inhibitor cocktail (P8340, Sigma, Taufkirchen, Germany), mouse monoclonal anti-TH antibody (Chemicon Int., Temecula, CA, USA), ABC-peroxidase kit (Vector Laboratories, Burlingame, CA, USA). ...
Article
In a previous study, a primary culture of midbrain cells was exposed to 9-methyl-beta-carboline for 48 h, which caused an increase in the number of tyrosine hydroxylase-positive cells. Quantitative RT-PCR revealed increased transcription of genes participating in the maturation of dopaminergic neurons. These in vitro findings prompted us to investigate the restorative actions of 9-methyl-beta-carboline in vivo. The compound was delivered for 14 days into the left cerebral ventricle of rats pretreated with the neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP+) for 28 days applying a dose which lowered dopamine by approximately 50%. Interestingly, 9-methyl-beta-carboline reversed the dopamine-lowering effect of the neurotoxin in the left striatum. Stereological counts of tyrosine hydroxylase-immunoreactive cells in the substantia nigra revealed that the neurotoxin caused a decrease in the number of those cells. However, when treated subsequently with 9-methyl-beta-carboline, the number reached normal values. In search of an explanation for the restorative activity, we analyzed the complexes that compose the respiratory chain in striatal mitochondria by 2-dimension gel electrophoresis followed by MALDI-TOF peptide mass fingerprinting.We found no changes in the overall composition of the complexes. However, the activity of complex I was increased by approximately 80% in mitochondria from rats treated with MPP+ and 9-methyl-beta-carboline compared to MPP+ and saline and to sham-operated rats, as determined by measurements of nicotinamide adenine dinucleotide dehydrogenase activity. Microarray technology and single RT-PCR revealed the induction of neurotrophins: brain-derived neurotrophic factor, conserved dopamine neurotrophic factor, cerebellin 1 precursor protein, and ciliary neurotrophic factor. Selected western blots yielded consistent results. The findings demonstrate restorative effects of 9-methyl-beta-carboline in an animal model of Parkinson's disease that improve the effectiveness of the respiratory chain and promote the transcription and expression of neurotrophin-related genes.
... Furthermore, it was observed that introducing certain moieties such as hydrazine (compound VIII), amide linkage, thiazole ring (compound IX), or indole scaffold to AD drugs, increased their activity through their choline esterase inhibitory activity, anti-Abaggregation properties, or anti-neuroinflammatory character [22][23][24][25][26][27] . ...
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A novel series of indole-based compounds was designed, synthesised, and evaluated as anti-Alzheimer’s and anti-neuroinflammatory agents. The designed compounds were in vitro evaluated for their AChE and BuChE inhibitory activities. The obtained results revealed that compound 3c had higher selectivity for AChE than BuChE, while, 4a, 4b, and 4d showed selectivity for BuChE over AChE. Compounds 5b, 6b, 7c, and 10b exerted dual AChE/BuChE inhibitory activities at nanomolar range. Compounds 5b and 6b had the ability to inhibit the self-induced Aβ amyloid aggregation. Different anti-inflammatory mediators (NO, COX-2, IL-1β, and TNF-α) were assessed for compounds 5b and 6b. Cytotoxic effect of 5b and 6b against human neuroblastoma (SH-SY5Y) and normal hepatic (THLE2) cell lines was screened in vitro. Molecular docking study inside rhAChE and hBuChE active sites, drug-likeness, and ADMET prediction were performed.
... 2-Me-βC + and 2,9-DiMe-βC + In the brain, norharman is converted by SAM to 2-Me-βC + and subsequently to 2,9-diMe-βC + [83]. The content of 2,9-diMe-βC + is 0.1 pmol/g tissue in the parietal association cortex and 0.77 pmol/g tissue in the substantia nigra [84]. 2,9-DiMe-βC + mediated cell death by apoptosis and necrosis and it is more toxic than 2-Me-βC + [85]. ...
Article
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Endogenous and exogenous neurotoxins are important factors leading to neurodegener-ative diseases. In the 1980s, the discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) contributes to Parkinson's disease (PD) symptoms led to new research investigations on neurotoxins. An abnormal metabolism of endogenous substances, such as condensation of bioam-ines with endogenous aldehydes, dopamine (DA) oxidation, and kynurenine pathway, can produce endogenous neurotoxins. Neurotoxins may damage the nervous system by inhibiting mitochondrial activity, increasing oxidative stress, increasing neuroinflammation, and up-regulating proteins related to cell death. This paper reviews the biological synthesis of various known endogenous neu-rotoxins and their toxic mechanisms.
... These compounds are good substrates for the dopamine transporter and thus may affect dopamine neurons. Although many β-carbolines are toxic for these neurons, one of these compounds, 9-methyl-βcarboline (Fig. 6), on the contrary, can promote survival and possibly stimulate neurite outgrowth from cultured dopamine neurons at 25-100 µM concentrations [182,183]. It was assumed that additional TH-immunoreactive neurons originated exclusively from non-proliferative, preexisting dopa decarboxylase-immunoreactive neurons [183]. ...
Article
Background: Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide, the lifetime risk of developing this disease is 1.5%.Motor diagnostic symptoms of PD are caused by degeneration of nigrostriatal dopamine neurons. There is no cure for PD and current therapy is limited to supportive care that partially alleviates disease signs and symptoms. As diagnostic symptoms of PD result from progressive degeneration of dopamine neurons, drugs restoring these neurons may significantly improve treatment of PD. Method: A literature search was performed using the PubMed, Web of Science and Scopus databases to discuss the progress achieved in development of neuroregenerative agents for PD. Papers published before early 2018 were taken into account. Results: Here, we review several groups of potential agents capable of protecting and restoring dopamine neurons in cultures or animal models of PD including neurotrophic factors and small molecular weight compounds. Conclusion: Despite the promising results of in vitro and in vivo experiments, none of the found agents have yet shown conclusive neurorestorative properties in PD patients. Meanwhile, a few promising biologicals and small molecules have been identified. Their further clinical development can eventually give rise to disease-modifying drugs for PD. Thus, intensive research in the field is justified.
... Dif--carboline studies found 3 false negative citations in 2 publications (Hamann et al, 2006;2008). All three were 9-methyl-beta-carboline xHCl, which belongs to the -carboline compound class, yet is not a neurotoxic, but a neuroprotective member of this class (Hamann et al, 2008). Hence, primary mesencephalon cells from mouse brain do not have any 'real' false negatives in the endpoint categories 'mitochondrial dysfunction/oxidative stress/apoptosis'. ...
... with a sample of over 1,400 people (Paterniti et al. 2002). However, a recent case-control study looking into the correlation between vascular dementia risk and chronic BZD use did not find an association between the two after controlling for prodromal BZD use (Hamann et al. 2008). ...
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Glutamatergic and cholinergic dysfunction are well‐attested features of Alzheimer's disease (AD), progressing with other pathological indices of the disorder and exacerbating neuronal and network dysfunction. However, relatively little attention has been paid to the inhibitory component of the excitatory/inhibitory (E/I) network, particularly dysfunction in the gamma‐aminobutyric acid (GABA) signaling system. There is growing evidence in support of GABAergic remodeling in the AD brain, potentially beginning in early stages of disease pathogenesis, and this could thus be a valid molecular target for drug development and pharmacological therapies. Several GABAergic drugs have been tested for efficacy in attenuating or reversing various features and symptoms of AD, and this could represent a novel path by which we might address the growing need for more effective and benign therapies. This article is protected by copyright. All rights reserved.
... Some β-carbolines like 2,9-dimethylβ-carbolines have exhibited mitochondrial damage leading to neurotoxicity, while 9-methyl-harmine has a neuroprotective effect. β-Carboline compounds are also known to reduce the expression of phosphorylated forms of the socalled tau protein, potently at multiple Alzheimer's diseaserelated sites [17][18][19][20]. ...
Article
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β-Carbolines (βCs) belong to the naturally occurring alkaloid family, derived from 9H-pyrido[3,4-b]indole, also known as norharmane (Hnor). Knowing the importance of the βCs alkaloid family in biological processes, a comprehensive binding study is reported of four Ag(I) compounds containing the ligand Hnor and having different counteranions, namely, NO3 −, ClO4 −, BF4 −, and PF6 −, with human serum albumin (HSA) as a model protein. Different approaches like UV-visible, fluorescence spectroscopy, circular dichroism (CD), and molecular docking studies have been used for this purpose. )efluorescence results establish that the phenomenon of binding of Ag(Hnor) complexes to HSA can be deduced from the static quenching mechanism. )e results showed a significant binding propensity of the used Ag(I) compounds towards HSA. )e role of the counteranion on the binding of Ag(I) compounds to HSA appeared to be remarkable. Compounds with (ClO4 −) and (NO3 −) were found to have the most efficient binding towards HSA as compared to BF4 −and PF6 −. Circular dichroism (CD) studies made clear that conformational changes in the secondary structure of HSA were induced by the presence of Ag(I) compounds. Also, the α-helical structure of HSA was found to get transformed into a β-sheeted structure. Interestingly, (ClO4 −) and (NO3 −) compounds were found to induce most substantial changes in the secondary structure of HSA. )e outcome of this study may contribute to understanding the propensity of proteins involved in neurological diseases (such as Alzheimer’s and Parkinson’s diseases) to undergo a similar transition in the presence of Ag-β-carboline compounds.
... (2) Normelinonine F and some bromo-derivatives (7-bromo-2-methyl-9H-pyrido [3,4-b]indole and 7-bromo-1,2-dimethyl-9H-pyrido [3,4-b]indole) isolated from the solitary ascidian Cnemidocarpa irene were found to be inhibitors of acetylcholinesterase (AchE), with activities similar to those of galantamine (an AChE inhibitor clinically used for the treatment of mild to moderate Alzheimer's disease and various other memory impairments, in particular those of vascular origin) (17). (3) 9-Methyl-norharmane (9-Me-nHo) could exert neuroprotective and neuron-differentiating effects (29). (4) bCs have shown to be a promising group of antimicrobial drugs (30)(31)(32)(33)(34)(35)(36)(37)(38)(39). ...
Article
In the present work, we have synthesized and fully characterized the photophysical and photochemical properties of a selected group of N-methyl-β-carboline derivatives (9-methyl-β-carbolines and iodine salts of 2-methyl- and 2,9-dimethyl-β-carbolinium) in aqueous solutions, in the pH-range 4.0 - 14.5. Moreover, despite the quite extensive studies reported in the literature regarding the overall photophysical behaviour of N-unsubstituted βCs, this work constitutes the first full and unambiguous characterization of anionic species of N-unsubstituted βCs (norharmane, harmane and harmine), present in aqueous solution under highly alkaline conditions (pH > 13.0). Acid dissociation constants (Ka ), thermal stabilities, room temperature UV-visible absorption and fluorescence emission and excitation spectra, fluorescence quantum yields (ФF ) and fluorescence lifetimes (τF ), as well as quantum yields of singlet oxygen production (ФΔ ) have been measured for all the studied compounds. Furthermore, for the first time to our knowledge, chemometric techniques (MCR-ALS and PARAFAC) were applied on these systems, providing relevant information about the equilibria and species involved. The impact of all the foregoing observations on the biological role, as well as, the potential biotechnological applications of these compounds is discussed. This article is protected by copyright. All rights reserved.
... As 2,9-dime-BC+ has been found elevated in the lumbar CSF idiopathic PD subjects and increased with the progression of the disease [214] bioactivated BC+s, might represent causative factors underlying PD. Interestingly, a different but closely related BC, 9-methyl--carboline (9me-BC), has been described to be beneficial in cellular and animal models of PD by providing a plethora of potentially valuable actions [211,215]. It has been reported to provide stimulatory effects to dopaminergic neurons by increasing the expression of TH and multiple of its transcription factors, by reducing inflammation and by lowering -synuclein levels. ...
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... As 2,9-dime-BC+ has been found elevated in the lumbar CSF idiopathic PD subjects and increased with the progression of the disease [214] bioactivated BC+s, might represent causative factors underlying PD. Interestingly, a different but closely related BC, 9-methyl--carboline (9me-BC), has been described to be beneficial in cellular and animal models of PD by providing a plethora of potentially valuable actions [211,215]. It has been reported to provide stimulatory effects to dopaminergic neurons by increasing the expression of TH and multiple of its transcription factors, by reducing inflammation and by lowering -synuclein levels. ...
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Parkinson's disease (PD) is a common chronic progressive neurodegenerative disorder in elderly people. A consistent neurochemical abnormality in PD is degeneration of dopaminergic neurons in substantia nigra pars compacta, leading to a reduction of striatal dopamine (DA) levels. As tyrosine hydroxylase (TH) catalyses the formation of L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of DA, the disease can be considered as a TH-deficiency syndrome of the striatum. Problems related to PD usually build up when vesicular storage of DA is altered by the presence of either α-synuclein protofibrils or oxidative stress. Phosphorylation of three physiologically-regulated specific sites of N-terminal domain of TH is vital in regulating its kinetic and protein interaction. The concept of physiological significance of TH isoforms is another interesting aspect to be explored further for a comprehensive understanding of its role in PD. Thus, a logical and efficient strategy for PD treatment is based on correcting or bypassing the enzyme deficiency by the treatment with L-DOPA, DA agonists, inhibitors of DA metabolism or brain grafts with cells expressing a high level of TH. Neurotrophic factors are also attracting the attention of neuroscientists because they provide the essential neuroprotective and neurorestorative properties to the nigrostriatal DA system. PPAR-γ, a key regulator of immune responses, is likewise a promising target for the treatment of PD, which can be achieved by the use of agonists with the potential to impact the expression of pro- and anti-inflammatory cytokines at the transcriptional level in immune cells via expression of TH. Herein, we review the primary biochemical and pathological features of PD, and describe both classical and developing approaches aimed to ameliorate disease symptoms and its progression.
... In this issue, Gruss et al. (2012) present an elegant and consistent set of in vivo experiments providing substantial evidence for 9-methyl-b-carboline as a potential cognitive enhancer. The starting point was the study of Hamann et al. (2008) showing up-regulation of differentiated dopaminergic neurones appearance in primary mesencephalic culture as well as protective and stimulatory effects of 9-methyl-bcarboline in primary dopaminergic culture reported by Polanski et al. (2010). Gruss et al. (2012) for the first time have demonstrated beneficial effects of 9-methyl-b-carboline on cognitive function in a hippocampus-dependent task, on hippocampal dopamine synthesis, and on synaptic and dendritic growth, thus showing its effects on behavioral, cellular/synaptic and neurotransmitter levels. ...
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β-Carboline sind heterozyklische Indolalkaloide, die ubiquitär in unserer Umwelt und Nahrung vorkommen, aber auch endogen aus Tryptophan gebildet werden können. Aufgrund der strukturellen Verwandtschaft bestimmter β-Carboline zu dem dopaminergen Neurotoxin MPP+ wird ein möglicher Beitrag zur Pathogenese der Parkinson-Krankheit diskutiert. MPP+ ist seit langem für seine selektive Toxizität gegenüber dopaminergen Neuronen und das Auslösen von Parkinsonsymptomen bekannt. Insbesondere 2,9-DiMe-BC wurde in erhöhter Konzentration in der lumbalen cerebrospinalen Flüssigkeit von Parkinsonpatienten detektiert, jedoch nicht in Kontrollprobanden. Eine Inhibierung von Komplex I der mitochondrialen Atmungskette und eine selektive Toxizität auf DA Neurone konnten nachgewiesen werden. Die genauen Mechanismen des Zelltodes bleiben jedoch ungeklärt. Im Rahmen dieser Arbeit wurden die Mechanismen des Zelltodes, ausgelöst durch 2,9-DiMe-BC, in dopaminergen Primärzellkulturen des Mesencephalons von C57Bl/6-Mäusen untersucht. Drei weitere BC 2-Me-BC, 9-Me-BC und 1,9-DiMe-BC standen für Untersuchungen zur Verfügung. In ersten Experimenten wies 9-Me-BC und 1,9-DiMe-BC keine Toxizität gegenüber DA Neuronen auf. Aufgrund der höheren Toxizität von 2,9-DiMe-BC verglichen mit 2-Me-BC wurden nachfolgende Experimente mit dem zweifach methylierten BC durchgeführt. Durch die Behandlung mit 2,9-DiMe-BC konnte ein höherer Verlust der DA Neurone gegenüber anderen neuralen Zellen festgestellt werden. Eine selektive Aufnahme über den Dopamintransporter und damit verbundene Schädigung der DA Neurone, wie bei MPP+, konnte nicht nachgewiesen werden. Für 2,9-DiMe-BC wurde eine LC50 der DA Neurone von 14,1 µM und für MPP+ von 4,4 µM bestimmt. 2,9-DiMe-BC verursachte in der Gesamtkultur eine erhöhte Entstehung von reaktiven Sauerstoffspezies und eine gesteigerte Laktatproduktion. In diesem Zusammenhang kann eine Hemmung von Komplex I der Atmungskette vermutet werden. Des Weiteren konnte eine Verringerung des mitochondrialen Membranpotentials und des ATP-Gehaltes gemessen werden. Eine Aktivierung des apoptotischen Zelltodes wurde mit einer erhöhten Aktivität von Caspase-3 nachgewiesen. Durch die Behandlung mit 2,9-DiMe-BC wurde in der Primärzellkultur jedoch auch in erhöhtem Maß Nekrose ausgelöst. Dabei wurde eine höhere Sensitivität von jüngeren Kulturen (8. DIV) gegenüber älteren (10. DIV) festgestellt. Genexpressionsanalysen konnten das Auslösen von oxidativem Stress und Apoptose durch 2,9-DiMe-BC bestätigen, da mehrere Gene dieser Prozesse hochreguliert wurden. Des Weiteren wurden Gene reguliert, die im Zusammenhang mit der Hitzeschock-Antwort, Entzündungsprozessen, DNA-Schädigung und Reparatur, Zellalterung und Proliferation stehen. Zusammenfassend lässt sich sagen, dass 2,9-DiMe-BC die Mitochondrienaktivität hemmt, sowohl nekrotische als auch apoptotische Prozesse in der dopaminergen mesencephalen Primärzellkultur auslöst und die Entstehung von oxidativem Stress eine zentrale Rolle spielt. Der zweite Teil dieser Arbeit beschäftigte sich mit der Untersuchung von unerwarteten neuroprotektiven Effekten von 9-Me-BC in der Primärzellkultur. Durch die Behandlung mit 9-Me-BC verringerte sich die LDH-Freisetzung und reduzierte sich die Anzahl der nekrotischen Zellen um 50 %. Nach 24 h konnte eine verminderte Caspase 3-Aktivität gemessen werden, die allerdings nach 48 h im Vergleich zur Kontrolle wieder zunahm. Hier wären längerfristige Untersuchungen zur Klärung dieser Frage anzuschließen. Des Weiteren erhöhte sich der intrazelluläre ATP-Gehalt. Möglicherweise fand eine energieabhängige Verschiebung von Nekrose zu Apoptose statt. Genexpressionsanalysen zeigten, dass verschiedene Gene von inflammatorischen und apoptotischen Signaltransduktionswegen herrunterreguliert wurden. Überraschenderweise erhöhte sich nach der Behandlung mit 9-Me-BC die Anzahl DA Neurone konzentrationsabhängig um bis zu 20 %. Diese Beobachtung ist neu und wurde über noch kein anderes BC berichtet. Der Effekt wurde durch die Inhibierung des DAT aufgehoben und lässt eine DAT-abhängige Aufnahme von 9-Me-BC vermuten. Die signifikante Erhöhung der Anzahl beschränkte sich nur auf DA Neurone, während sich der Gesamtanteil der Neurone nur geringfügig erhöhte und die übrigen Zellen unbeeinflusst blieben. Zusätzlich wurden ein erhöhter intrazellulärer DA-Gehalt und eine gesteigerte Aufnahme von [3H]DA um 20 % nachgewiesen. Die [3H]DA-Aufnahme und morphologische Untersuchungen zeigten funktionale und reife DA Neurone, es wurde aber auch die Theorie der Neuentstehung durch mögliche Differenzierungsprozesse untersucht. Interessanterweise wurde die Genexpression von einem breiten Spektrum neurotropher Faktoren (Shh, Wnt1, Wnt5a) und Transkriptionsfaktoren (En1, Nurr1, Pitx3), die für die Differenzierung und Entwicklung DA Neurone entscheidend sind, durch die Behandlung mit 9-Me-BC hochreguliert. Zusätzlich erhöhte sich die Expression der DA Markergene Aldh1a1, Dat und Th. Dabei war die Hochregulierung der Genexpression bei allen Faktoren bis auf Shh und Wnt1 von der Anwesenheit des BC abhängig. Ein weiterer Aspekt, der auf eine Differenzierung hindeuten könnte, war die verringerte Anzahl mitotischer BrdU-positiver Zellen. Das Erscheinen DA Neurone könnte also auf Differenzierung und Entwicklung von undifferenzierten Zellen oder Vorläuferzellen beruhen. Jedoch wäre auch eine Induktion der TH von vorher TH-negativen Zellen denkbar. Eine weitere Erklärung könnte das Vorherrschen eines dynamischen Gleichgewichts von Absterben und Neuentstehung DA Neurone innerhalb der Primärzellkultur sein und der Absterbeprozess durch protektive Effekte von 9-Me-BC unterbunden wurde. Zukünftige Experimente sollten zu einer weiteren Aufklärung, der diesem Phänomen zu Grunde liegendenen Mechanismen beitragen. Auch durch die Behandlung mit dem Harman 1,9-DiMe-BC erhöhte sich die Anzahl der DA Neurone konzentrationsabhängig, jedoch erwies sich im Vergleich zu 9-Me-BC nur eine Konzentration von 50 µM als signifikant. Innerhalb dieser Arbeit wurden auf Genexpressionsebene mit Hilfe von Microarrays und qRT-PCR mögliche neuroprotektive Effekte des Dopaminagonisten Lisurid im gleichen Zellkulturmodell untersucht. Lisurid gehört zur Substanzklasse der Ergotalkaloide und wird zur Behandlung der Parkinson-Krankheit eingesetzt. Bei Voruntersuchungen in der DA mesencephalen Primärzellkultur wies Lisurid eine protektive Wirkung für DA Neurone gegen Glutamattoxizität auf. Durch qRT-PCR konnten nur 50 % der ausgewählten Gene der Microarraydaten validiert werden. Nach 24 h Behandlung mit Lisurid wurde die Genexpression von dem Transportprotein Transthyretin (Ttr) hochreguliert, dessen erhöhte Biosynthese und Sekretion interessanterweise mit einer Verminderung der Aggregation des Amyloid-β-Proteins assoziiert wird. Die Genexpression der Aldoketoreduktase 1c20 (Ark1c20) wurde um 50 % herrunterreguliert. Die Bedeutung dieses Ergebnisses bedarf weiterer Abklärung, da eine gewebspezifische Expression bisher nur für die Leber gefunden wurde. Das Thyroidhormonrezeptorbindende Protein 3 (Thrap3), die Mitogen aktivierte Kinase Kinase Kinase 12 (Map3k12) und der G-Protein gekoppelte Rezeptor 27 (Gpr27) waren in ihrer Genexpression hochreguliert. Ein Einfluss von Lisurid auf Signaltransduktionswege konnte somit nachgewiesen werden. Des Weiteren wurde durch Lisurid die Expression der Transkriptionsfaktoren NeuroD1 und Tcf3 hochreguliert, die in Differenzierungsprozesse involviert sind. NeuroD1 gilt dabei als proneurales Gen und ist somit möglicherweise an Vorgängen der Neuroprotektion beteiligt. Keines der validierten differentiell exprimierten Gene des 24 h Experimentes war nach einem Behandlungszeitraum von 6 h reguliert. Die Änderungen der Genexpression nach Preinkubation mit Lisurid und anschließender Glutamatbehandlung und Behandlung mit Glutamat allein überschnitten sich weitestgehend. Demnach war vor allem die Glutamatbehandlung für die differentielle Genexpression verantwortlich. Eine zusätzliche Neusynthese von radikalfangenden Proteinen durch Preinkubation mit Lisurid konnte auf Genebene nicht gefunden werden. Es ist jedoch nicht auszuschließen, da eine Regulation auf post-transkriptioneller Ebene möglich ist.
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Alzheimer's disease (AD) is a prevalent neurodegenerative disorder with multifactorial causes that requires multitargeted treatment. Inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) improve cholinergic signaling in the central nervous system and thus AChE inhibitors are well established in the therapy of AD to improve memory disturbances and other cognitive symptoms. On the other hand, AD patients benefit from reduction of pathologic glutamate-induced, Ca(2+)-mediated excitotoxicity by the N-methyl-d-aspartate receptor (NR) antagonist memantine. New drugs that simultaneously affect both cholinergic transmission and glutamate-induced excitotoxicity may further improve AD treatment. While connecting beta-carboline units by alkylene spacers in two different series of compounds and subsequent evaluation of their AChE/BChE-inhibitory potential, we found that several of these bivalent beta-carbolines were potent NR blockers. The most promising compound was a N(9)-homobivalent beta-carboline with a nonylene spacer, which displayed IC(50) values of 0.5 nM for AChE, 5.7 nM for BChE, and 1.4 microM for NR, respectively.
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Dopamine cells are generated in the ventral midbrain during embryonic development. The progressive degeneration of these cells in patients with Parkinson's disease, and the potential therapeutic benefit by transplantation of in vitrogenerated dopamine cells, has triggered intense interest in understanding the process whereby these cells develop. Nurr1 is an orphan nuclear receptor essential for the development of midbrain dopaminergic neurons. However, the mechanism by which Nurr1 promotes dopamine cell differentiation has remained unknown. In this study we have used a dopamine-synthesizing cell line (MN9D) with immature characteristics to analyze the function of Nurr1 in dopamine cell development. The results demonstrate that Nurr1 can induce cell cycle arrest and a highly differentiated cell morphology in these cells. These two functions were both mediated through a DNA binding-dependent mechanism that did not require Nurr1 interaction with the heterodimerization partner retinoid X receptor. However, retinoids can promote the differentiation of MN9D cells independently of Nurr1. Importantly, the closely related orphan receptors NGFI-B and Nor1 were also able to induce cell cycle arrest and differentiation. Thus, the growth inhibitory activities of the NGFI-B/Nurr1/Nor1 orphan receptors, along with their widespread expression patterns both during development and in the adult, suggest a more general role in control of cell proliferation in the developing embryo and in adult tissues.
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Previous [3H]thymidine studies in Nisslstained sections in rats established that the substantia nigra pars compacta and the ventral tegmental area originate sequentially according to an anterolateral to posteromedial neurogenetic gradient. We investigated whether that same pattern is found in mice in the dopaminergic neurons in each of these structures. Using tyrosine hydroxylase immunostaining combined with [3H]thymidine autoradiography, the time of origin of dopaminergic midbrain neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus was determined in postnatal day 20 mice. The dams of the experimental animals were injected with [3H]thymidine on embryonic days (E) 11–E12, E12–E13, E13–E14, and E14–E15. The time of origin profiles for each group indicated significant differences between populations. The retrorubral field and the substantia nigra pars compacta arose nearly simultaneously and contained the highest proportion of neurons, 49 to 37%, generated on or before E11. Progressively fewer early-generated neurons were found in the ventral tegmental area (20%), and the interfascicular nucleus (8.5%). In addition, anterior dorsolateral neurons in the substantia nigra and ventral tegmental area were more likely to be generated early than the posterior ventromedial neurons. These findings indicate that mouse and rat brains have nearly identical developmental patterns in the midbrain, and neurogenetic gradients in dopaminergic neurons are similar to those found in Nissl studies in rats.
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Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing and mature dopaminergic neurons. Recent studies have demonstrated that Nurr1 is essential for the induction of phenotypic markers of ventral mid-brain dopaminergic neurons whose generation is specified by the floor plate-derived morphogenic signal sonic hedgehog (SHH), but the precise role of Nurr1 in this differentiative pathway has not been established. To provide further insights into the role of Nurr1 in the final differentiation pathway, we have examined the fate of dopamine cell precursors in Nurr1 null mutant mice. Here we demonstrate that Nurr1 functions at the later stages of dopamine cell development to drive differentiation of ventral mesencephalic late dopaminergic precursor neurons. In the absence of Nurr1, neuroepithelial cells that give rise to dopaminergic neurons adopt a normal ventral localization and neuronal phenotype characterized by expression of the homeodomain transcription factor and mesencephalic marker, Ptx-3, at embryonic day 11.5. However, these late precursors fail to induce a dopaminergic phenotype, indicating that Nurr1 is essential for specifying commitment of mesencephalic precursors to the full dopaminergic phenotype. Further, as development progresses, these mid-brain dopamine precursor cells degenerate in the absence of Nurr1, resulting in loss of Ptx-3 expression and a concomitant increase in apoptosis of ventral midbrain neurons in newborn null mutant mice. Taken together, these data indicate that Nurr1 is essential for both survival and final differentiation of ventral mesencephalic late dopaminergic precursor neurons into a complete dopaminergic phenotype.
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Mitochondrial accumulation and respiratory inhibition are critical steps in the actions of N-methyl-4-phenylpyridinium ion (MPP+), the toxic metabolite of the parkinsonism-inducing agent, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We examined the respiratory characteristics of 2-methylated beta-carbolines (2-Me beta Cs) and 2-methylated 3,4-dihydro-beta-carbolines (2-MeDH beta Cs), which encompass the MPP+ structure. As indoleamine derivatives, they could have endogenous roles in idiopathic parkinsonism. With rat liver mitochondria, the order for inhibition of NAD(+)-linked O2 consumption (6-min preincubations) was as follows: MPP+ = 2-methylharmine greater than 2-methylharmol = 2-methylharmaline much greater than 2-methylharmalol greater than 2-methylnorharman greater than 6-OH-2-methylharmalan much greater than 2-methylharman. Similar to MPP+, 2-MeDH beta C/2-Me beta C inhibition was potentiated by tetraphenylboron and reversed by dinitrophenol, consistent with the involvement of cationic forms. However, the participation of neutral forms was indicated by the 2-MeDH beta C/2-Me beta C inhibitory time courses, which were unlike MPP+. The neutral forms probably arise via indolic nitrogen deprotonation because the characteristics of a cationic beta-carboline that cannot N-deprotonate, 2,9-dimethylnorharman, mirrored MPP+ rather than 2-Me beta Cs. Succinate-supported respiration was also significantly blocked by 2-MeDH beta Cs/2-Me beta Cs, but results with tetraphenylboron and 2,9-dimethylnorharman indicated that cationic forms were less important than in the inhibition of NAD(+)-linked respiration. We suggest that the relatively potent inhibition by certain 2-MeDH beta Cs/2-Me beta Cs involves neutral forms for passive mitochondrial entry and cationic as well as neutral forms that act at several respiratory sites. Respiratory inhibition could reasonably underlie the reported neurotoxicity of 2-Me beta Cs.
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Intrinsic, striatal tyrosine hydroxylase-immunoreactive (TH-i) cells have received little consideration. In this study we have characterized these neurons and their regulatory response to nigrostriatal dopaminergic deafferentation. TH-i cells were observed in the striatum of both control and 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-treated monkeys; TH-i cell counts, however, were 3.5-fold higher in the striatum of MPTP-lesioned monkeys. To establish the dopaminergic nature of the TH-i cells, sections were double-labeled with antibodies to dopamine transporter (DAT). Immunofluorescence studies demonstrated that nearly all TH-i cells were double-labeled with DAT, suggesting that they contain the machinery to be functional dopaminergic neurons. Two types of TH-i cells were identified in the striatum: small, aspiny, bipolar cells with varicose dendrites and larger spiny, multipolar cells. The aspiny cells, which were more prevalent, corresponded morphologically to the GABAergic interneurons of the striatum. Double-label immunofluorescence studies using antibodies to TH and glutamate decarboxylase (GAD67), the synthetic enzyme for GABA, showed that 99% of the TH-i cells were GAD67-positive. Very few (<1%) of the TH-i cells, however, were immunoreactive for the calcium-binding proteins calbindin and parvalbumin. In summary, these results demonstrate that the dopaminergic cell population of the striatum responds to dopamine denervation by increasing in number, apparently to compensate for loss of extrinsic dopaminergic innervation. Moreover, this population of cells corresponds largely with the intrinsic GABAergic cells of the striatum. This study also suggests that the adult primate striatum does retain some intrinsic capacity to compensate for dopaminergic cell loss.
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The mesencephalic dopaminergic (mesDA) system regulates behavior and movement control and has been implicated in psychiatric and affective disorders. We have identified a bicoid-related homeobox gene, Ptx3, a member of the Ptx-subfamily, that is uniquely expressed in these neurons. Its expression starting at E11.5 in the developing mouse midbrain correlates with the appearance of mesDA neurons. The number of Ptx3-expressing neurons is reduced in Parkinson patients, and these neurons are absent from 6-hydroxydopamine-lesioned rats, an animal model for this disease. Thus, Ptx3 is a unique transcription factor marking the mesDA neurons at the exclusion of other dopaminergic neurons, and it may be involved in developmental determination of this neuronal lineage.
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Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. We have investigated in mice the role of the engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of midbrain dopaminergic neurons. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a link between engrailed and Parkinson's disease.
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In Parkinson's disease, progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SN) leads to debilitating motor dysfunction. One current therapy aims at exogenous cellular replacement of dopaminergic function by transplanting fetal midbrain cells into the striatum, the main projection area of the SN. However, results using this approach have shown variable success. It has been proposed that cellular replacement by endogenous stem/progenitor cells may be useful for therapeutic interventions in neurodegenerative diseases, including Parkinson's disease. Although it is widely accepted that progenitor cells are present in different areas of the adult CNS, it is unclear whether such cells reside in the adult SN and whether they have the potential to replace degenerating neurons. Here, we describe a population of actively dividing progenitor cells in the adult SN, which in situ give rise to new mature glial cells but not to neurons. However, after removal from the SN, these progenitor cells immediately have the potential to differentiate into neurons. Transplantation of freshly isolated SN progenitor cells into the adult hippocampus showed that these cells also have a neuronal potential under in vivo conditions. These results suggest that progenitor cells reside in the adult SN and can give rise to new neurons when exposed to appropriate environmental signals. This developmental potential of SN progenitor cells might be useful for future endogenous cell replacement strategies in Parkinson's disease.
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Dopaminergic (DA) neurons of substantia nigra in the midbrain control voluntary movement, and their degeneration is the cause of Parkinson's disease. The complete set of genes required to specifically determine the development of midbrain DA subgroups is not known yet. We report here that mice lacking the bicoid-related homeoprotein Pitx3 fail to develop DA neurons of the substantia nigra. Other mesencephalic DA neurons of the ventral tegmental area and retrorubral field are unaltered in their dopamine expression and histological organization. These data suggest that Pitx3-dependent gene expression is specifically required for the differentiation of DA progenitors within the mesencephalic DA system.
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New neurons are generated from stem cells in a few regions of the adult mammalian brain. Here we provide evidence for the generation of dopaminergic projection neurons of the type that are lost in Parkinson's disease from stem cells in the adult rodent brain and show that the rate of neurogenesis is increased after a lesion. The number of new neurons generated under physiological conditions in substantia nigra pars compacta was found to be several orders of magnitude smaller than in the granular cell layer of the dentate gyrus of the hippocampus. However, if the rate of neuronal turnover is constant, the entire population of dopaminergic neurons in substantia nigra could be replaced during the lifespan of a mouse. These data indicate that neurogenesis in the adult brain is more widespread than previously thought and may have implications for our understanding of the pathogenesis and treatment of neurodegenerative disorders such as Parkinson's disease.
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The neuropathological hallmark of Parkinson's disease is the loss of dopaminergic neurons in the substantia nigra pars compacta, presumably mediated by apoptosis. The homeobox transcription factors engrailed 1 and engrailed 2 are expressed by this neuronal population from early in development to adulthood. Despite a large mid-hindbrain deletion in double mutants null for both genes, mesencephalic dopaminergic (mDA) neurons are induced, become postmitotic and acquire their neurotransmitter phenotype. However, at birth, no mDA neurons are left. We show that the entire population of these neurons is lost by E14 in the mutant animals, earlier than in any other described genetic model system for Parkinson's disease. This disappearance is caused by apoptosis revealed by the presence of activated caspase 3 in the dying tyrosine hydroxylase-positive mutant cells. Furthermore, using in vitro cell mixing experiments and RNA interference on primary cell culture of ventral midbrain we were able to show that the demise of mDA neurons in the mutant mice is due to a cell-autonomously requirement of the engrailed genes and not a result of the missing mid-hindbrain tissue. Gene silencing in the postmitotic neurons by RNA interference activates caspase 3 and induces apoptosis in less than 24 hours. This rapid induction of cell death in mDA neurons suggests that the engrailed genes participate directly in the regulation of apoptosis, a proposed mechanism for Parkinson's disease.
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During development of the cerebellum, sonic hedgehog (Shh) is directly responsible for the proliferation of granule cell precursors in the external germinal layer. We have looked for signals able to regulate a switch from the Shh-mediated proliferative response to one that directs differentiation of granule neurones. Bone morphogenetic proteins (BMPs) are expressed in distinct neuronal populations within the developing cerebellar cortex. Bmp2 and Bmp4 are expressed in the proliferating precursors and subsequently in differentiated granule neurones of the internal granular layer, whereas Bmp7 is expressed by Purkinje neurones. In primary cultures, Bmp2 and Bmp4, but not Bmp7, are able to prevent Shh-induced proliferation, thereby allowing granule neuron differentiation. Furthermore, Bmp2 treatment downregulates components of the Shh pathway in proliferating granule cell precursors. Smad proteins, the only known BMP receptor substrates capable of transducing the signal, are also differentially expressed in the developing cerebellum: Smad1 in the external germinal layer and Smad5 in newly differentiated granule neurones. Among them, only Smad5 is phosphorylated in vivo and in primary cultures treated with Bmp2, and overexpression of Smad5 is sufficient to induce granule cell differentiation in the presence of Shh. We propose a model in which Bmp2-mediated Smad5 signalling suppresses the proliferative response to Shh by downregulation of the pathway, and allows granule cell precursor to enter their differentiation programme.
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The loss of dopaminergic (DA) neurons in the ventral midbrain is the principal cause of Parkinson's disease. The search for candidate molecules that promote the genesis and survival capacities of DA neurons is a major area of investigation and hope. A better characterization of the developmental pathways that govern the specification, differentiation, and survival of these neurons will be essential in devising therapies aimed to rescue or replace midbrain DA neurons in Parkinson's patients. In this brief review, we will discuss the major steps in the normal development of midbrain DA neurons.
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Antioxidants have concentration-dependent neuroprotective and proapoptotic activities in models of Parkinson's disease. The aim of our study was to determine gene-protein pathways of the antioxidants, dopamine (DA), R-apomorphine (R-APO), melatonin, and green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG), in neuroblastoma cells, using a customized cDNA microarray and quantitative reverse transcriptase-polymerase chain reaction gene expression techniques. We demonstrate a concentration-dependent correlation between these compounds and modulation of cell survival/cell death-related gene pathways. High toxic concentration of DA (500 microM), R-APO (50 microM), melatonin (50 microM), and EGCG (50 microM) exhibited a similar profile of proapoptotic gene expression, increasing the level of bax, caspase-6, fas ligand, and the cell-cycle inhibitor gadd45 genes, while decreasing antiapoptotic bcl-2 and bcl-xL. Conversely, the low neuroprotective concentrations (1-10 microM) of these compounds induced an antiapoptotic response. Melatonin displayed an extremely low index of mortality, which may be partially explained by the observation that a high concentration did not significantly affect the expression of mitochondrial Bcl-2 family members, bcl-2 and bax. Protein analysis of Bcl-2, Bax, and activated caspase-3 correlated with the gene expression pattern. Our results provide for the first time new insights into the molecular events involved in the dose-dependent neuroprotective and neurotoxic activities of catechols and indole amine compounds.
Chapter
Carbolines (pyrido-indoles) are prevalent tryptophan-derived heterocyclics in the diet and environment that have been linked since the late 1970’s to carcinogenesis. Evidence from our and other laboratories suggests the possibility that the most common pyrido-indole isomers, the ß-carbolines, also might be “protoxicants” leading to neuronal damage and resultant idiopathic parkinsonism in susceptible individuals. There is a close structural similarity between the N-methylated cationic products of simple ß-carbolines (e.g., norharman or harman) and the parkinsonian neurotoxin, MPTP, or specifically, its active cationic metabolite, MPP+. The N-methylated ß-carbolinium species are formed in mammalian brain via one or possibly two enzymatic S-adenosylmethionine-dependent N-methylations. These chemical alterations metabolically bioactivate the molecules, greatly increasing both the mitochondrial inhibitory activity and the in vitro and in vivo dopaminergic toxicity of the ß-carboline nucleus. Furthermore, the second (indole nitrogen) methylating activity as well as the product (2,9-di-N-methylated) carbolinium cations appear to be elevated in parkinsonian brain. Matsubara’s studies (this volume) demonstrate nigrostriatal neurodegeneration and neurobehavioral impairment in mice given precursors of the ß-carbolinium cations. In view of these findings, epidemiological studies on the possible relationship between environmental carboline alkaloid exposure and idiopathic parkinsonism could be revealing.
Chapter
1,2,3,4-Tetrahydro-β-carboline
Article
A series of 9-alkyl aromatic-β-carbolines was synthesized and evaluated as inhibitors of mitrochondrial monoamine oxidase. The possible existence of a hydrophobic or hydrophilic region on the enzyme was explored. Substitution of an electron-withdrawing group such as acetyl on N-9 position reduced the inhibitory activity. This suggested that the increase in the inhibitory activity of 9-methyl-β-carboline was at least partially due to the increase of its electron density by the methyl group, thus making the β-carboline bind better to the enzyme.
Article
Tritium labelled 1-carboxy-tetrahydroharman was identified in rat brain following i.c.v.-injection of [3H]tryptamine and pyruvic acid. The animals had been treated with the MAO inhibitor pargyline (40 mg/kg) 30 min before i.c.v. injection. Under these conditions, only trace amounts of [3H]indole acetic acid could be detected in the brain. The formation of 1-CTHH was time-dependent. Five minutes following the i.c.v. injection, approximately 0.45% of the administered tryptamine was converted into 1-CTHH and 23% were still unchanged. The amount of the radioactive 1-CTHH increased slightly within 1 h (0.8%; [3H] tryptamine: 6%). Pretreatment of the rats with high doses of pargyline (75 mg/kg; 90 min before i.c.v. injection) prevented the formation of both [3H]1-CTHH and [3H]indole acetic acid (IAA) suggesting that high doses of pargyline inhibit the formation of 1-CTHH. As control for a possible non-enzymatic formation of 1-CTHH, [3H]tryptamine and various concentrations of pyruvic acid were incubated in phosphate buffer at pH 7.4. 1-CTHH was not detected under these conditions. However, the formation of 1-CTHH was observed at high pyruvic acid concentrations (final concentration = 100 mM) and low pH values (less than pH4). To support the assumption that the observed condensation of both precursors to 1-CTHH occurred intracellularly, the metabolism of tryptamine was studied. Two minutes after i.c.v. injection of [3H]tryptamine approximately 4% of the injected dose remained unchanged and 10% were metabolized to [3H]IAA. These findings suggest a rapid disappearance of [3H]tryptamine from the cerebrospinal fluid as well as a rapid penetration into the cerebral tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
The distribution, metabolism and elimination into the urine of (14C)-tetrahydronorharmane (THN) as well as of (14C)-6-hydroxy-tetrahydronorharmane (6-OH-THN) are investigated in female and male rats. Following intravenous injection of (14C)-THN radioactivity was detected in all organs examined, namely blood, brain, lung, adrenal gland, small intestine, fat tissue, kidney and liver. In the brain the elimination half life of THN was calculated to be 1.8 h, the elimination half life of the radioactivity in the blood 6.24 h, and the accumulation half life in the urine 9.24 h. The elimination of 6-OH-THN into the urine is faster than that of THN. At least four metabolites of (14C)-THN were found in the urine of female rats. Two different metabolic pathways are discussed, firstly, hydroxylation followed by conjugation with glucuronic and sulfuric acids and secondly, dehydrogenation, followed by oxygenation. In female rats only traces of the conjugated metabolites are hydrolysed by arylsulfatase, whereas in male rats approximately 2/5 are cleaved by this enzyme. Pretreatment of male rats with 3-methylcholanthrene induced conjugation, whereas phenobarbital had no obvious effect on the pattern of metabolites. SKF 525 A and CFT 1201 both prevented almost completely the formation of conjugates from THN.
Article
Death of dopaminergic neurons in Parkinson's disease (PD) may partially be caused by synthesis and accumulation of endogenous and exogenous toxins. Because of structural similarity to MPTP, -carbolines, like norharman and harman, have been proposed as putative neurotoxins. In vivo they may easily be formed by cyclization of indoleamines with e.g. aldehydes. For further elucidation of the role of -carbolines in neurodegenerative disorders harman and norharman levels in cerebrospinal fluid (CSF) were measured in 14 patients with PD and compared to an age- and sex-matched control group (n=14). CSF levels of norharman and harman in PD were significantly higher compared to controls. These results may suggest a possible role of harman and norharman or its N-methylated carbolinium ions in the pathophysiological processes initiating PD. However the origin of increased levels of these -carbolines remains unclear. On the one hand one may speculate, that unknown metabolic processes induce the increased synthesis of harman and norharman in PD. On the other hand a possible impact of exogenous sources may also be possible.
Article
The interaction of several -carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several hundred fold higher affinity for the benodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related -carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.
Article
N-Methyl-4-phenylpyridinium ion (MPP+), a highly toxic metabolite produced in the brain from a street drug contaminant, is selectively taken up by nigrostriatal dopaminergic neurons and accumulated intraneuronally in mitochondria. There it inhibits respiration, causes neuronal death and, in primates, provokes a parkinsonian condition. It has been suggested that endogenously generated or activated agents resembling MPP+ may contribute to the development of Parkinson's disease. We report here that simple β-carbolines derived from tryptophan or related open chain indoles, when specifically methyl-substituted on both (2[β] and 9[indole]) available nitrogens, display mitochondrial inhibitory potencies and neurotoxic effects in vitro (PC12 cultures) and in vivo (striatal microdialysis) which approach or even surpass MPP+. These results take on physiological significance with our finding that brain enzyme activity catalyzesS-adenosylmethionine-dependent methylations of the β- and indole-nitrogens in β-carbolines that have been detected in vivo. The unusual 9[indole]-N-methyl transfer, previously unrecognized in animals, apparently requires prior methylation of the 2[β]-nitrogen. Sequential di-N-methylation of endogenous or xenobiotic β-carbolines to form unique, neurotoxic 2, 9-N, N′-dimethyl-β-carbolinium ions may serve as a brain bioactivation route in chronic neurodegenerative conditions such as Parkinson's disease.
Article
In the present study, we examined whether the bone morphogenetic proteins (BMPs), which are important in the developmental specification of transmitter type in certain classes of neurons, might also play a role in signaling the differentiation of a dopaminergic (DA) phenotype. We found that BMP-2, -4 and -6 were each capable of inducing, in a dose and time dependent manner, moderate levels of the DA enzyme tyrosine hydroxylase (TH) in cultured neurons from the mouse embryonic striatum. In contradistinction to other TH-inducing agents, BMPs initiated de novo TH expression without the required synergy of exogenous growth factors or co-activating substances and in neurons presumably aged (E16) beyond the critical period for induction. However, the appearance of TH in induced cells was short-lived (24 h) and could not be prolonged by repeated supplementation with the BMPs. Inhibitors of the mitogen-activated protein kinase (MAPK/ERK) signaling pathway, PD98059 and apigenin, did not prevent TH induction by BMP-4, as they did other TH inducing agents, indicating that the MAPK/ERK pathway does not mediate BMPs effects on TH expression. We conclude that BMP-2, -4 and -6 can be added to the expanding inventory of agents capable of inducing TH, making them potentially important in the specification of a DA phenotype in stem/precursor cells for the treatment of Parkinson’s disease.
Article
Enzymatic β-carboline N-methyltransferase activities generate N-methylated β-carbolinium cations that are analogs of the parkinsonian-producing neurotoxin MPP+. We measured β-carboline-2N-methyltransferase and β-carboline-9N-methyltransferase activities in the supernatant and particulate fractions from postmortem human brains. These N-methyltransferase activities were assessed in the substantia nigra, putamen, and frontal cortex from control and Parkinson's disease cases. No significant differences were measured in any brain region in particulate and supernatant fraction β-carboline 2N-methyltransferase activity or particulate fraction β-carboline 9N-methyltransferase activity. Likewise, supernatant fraction β-carboline 9N-methyltransferase activity was similar in the putamen and substantia nigra from Parkinson's disease and control cases. Unexpectedly, supernatant fraction β-carboline 9N-methyltransferase activity was increased fourfold in Parkinson's disease frontal cortex (P < 0.05), suggesting that β-carboline N-methylation may play a role in Parkinson's disease.
Article
Abuse of 4-propyloxy-4-phenyl-N-methylpiperidine, a meperidine congener, produced parkinsonism in a 23-year-old man. Unlike other drug-induced motor disturbances, the syndrome persisted for 18 months and responded to drugs that stimulate dopamine receptors. Biogenic amines and metabolites in the cerebrospinal fluid and microscopic evaluation of the brain at necropsy were consistent with damage to aminergic neurons in the substantia nigra.
Article
In the present paper a sensitive method is described to measure tetrahydronorharmane (THN) in the urine of man and rats as well as in the forebrain of rats. The compound is extracted into diethyl ether, separated by thin layer chromatography (TLC), acetylated with radiolabelled acetic anhydride and further isolated by two-dimensional TLC development. The existence of THN in urine of man was proven by using chromatography with different solvent systems, cocristallisation, isotope dilution technique as well as mass-spectrometry. The amount of THN in the urine varied over a wide range. With the same method it was demonstrated that THN occurs also in the forebrain of rats. The concentration increases after loading with tryptamine. The findings are discussed in view of the hypothesis that THN acts as a compound modulating neuronal mechanism.
Article
beta-Carboline alkaloids are derived as a result of condensation between indoleamine (e.g. tryptamine) and short-chain carboxylic acid (e.g. pyruvic acid) or aldehyde (e.g. acetaldehyde), a reaction that occurs readily at room temperature. These compounds have been found endogenously in human and animal tissues and may be formed as a byproduct of secondary metabolism: their endogenous functions however, are not well understood. Indoles and tryptophan derivatives exhibit antioxidative actions by scavenging free radicals and forming resonance stabilized indolyl radicals. Harmane and related compounds exhibited concentration-dependent inhibition of lipid peroxidation (measured as thiobarbiturate reactive products) in a hepatic microsomal preparation incubated with either enzymatic dependent (Fe3+ ADP/NADPH) or non-enzymatic dependent (Fe3+ ADP/dihydroxyfumarate) oxygen radical producing systems. Alkaloids with hydroxyl substitution and a partially desaturated pyridyl ring were found to have the highest antioxidative potencies. Substitution of a hydroxyl group by a methoxyl group at the 6-position resulted in a decrease of greater than 10-fold in the antioxidative activities. Harmane showed high efficacy in an enzymatic system but low efficacy in a non-enzymatic system. The antioxidative effects of harmane in the former system may be attributed to its ability to inhibit oxidative enzymes in the microsomal system. These results suggest that beta-carbolines may also serve as endogenous antioxidants.
Article
Abecarnil (isopropyl 6-benzyloxy-4-methoxymethyl-beta-carboline-3-carboxylate) is a novel ligand for central benzodiazepine (BZ) receptors, possessing anxiolytic and anticonvulsant properties, but with considerably reduced muscle relaxant effects in comparison to diazepam (DZP). In vitro, abecarnil inhibited the binding of the BZ [3H]lormetazepam to rat cerebral cortex membranes with an IC50 value of 0.82 nM in comparison to 56 nM for DZP. The ability of abecarnil to displace [3H]lormetazepam was enhanced 1.24-fold in the presence of 30 microM gamma-aminobutyric acid; the corresponding value for DZP was 2.8-fold. DZP and abecarnil were equally effective in enhancing the binding of t-[35S]butylbicyclophosphorothionate to rat cortical membranes. In vivo, abecarnil exhibited a 3- to 6-fold higher affinity to forebrain BZ receptors than DZP. Abecarnil was from 2 to 10 times more potent than DZP in most rodent tests of anxiolytic activity, and in reducing locomotor activity in mice and rats thoroughly habituated to the test chamber. However, in rats newly exposed to a novel cage, abecarnil was less potent than DZP in reducing locomotor activity. In tests of motor coordination, abecarnil, in contrast to DZP, showed no or only weak activity, and in potentiating the effects of ethanol and hexobarbital on motor performance abecarnil was 4 to 25 times less potent than DZP. Abecarnil antagonized the effects of BZs in the chimney and loss of righting reflex tests, but not in the rotarod test.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Humans are continuously exposed to naturally occurring and industrial xenobiotics in their daily lives. Heterocyclic amines, which are formed during the cooking of proteinaceous foods, have been categorized as a new class of naturally occurring xenobiotics. They are divided into 2-amino-3-methylimidazo[4,5-f] quinoline (IQ)- and non-IQ-types. The amounts and proportion of total mutagenicity contributed by the IQ-type heterocyclic amines in cooked food are greater than those of the non-IQ-types. Precursors of the IQ-type heterocyclic amines including IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are creatinine, amino acids and sugars in meat and fish. Both types of heterocyclic amines are carcinogenic in mice and rats. All heterocyclic amines except PhIP frequently induce cancers in the liver, while PhIP induces lymphomas in mice and carcinomas of the colon and mammary gland in rats. Based on quantitative analysis of heterocyclic amines in cooked food and levels of excretion of unchanged heterocyclic amines in human urine, total heterocyclic amine intake was calculated to be around 0.4-16 micrograms/person per day. As in the case of other naturally occurring xenobiotics, and degree of exposure is small and is presumably insufficient alone to account for the development of human cancer. Nevertheless, a linear relationship has been demonstrated between DNA adduct levels and a wide range of doses of MeIQx in animals. In addition, combined treatment with five heterocyclic amines yielded additive or synergistic effects in the development of glutathione S-transferase placental form (GST-P) positive foci. Taking these results and current observations of multiple genetic alterations in human cancers into consideration together, heterocyclic amines are probably involved in the development of human cancer in the presence of other carcinogens, tumor promoters and factors stimulating cancer progression.
Article
High-affinity binding of 3H-diazepam and 3H-flunitrazepam has provided evidence for the presence of benzodiazepine receptors on brain neurones. Pharmacological evidence showing a clear correlation between receptor affinity and in vivo pharmacological potency for several benzodiazepines and a link between benzodiazepine receptors and GABA (gamma-amino-butyric acid) receptors at the molecular level, indicates that these receptors are relevant to the pharmacological and clinical effects of benzodiazepines. In searching for possible endogeneous ligands for benzodiazepine receptors we have recently isolated ethyl beta-carboline-3-carboxylate (beta-CCE) found human urine and brain, and shown that beta-CCE has a higher affinity than diazepam for brain benzodiazepine receptors. beta-CCE itself is probably not present in the brain, but may be closely related to an endogenous benzodiazepine receptor ligand. We report here that beta-CCE, in contrast to benzodiazepines, can distinguish clearly between benzodiazepine receptors in cerebellum and hippocampus. This result strongly indicates that benzodiazepine receptors are not a single class of non-interacting entities. It has not been possible to determine whether two distinct receptors are present and/or whether true negative cooperativity exists among hippocampal, but not cerebellar, benzodiazepine receptors.
Article
The gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex, which is composed of distinct proteins embedded in the neuronal plasma membrane, is important for several effects of benzodiazepines, including protection afforded against convulsions. During structural modification of ethyl beta-carboline-3-carboxylate an agent was discovered which has high affinity for brain benzodiazepine receptors but which is a potent convulsant. Also in contrast to benzodiazepines, this type of benzodiazepine receptor ligand favors benzodiazepine receptors in the non-GABA-stimulated conformation, which may explain the convulsive properties.
Article
Four persons developed marked parkinsonism after using an illicit drug intravenously. Analysis of the substance injected by two of these patients revealed primarily 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) with trace amounts of 1-methyl-4-phenyl-4-propionoxy-piperidine (MPPP). On the basis of the striking parkinsonian features observed in our patients, and additional pathological data from one previously reported case, it is proposed that this chemical selectively damages cells in the substantia nigra.
Article
The beta-carboline norharman was determined in plasma, brain, liver, kidney, spleen, heart and lung of the rat using HPLC with fluorescence detection. In order to improve the speed and sensitivity of this assay an earlier published sample clean-up extraction procedure and HPLC method were adjusted. Norharman was found to be present in plasma as well as in all organs tested, concentrations in organs being about 80 times higher than those in plasma. Intraperitoneal injections of 2 and 100 mg/kg norharman showed that the partition of norharman between organs and plasma is about 3. Only the highest dose was found to have behavioural effects, viz. alerting reactions, a decrease in motor and exploratory activity, sedation, loss of righting reflex and after 30 min complete muscle relaxation, but no catatonia was observed. Norharman was found to be metabolized by the liver with a half live of about 20 min, whereas all other organs tested did not show any norharman clearing capacity. The results suggest that norharman is not likely the cause of psychosis, but a natural sedative and by- or coproduct of a more primary biochemical derangement.
Article
Potential bioactivated neurotoxicants, 2-N-methyl-beta-carbolinium and 2,9-N,N'-dimethyl-beta-carbolinium ions, as well as N-methylation activities which form these charged species, were analyzed for the first time in the parietal association cortex and the substantia nigra of human brain using GC/MS and HPLC. The brains were taken during forensic autopsies from corpses without obvious degeneration of substantia nigra. In the cortex, 2-methyl-norharmanium ion (2-MeNH) and 2,9-dimethyl-norharmanium ion (2,9-Me2NH) were detected in almost all samples. 2-Methyl-harmanium ions (2-MeHA) and 2,9-dimethyl-harmanium ions (2,9-Me2HA) were detectable in only two samples. In substantia nigra samples pooled from 3 or 4 brains for analysis, 2-MeNH and 2,9-Me2NH levels were higher than those in the cortex, whereas 2-MeHA and 2,9-Me2HA were below detection limits. Their precursors, norharman (NH) and harman (HA), were also measured using HPLC/fluorescence detection. In both regions, NH and HA were present in almost all samples; levels of NH and HA were also significantly higher in the nigra than in the cortex. Using 9-methyl-NH and 2-MeNH as substrates, in vitro N-methylation of the 2[beta] and 9[indole] nitrogens toward beta-carbolines was measured both in the cortex and in the nigra. 2[beta]-N-Methylation activity was significantly higher than 9[indole]-N-methylation activity in both regions. Recent studies show that beta-carbolinium ions resemble the synthetic parkinsonian toxicant, MPP+, with respect to structure and neurotoxic activity. Such 'bioactivated' carbolinium ions could be endogenous causative factors in Parkinson's disease.
Article
Mice unable to synthesize dopamine (DA) specifically in dopaminergic neurons were created by inactivating the tyrosine hydroxylase (TH) gene then by restoring TH function in noradrenergic cells. These DA-deficient (DA-/-) mice were born at expected frequency but became hypoactive and stopped feeding a few weeks after birth. Midbrain dopaminergic neurons, their projections, and most characteristics of their target neurons in the striatum appeared normal. Within a few minutes of being injected with L-dihdroxyphenylalanine (L-DOPA), the product of TH, the DA-/- mice became more active and consumed more food than control mice. With continued administration of L-DOPA, nearly normal growth was achieved. These studies indicate that DA is essential for movement and feeding, but is not required for the development of neural circuits that control these behaviors.
Article
We measured beta-carbolinium cations (BC plus s), endogenous analogs of the N-methyl-4-phenylpyridinium ion (MPP plus ), in the lumbar CSF of 22 patients with idiopathic Parkinson's disease (PD) and 11 age-matched controls without any symptoms of parkinsonism. Among the BC plus s, 2,9-dimethylnorharmanium cation (2,9-Me 2 NH plus ), the most potent neurotoxicant that mirrors MPP plus in mitochondria toxicity, was present in 12 patients with PD but not in controls. Although the 2-monomethylated beta-carbolinium cations (2-MeBC plus s), which were present in almost all subjects, registered a slightly higher level in PD patients than in controls, the difference was not significant. The total BC plus content, sum of 2-MeBC plus and 2,9-Me 2 NH plus levels, was significantly higher in PD patients than in controls. The 2-MeBC plus contents significantly increased with the progression of the PD, but 2,9-ME 2 NH plus decreased as the disease exacerbated, although levels varied within a wide range. The present results strongly support the hypothesis that ``bioactivated'' BC plus s, especially 2,9-Me 2 NH plus s, may be the endogenous causative factors underlying PD. NEUROLOGY 1995;45: 2240-2245
Article
Dopamine neurons of the substantia nigra and ventral tegmental area regulate movement and affective behavior and degenerate in Parkinson's disease. The orphan nuclear receptor Nurr1 was shown to be expressed in developing dopamine neurons before the appearance of known phenotypic markers for these cells. Mice lacking Nurr1 failed to generate midbrain dopaminergic neurons, were hypoactive, and died soon after birth. Nurr1 expression continued into adulthood, and brains of heterozygous animals, otherwise apparently healthy, contained reduced dopamine levels. These results suggest that putative Nurr1 ligands may be useful for treatment of Parkinson's disease and other disorders of midbrain dopamine circuitry.
Article
Bone morphogenetic proteins (BMPs) are a family of growth differentiation factors which induce bone formation from mesenchymal cells. These proteins are members of the transforming growth factor-beta super-family. The expression of BMPs in the nervous system as well as in other tissues has been reported. In this study, we show that the presence of BMP-2 resulted in a dose-dependent increase in the number of tyrosine hydroxylase-immunoreactive ventral mesencephalic cells after 7 days in serum-free medium cultures. A maximal response was elicited at 10 ng/mL. BMP-2 also increased the number of primary neurites and branch points as well as the length of the longest neurite in a dose-dependent manner, with a maximal effect at 1 ng/mL. In contrast, BMP-2 did not modify the number or the function of GABAergic neurons. On the other hand, we observed stimulation of proliferation and morphological changes in glial cells (astrocytes become more fibrous shaped) in the presence of a high BMP-2 concentration (100 ng/mL), but not with lower doses, suggesting that the neurotrophic effect in dopaminergic neurons is not mediated by astroglial cells. This is consistent with the fact that the BMP-2 effect on dopaminergic neurons was observed even when the cultures were treated with alpha-aminoadipic acid to exclude the presence of glial cells. In summary, our data indicate that BMP-2 is a potent neurotrophic factor for ventral mesencephalic dopaminergic cells in culture.
Article
The Hedgehog signalling pathway is essential for the development of diverse tissues during embryogenesis. Signalling is activated by binding of Hedgehog protein to the multipass membrane protein Patched (Ptc). We have now identified a novel component in the vertebrate signalling pathway, which we name Hip (for Hedgehog-interacting protein) because of its ability to bind Hedgehog proteins. Hip encodes a membrane glycoprotein that binds to all three mammalian Hedgehog proteins with an affinity comparable to that of Ptc-1. Hip-expressing cells are located next to cells that express each Hedgehog gene. Hip expression is induced by ectopic Hedgehog signalling and is lost in Hedgehog mutants. Thus, Hip, like Ptc-1, is a general transcriptional target of Hedgehog signalling. Overexpression of Hip in cartilage, where Indian hedgehog (Ihh) controls growth, leads to a shortened skeleton that resembles that seen when Ihh function is lost (B. St-Jacques, M. Hammerschmidt & A.P.M., in preparation). Our findings support a model in which Hip attenuates Hedgehog signalling as a result of binding to Hedgehog proteins: a negative regulatory feedback loop established in this way could thus modulate the responses to any Hedgehog signal.
Article
A new population of dopaminergic neurons has been identified in Parkinson's disease striatum. These neurons are sufficiently numerous to have an important effect on dopaminergic function in the striatum.
Article
Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine that plays an important role in immunity and inflammation, and in the control of cell proliferation, differentiation and apoptosis. TNFalpha is also the founding member of a still growing family of cytokines with diverse bioregulatory functions. Considerable progress has been made in understanding the molecular mechanisms that mediate TNFalpha-induced cellular responses. Binding of TNFalpha to its two receptors, TNFR1 and TNFR2, results in recruitment of signal transducers that activate at least three distinct effectors. Through complex signaling cascades and networks, these effectors lead to the activation of caspases and two transcription factors, AP-1 and NF-kappaB. Similar signaling mechanisms are likely to be used by other members of the TNF family. This review focuses on proteins that transduce the signals generated at TNF receptors to nuclear targets such as AP-1 and NF-kappaB.
Article
The hypothesized role of the beta-carboline norharman in processes of drug dependence forms the basis for several studies on plasma levels of norharman among substance-using populations, particularly among alcoholics and smokers. However, it is not clear whether norharman is implicated in processes of dependence to both substances, or only to tobacco smoke. In the present study plasma concentrations of norharman were measured among four groups of participants regarding heavy smokers who do or do not drink alcohol excessively and nonsmokers who do or do not drink alcohol excessively. All measurements were conducted on three different days with an interval of 2 months in between and at three times during the day to account for possible circadian or seasonal variations. Results showed that elevated plasma levels of norharman appear only in heavy smokers regardless of their drinking profile. The norharman plasma levels of nonsmoking excessive drinkers showed a similar pattern to that of the control group. The findings indicate that elevated plasma levels of norharman are due to heavy smoking and not to excessive drinking.
Article
Mammalian brain has a beta-carboline 2N-methyltransferase activity that converts beta-carbolines, such as norharman and harman, into 2N-methylated beta-carbolinium cations, which are structural and functional analogs of the Parkinsonian-inducing toxin 1-methyl-4-phenylpyridinium cation (MPP+). The identity and physiological function of this beta-carboline 2N-methylation activity was previously unknown. We report pharmacological and biochemical evidence that phenylethanolamine N-methyltransferase (EC 2.1.1.28) has beta-carboline 2N-methyltransferase activity. Specifically, purified phenylethanolamine N-methyltransferase (PNMT) catalyzes the 2N-methylation (21.1 pmol/h per unit PNMT) of 9-methylnorharman, but not the 9N-methylation of 2-methylnorharmanium cation. LY134046, a selective inhibitor of phenylethanolamine N-methyltransferase, inhibits (IC50 1.9 microM) the 2N-methylation of 9-methylnorharman, a substrate for beta-carboline 2N-methyltransferase. Substrates of phenylethanolamine N-methyltransferase also inhibit beta-carboline 2N-methyltransferase activity in a concentration-dependent manner. beta-Carboline 2N-methyltransferase activity (43.7pmol/h/mg protein) is present in human adrenal medulla, a tissue with high phenylethanolamine N-methyltransferase activity. We are investigating the potential role of N-methylated beta-carbolinium cations in the pathogenesis of idiopathic Parkinson's disease. Presuming that phenylethanolamine N-methyltransferase activity forms toxic 2N-methylated beta-carbolinium cations, we propose a novel hypothesis regarding Parkinson's disease-a hypothesis that includes a role for phenylethanolamine N-methyltransferase-catalyzed formation of MPP+ -like 2N-methylated beta-carbolinium cations.
Article
The natural beta-carbolines (BC) closely resemble the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in structure. The N-methylated beta-carbolinium ions (BC+) are potent inhibitors of mitochondrial respiration and are nigrostriatal neurotoxins. Utilizing [3H]BC, we have identified several proteins to which BC binds with high affinity (e.g. the chaperone member glucose regulated protein 78, the enzyme carboxylesterase, the cytochrome P450 2E1, the enzyme monoamine oxidase B and a small G-protein of the Rho subfamily). In the present study we isolated a protein from bovine brain to which [3H]BC binds with high affinity and identified it being the enzyme triosephosphate isomerase (TPI; EC 5.3.1.1.). 2,9-Dimethyl-BC+ was the most potent inhibitor of TPI, clearly more potent than the known inhibitors. TPI deficiency is a rare disorder in humans characterized by a severe progressive extrapyramidal course. Thus, TPI inhibition could contribute to neurodegeneration observed after injection of BCs into substantia nigra. Furthermore, the findings fit into the hypothesis of BCs as endogenous toxins responsible for neurodegeneration.
Article
The putative dopaminergic (DA) neurons intrinsic to human striatum were studied to determine their similarity with DA neurons of the substantia nigra pars compacta (SNpc). The comparison was based on morphological features and on the presence or absence of Nurr1, an orphan receptor of the nuclear receptor family that is essential for the expression of DA phenotype by developing SNpc neurons. Immunohistochemistry for the neuronal nuclear protein (NeuN; a neuronal marker) and in situ hybridization for tyrosine hydroxylase (TH) and/or Nurr1 were applied to post-mortem tissue obtained from seven normal individuals. On one hand, the TH-positive multipolar neurons in the human striatum, which were subdivided into three groups according to their size and pattern of dendritic arborization, were found to be morphologically similar to TH-positive neurons of the SNpc. The distribution frequency of striatal TH-positive neurons, according to their diameter, closely matches the frequency observed for multipolar TH-positive cells in the SNpc. On the other hand, the proportion of neurons expressing Nurr1 and TH mRNA transcripts on single striatal section was similar to the proportion of TH-immunoreactive neurons observed on adjacent sections. More importantly, in each striatum analysed, virtually all cells that stained for TH also expressed NeuN and Nurr1. This study provides novel data that confirm the existence of DA neurons intrinsic to the human striatum. It also provides the first evidence for the existence of striking morphological and chemical similarities between the DA neurons present at striatal level and those that populate the SNpc.
Article
The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.
Article
The transcription factor/nuclear receptor Nurr1 is essential for the differentiation of midbrain dopaminergic neurones. Here we demonstrate that, during the ontogeny of rat ventral mesencephalon, nurr1 gene expression is developmentally regulated and its levels show a sharp peak between embryonic day E13 and E15, when most dopaminergic neurones differentiate. In addition, in primary cultures from embryonic rat mesencephalon, nurr1 gene follows a temporal pattern of expression comparable to that observed in vivo. We also report that exposure of embryonic mesencephalic cultures to depolarizing stimuli leads to a robust increase in nurr1 mRNA and protein. The depolarizing effect is also detected in mesencephalic cultures enriched in dopaminergic neurones by using a combination of bFGF and Sonic hedgehog. The latter further increases the number of dopaminergic neurones in these 'expanded' cultures, an effect abolished in the presence of anti-Sonic hedgehog antibodies. Our data show that nurr1 gene is highly expressed in midbrain dopaminergic neurones in a sharp temporal window and that its expression is plastic, both in vivo and in vitro. In addition we show that Sonic hedgehog can direct dopaminergic differentiation in proliferating dopaminergic neuroblasts in vitro.