Abstract The novel multifunctional brain permeable iron, chelator M30 5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline was shown to possess neuroprotective activities in vitro and in vivo , ...against several insults applicable to various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present study, we demonstrate that systemic chronic administration of M30 resulted in up-regulation of hypoxia-inducible factor (HIF)-1α protein levels in various brain regions (e.g. cortex, striatum, and hippocampus) and spinal cord of adult mice. Real-time RT-PCR revealed that M30 differentially induced HIF-1α-dependent target genes, including vascular endothelial growth factor (VEGF), erythropoietin (EPO), enolase-1, transferrin receptor (TfR), heme oxygenase-1 (HO-1), inducible nitric oxide synthase (iNOS), and glucose transporter (GLUT)-1. In addition, mRNA expression levels of the growth factors, brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF) and three antioxidant enzymes (catalase, superoxide dismutase (SOD)-1, and glutathione peroxidase (GPx)) were up-regulated by M30 treatment in a brain-region-dependent manner. Signal transduction immunoblotting studies revealed that M30 induced a differential enhanced phosphorylation of protein kinase C (PKC), mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), protein kinase B (PKB/Akt), and glycogen synthase kinase-3β (GSK-3β). Together, these results suggest that the multifunctional iron chelator M30 can up-regulate a number of neuroprotective-adaptive mechanisms and pro-survival signaling pathways in the brain that might function as important therapeutic targets for the drug in the context of neurodegenerative disease therapy.
Dysfunction of the ubiquitin-proteasome system (UPS) and accumulation of iron in substantia nigra (SN) are implicated in the pathogenesis of Parkinson's disease (PD). UPS dysfunction and iron ...misregulation may reinforce each other's contribution to the degeneration of dopamine (DA) neurons. In the present study, we use a new brain-permeable iron chelator, VK-28 5-(4-(2-hydroxyethyl) piperazin-1-yl (methyl)-8-hydroxyquinoline, and its derivative M30 5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline in vivo to test their neuroprotective and neurorestorative properties against proteasome inhibitor (lactacystin) -induced nigrostriatal degeneration. Bilateral microinjections of lactacystin (1.25 μg/side) into the mouse medial forebrain bundle were performed. Administration of VK-28 (5 mg/kg, once a day) or M30 (5 mg/kg, once a day) was applied intraperitoneally 7 days before or after the lactacystin microinjection until the mice were sacrificed 28 days after microinjection. We found that VK-28 and M30 both significantly improved behavioral performances and attenuated lactacystin-induced DA neuron loss, proteasomal inhibition, iron accumulation, and microglial activation in SN. In addition, M30 restored the Bcl-2 level, which was suppressed after lactacystin injection. These findings suggest that brain-permeable iron chelators can improve DA neuron survival under UPS impairment. Furthermore, M30, a derivative of VK-28 and neuroprotective agent rasagiline, may serve as a better neuroprotective therapy for PD.--Zhu, W., Xie, W., Pan, T., Xu, P., Fridkin, M., Zheng, H., Jankovic, J., Youdim, M. B. H., Le, W. Prevention and restoration of lactacystin-induced nigrostriatal dopamine neuron degeneration by novel brain-permeable iron chelators.
Since the description of some peculiar symptoms by James Parkinson in 1817, attempts have been made to define its cause or at least to enlighten the pathology of “Parkinson’s disease (PD).” The vast ...majority of PD subtypes and most cases of sporadic PD share Lewy bodies (LBs) as a characteristic pathological hallmark. However, the processes underlying LBs generation and its causal triggers are still unknown. ɑ-Synuclein (ɑ-syn, encoded by the
SNCA
gene) is a major component of LBs, and
SNCA
missense mutations or duplications/triplications are causal for rare hereditary forms of PD. Thus, it is imperative to study ɑ-syn protein and its pathology, including oligomerization, fibril formation, aggregation, and spreading mechanisms. Furthermore, there are synergistic effects in the underlying pathogenic mechanisms of PD, and multiple factors—contributing with different ratios—appear to be causal pathological triggers and progression factors. For example, oxidative stress, reduced antioxidative capacity, mitochondrial dysfunction, and proteasomal disturbances have each been suggested to be causal for ɑ-syn fibril formation and aggregation and to contribute to neuroinflammation and neural cell death. Aging is also a major risk factor for PD. Iron, as well as neuromelanin (NM), show age-dependent increases, and iron is significantly increased in the Parkinsonian substantia nigra (SN). Iron-induced pathological mechanisms include changes of the molecular structure of ɑ-syn. However, more recent PD research demonstrates that (i) LBs are detected not only in dopaminergic neurons and glia but in various neurotransmitter systems, (ii) sympathetic nerve fibres degenerate first, and (iii) at least in “brain-first” cases dopaminergic deficiency is evident before pathology induced by iron and NM. These recent findings support that the ɑ-syn/LBs pathology as well as iron- and NM-induced pathology in “brain-first” cases are important facts of PD pathology and via their interaction potentiate the disease process in the SN. As such, multifactorial toxic processes posted on a personal genetic risk are assumed to be causal for the neurodegenerative processes underlying PD. Differences in ratios of multiple factors and their spatiotemporal development, and the fact that common triggers of PD are hard to identify, imply the existence of several phenotypical subtypes, which is supported by arguments from both the “bottom-up/dual-hit” and “brain-first” models. Therapeutic strategies are necessary to avoid single initiation triggers leading to PD.
Iron‐dependent oxidative stress, elevated levels of iron and of monoamine oxidase (MAO)‐B activity, and depletion of antioxidants in the brain may be major pathogenic factors in Parkinson's disease, ...Alzheimer's disease and related neurodegenerative diseases. Accordingly, iron chelators, antioxidants and MAO‐B inhibitors have shown efficacy in a variety of cellular and animal models of CNS injury. In searching for novel antioxidant iron chelators with potential MAO‐B inhibitory activity, a series of new iron chelators has been designed, synthesized and investigated. In this study, the novel chelators were further examined for their activity as antioxidants, MAO‐B inhibitors and neuroprotective agents in vitro. Three of the selected chelators (M30, HLA20 and M32) were the most effective in inhibiting iron‐dependent lipid peroxidation in rat brain homogenates with IC50 values (12–16 µm), which is comparable with that of desferal, a prototype iron chelator that is not has orally active. Their antioxidant activities were further confirmed using electron paramagnetic resonance spectroscopy. In PC12 cell culture, the three novel chelators at 0.1 µm were able to attenuate cell death induced by serum deprivation and by 6‐hydroxydopamine. M30 possessing propargyl, the MAO inhibitory moiety of the anti‐Parkinson drug rasagiline, displayed greater neuroprotective potency than that of rasagiline. In addition, in vitro, M30 was a highly potent non‐selective MAO‐A and MAO‐B inhibitor (IC50 < 0.1 µm). However, HLA20 was more selective for MAO‐B but had poor MAO inhibition, with an IC50 value of 64.2 µm. The data suggest that M30 and HLA20 might serve as leads in developing drugs with multifunctional activities for the treatment of various neurodegenerative disorders.
Our previous studies have shown that the green tea polyphenol (–)‐epigallocatechin‐3‐gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine ...the neuroprotective effect of EGCG when it is administered after the induction of cell damage (‘neurorescue’). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum‐starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1–10 µm) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 µm) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre‐treatment with the protein kinase C inhibitor GF109203X (2.5 µm), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.
In the present study we demonstrate neuroprotective property of green tea extract and (–)‐epigallocatechin‐3‐gallate in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mice model of Parkinson's disease. ...N‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (–)‐epigallocatechin‐3‐gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (–)‐epigallocatechin‐3‐gallate. (–)‐Epigallocatechin‐3‐gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine conversion to its active metabolite 1‐methyl‐4‐phenylpyridinium by monoamine oxidase‐B, as both green tea and (–)‐epigallocatechin‐3‐gallate are very poor inhibitors of this enzyme in vitro (770 µg/mL and 660 µM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron‐chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.
Brain iron dysregulation and its association with amyloid precursor protein (APP) plaque formation are implicated in Alzheimer's disease (AD) pathology and so iron chelation could be considered a ...rational therapeutic strategy for AD. Here we analyzed the effect of the main polyphenol constituent of green tea, (–)‐epigallocatechin‐3‐gallate (EGCG), which possesses metal‐chelating and radical‐scavenging properties, on the regulation of the iron metabolism‐related proteins APP and transferrin receptor (TfR). EGCG exhibited potent iron‐chelating activity comparable to that of the prototype iron chelator desferrioxamine, and dose dependently (1–10 µm) increased TfR protein and mRNA levels in human SH‐SY5Y neuroblastoma cells. Both the immature and full‐length cellular holo‐APP were significantly reduced by EGCG, as shown by two‐dimensional gel electrophoresis, without altering APP mRNA levels, suggesting a post‐transcriptional action. Indeed, EGCG suppressed the translation of a luciferase reporter gene fused to the APP mRNA 5′‐untranslated region, encompassing the APP iron‐responsive element. The finding that Fe2SO4 reversed the action of EGCG on APP and TfR proteins reinforces the likelihood that these effects are mediated through modulation of the intracellular iron pool. Furthermore, EGCG reduced toxic β‐amyloid peptide generation in Chinese hamster ovary cells overexpressing the APP ‘Swedish’ mutation. Thus, the natural non‐toxic brain‐permeable EGCG may provide a potential therapeutic approach for AD and other iron‐associated disorders.
Several multifunctional iron chelators have been synthesized from hydroxyquinoline pharmacophore of the iron chelator, VK‐28, possessing the monoamine oxidase (MAO) and neuroprotective ...N‐propargylamine moiety. They have iron chelating potency similar to desferal. M30 is a potent irreversible rat brain mitochondrial MAO‐A and ‐B inhibitor in vitro (IC50, MAO‐A, 0.037 ± 0.02; MAO‐B, 0.057 ± 0.01). Acute (1–5 mg/kg) and chronic 5–10 mg/kg intraperitoneally (i.p.) or orally (p.o.) once daily for 14 daysin vivo studies have shown M30 to be a potent brain selective (striatum, hippocampus and cerebellum) MAO‐A and ‐B inhibitor. It has little effects on the enzyme activities of the liver and small intestine. Its N‐desmethylated derivative, M30A is significantly less active. Acute and chronic treatment with M30 results in increased levels of dopamine (DA), serotonin(5‐HT), noradrenaline (NA) and decreases in DOPAC (dihydroxyphenylacetic acid), HVA (homovanillic acid) and 5‐HIAA (5‐hydroxyindole acetic acid) as determined in striatum and hypothalamus. In the mouse MPTP (N‐methy‐4‐phenyl‐1,2,3,6‐tetrahydropyridine) model of Parkinson's disease (PD) it attenuates the DA depleting action of the neurotoxin and increases striatal levels of DA, 5‐HT and NA, while decreasing their metabolites. As DA is equally well metabolized by MAO‐A and ‐B, it is expected that M30 would have a greater DA neurotransmission potentiation in PD than selective MAO‐B inhibitors, for which it is being developed, as MAO‐B inhibitors do not alter brain dopamine.
The recent therapeutic approach in which drug candidates are designed to possess diverse pharmacological properties and act on multiple targets has stimulated the development of the bifunctional drug ...ladostigil (TV3326) (N-propargyl-(3R) aminoindan-5yl)-ethyl methyl carbamate. Ladostigil combines the neuroprotective effects of the antiparkinson drug rasagiline, a selective monoamine oxidase (MAO)-B inhibitor, with the cholinesterase (ChE) inhibitory activity of rivastigmine in a single molecule, as a potential treatment for Alzheimer's disease (AD) and Lewy Body disease. Here, we assessed the dual effects of lodostigil in terms of the molecular mechanism of neuroprotection and amyloid precursor protein (APP) regulation/processing by using an apoptotic model of neuroblastoma SK-N-SH cells. Ladostigil dose-dependently decreased cell death via inhibition of the cleavage and prevention of caspase-3 activation (IC50=1.05 μM) through a mechanism related to regulation of the Bcl-2 family proteins, which resulted in reduced levels of Bad and Bax and induced levels of Bcl-2 gene and protein expression. We have also followed APP regulation/processing and found that ladostigil markedly decreased apoptotic-induced levels of holo-APP protein without altering APP mRNA levels, suggesting a posttranscriptional mechanism. In addition, the drug-elevated phosphorylated protein kinase C (pPKC) levels and stimulated the release of the nonamyloidogenic α-secretase proteolytic pathway. Similar to ladostigil, its S-isomer, TV3279, which is a ChE inhibitor but lacks MAO inhibitory activity, exerted neuroprotective properties and regulated APP processing, indicating that these effects are independent of MAO inhibition.--Yogev-Falach, M., Bar-Am, O., Amit, T., Weinreb, O., Youdim, M. B. H. The multifunctional neuroprotective anti-Alzheimer/anti-Parkinson drug ladostigil (TV3326) regulates holo-APP translation and processing.
: Investigations that revealed increased levels of iron in postmortem brains from patients with Parkinson's disease (PD) as compared to those from individuals not suffering from neurological ...disorders are reported. The chemical natures in which iron predominates in the brain and the relevance of neuromelanin for neuronal iron binding are discussed. Major findings have been that iron levels increase with the severity of neuropathological changes in PD, presumably due to increased transport through the blood‐brain barrier in late stages of parkinsonism. Glial iron is mainly stored as ferric iron in ferritin, while neuronal iron is predominantly bound to neuromelanin. Iron overload may induce progressive degeneration of nigrostriatal neurons by facilitating the formation of reactive biological intermediates, including reactive oxygen species, and the formation of cytotoxic protein aggregates. There are indications that iron‐mediated neuronal death in PD proceeds retrogradely. These results are also discussed with respect to their relevance for disease progression in relation to cytotoxic α‐synuclein protofibril formation.
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