The role of iron in neurodegeneration--M?ssbauer spectroscopy, electron microscopy, enzyme-linked immunosorbent assay and neuroimaging studies

The possible role of iron in neurodegeneration was studied by various techniques: electron microscopy, enzyme-linked immunosorbent assay, M?ssbauer spectroscopy, atomic absorption, ultrasonography and magnetic resonance imaging. The measurements were made on human tissues extracted from liver and from brain structures involved in diseases of the human brain: substantia nigra (Parkinson's, PD), hippocampal cortex (Alzheimer's, AD) and globus pallidus (progressive supranuclear palsy, PSP). The sizes of the iron cores of ferritin, the main iron storage compound in tissues, were found to be smaller in brain than in liver. Brain ferritin has a higher proportion of H to L chains compared to liver. A significant decrease of the concentration of L chains in PD compared to control was found. No increase in the concentration of iron in PD versus control was detected; however, there was an increase of labile iron, which constitutes only 2‰ of brain iron. In AD an increase in the concentration of ferritin was noticed, without a significant increase in iron concentration. In PSP an increase of total iron was observed. Our findings suggest that the mechanisms leading to the death of nerve cells in these three diseases may be different, although all may be related to iron mediated oxidative stress.     

Iron in typical and atypical parkinsonism – Mössbauer spectroscopy and MRI studies

Iron may play important role in neurodegeneration. The results of comparative studies of human brain areas (control and pathological) performed by Mössbauer spectroscopy (MS) and magnetic resonance imaging (MRI) techniques are presented. Mössbauer spectroscopy demonstrated a higher concentration of iron in atypical parkinsonism (progressive supranuclear palsy PSP) in the brain areas Substantia Nigra (SN) and Globus Pallidus (GP) involved in this pathological process, compared to control, while the concentration of iron in pathological tissues in typical parkinsonism (Parkinson’s disease - PD) did not differ from that in control. These results were compared with the changes in 1/T1 and 1/T2 (T1 and T2 being the relaxation times determined by MRI). A good linear correlation curve was found between the concentration of iron as determined by MS in different areas of control human brains and between 1/T1 and 1/T2. Whereas the finding in PSP-GP (the brain area involved in PSP) also fitted to such a correlation, this was not so for the correlation between pathological SN – the brain area involved in both diseases – and 1/T2, indicating a dependence of T2 on other factors than just the concentration of iron.     

Iron in Parkinson's Disease Revisited

Hyperfine Interactions - Mössbauer studies of fresh frozen samples taken at autopsy from different parts of the human brain (globus pallidus (GP), substantia nigra (NS), and hippocamp (Hip))...     

Iron in the brain

The results of our studies of iron in three brain structures, substantia nigra (SN), globus pallidus (GP), and hippocampus (Hip), are presented. Mössbauer spectroscopy, electron microscopy and ELISA (enzyme-linked immuno-absorbent assay) were applied. Mössbauer studies show that most of the iron in the brain is ferritin-like. The concentration of iron is similar in SN and GP, but less than half of this in Hip. ELISA studies showed that the H/L ratio of ferritin in SN and GP is also similar, but is about three times higher in Hip. These results suggest that the role of iron in SN and GP may be different from that in Hip. Electron microscopy shows that the diameters of the ferritin iron cores in the brain are smaller that in the liver (3.5 ± 0.5 nm vs. 6.0 ± 0.5 nm). Mössbauer studies yield the ratio between the concentration of iron in control and parkinsonian SN as 1.00 ± 0.13.     

ESR study on scavenging effect of nicotine on free radicals

Smokers have less risk to catch degenerative diseases such as Alzheimer’s disease and Parkinson’s disease that are both correlated with enhanced oxidative stress. The scavenging effect of nicotine on free radicals was investigated by electron spin resonance techniques. It was found that the scavenging effect of nicotine on hydroxyl radicals and superoxide free radicals was higher than that of vitamin C. It was also found that nicotine could scavenge free radicals in gas-phase cigarette smoke. The data indicate that nicotine may be a potential antioxidant.     

MRI evaluation of brain iron in earlier- and later-onset Parkinson's disease and normal subjects

Tissue iron levels in the extrapyramidal system of earlier- and later-onset Parkinson's disease (PD) subjects were evaluated in vivo using a magnetic resonance imaging (MRI) method. The method involves scanning subjects in both high- and low-field MRI instruments, measuring tissue relaxation rate (R2), and calculating the field-dependent R2 increase (FDRI) which is the difference between the R2 measured with the two MRI instruments. In tissue, only ferritin iron is known to increase R2 in a field-dependent manner and the FDRI measure is a specific measure of this tissue iron pool. Two groups of male subjects with PD and two age-matched groups of normal control males were studied. The two groups of six subjects with PD consisted of subjects with earlier- or later-onset (before or after age 60) PD. FDRI was measured in five subcortical structures: the substantia nigra reticulata (SNR), substantia nigra compacta (SNC), globus pallidus, putamen, and caudate nucleus, and in one comparison region; the frontal white matter. Earlier-onset PD subjects had significant (p < 0.05) increases in FDRI in the SNR, SNC, putamen, and globus pallidus, while later-onset PD subjects had significantly decreased FDRI in the SNR when compared to their respective age-matched controls. Controlling for illness duration or structure size did not meaningfully alter the results. Published post-mortem studies on SN iron levels indicate decreased ferritin levels and increased free iron levels in the SN of older PD subjects, consistent with the decreased FDRI observed in our later-onset PD sample, which was closely matched in age to the post-mortem PD samples. The FDRI results suggest that disregulation of iron metabolism occurs in PD and that this disregulation may differ in earlier- versus later-onset PD.     

The correlation between phase shifts in gradient-echo MR images and regional brain iron concentration.

The purpose of this study was to investigate the relationship between the magnetic susceptibility of brain tissue and iron concentration. Phase shifts in gradient-echo images (TE = 60 ms) were measured in 21 human subjects, (age 0.7-45 years) and compared with published values of regional brain iron concentration. Phase was correlated with brain iron concentration in putamen (R2 = 0.76), caudate (0.72), motor cortex (0.68), globus pallidus (0.59) (all p < 0.001), and frontal cortex (R2 = 0.19, p = 0.05), but not in white matter (R2 = 0.05,p = 0.34). The slope of the regression (degrees/mg iron/g tissue wet weight) varied over a narrow range from -1.2 in the globus pallidus and frontal cortex to -2.1 in the caudate. These results suggest that magnetic resonance phase reflects iron-induced differences in brain tissue susceptibility in gray matter. The lack of correlation in white matter may reflect important differences between gray and white matter in the cellular distribution and the metabolic functions of iron. Magnetic resonance phase images provide insight into the magnetic state of brain tissue and may prove to be useful in elucidating the relationship between brain iron and tissue relaxation properties.     

Iron in Parkinson disease, blood diseases, malaria and ferritin

The concentration of iron in Substantia nigra, the part of the brain which is involved in Parkinson disease, has been found by Mössbauer spectroscopy (MS) to be ~ 160 μg/g wet tissue and ~ 670 μg/g dry weight, both in control and Parkinson samples. All the iron observed by MS in these samples is ferritin-like iron. In several blood diseases, large amounts of ferritin-like iron have been observed in red blood cells. Desferral removed iron from serum, but not from red blood cells. The iron compound in the malarial pigment of human blood infected by P. falciparum was found to be hemin-like, whereas the pigment iron in rats infected by P. berghei was different from any known iron porphyrin.     

The measurement of R2, and R2′ in HIV-infected patients using the prime sequence as a measure of brain iron deposition

Brain iron deposition was assessed at 1.5 T in the caudate nucleus, globus pallidus and frontal and parietooccipital white matter in 28 human immunodeficiency virus (HIV)-infected patients and 15 control subjects with a new Partially Refocussed Interleaved Multi-Echo sequence by measuring 1/T₂, 1/T₂^* and 1/T2′ (i.e., R₂, and R₂′). There were significant differences in the R₂ and of the caudate nucleus (p < 0.0001 and p < 0.05) and the R₂, and R₂′ of the globus pallidus (p < 0.001, p < 0.005 and p < 0.05) in HIV-infected patients compared to control subjects. There was a trend for higher values of R₂, and R₂′ in the globus pallidus and caudate nucleus in HIV-infected patients with later stage HIV disease. These results suggest that there is greater iron deposition in the basal ganglia of HIV-infected patients compared with control subjects, with a predilection for the globus pallidus. The relationship between iron deposition in the brain and various parameters of severity of HIV infection remains uncertain.     

Selective MAO-B inhibitors: a lesson from natural products

Monoamine oxidases (MAOs) are mitochondrial bound enzymes, which catalyze the oxidative deamination of monoamine neurotransmitters. Inside the brain, MAOs are present in two isoforms: MAO-A and MAO-B. The activity of MAO-B is generally higher in patients affected by neurodegenerative diseases like Alzheimer’s and Parkinson’s. Therefore, the search for potent and selective MAO-B inhibitors is still a challenge for medicinal chemists. Nature has always been a source of inspiration for the discovery of new lead compounds. Moreover, natural medicine is a major component in all traditional medicine systems. In this review, we present the latest discoveries in the search for selective MAO-B inhibitors from natural sources. For clarity, compounds have been classified on the basis of structural analogy or source: flavonoids, xanthones, tannins, proanthocyanidins, iridoid glucosides, curcumin, alkaloids, cannabinoids, and natural sources extracts. MAO inhibition values reported in the text are not always consistent due to the high variability of MAO sources (bovine, pig, rat brain or liver, and human) and to the heterogeneity of the experimental protocols used.     

9.4T下TX模型小鼠脑组织的扩散张量成像研究

本文首次应用9.4 T高场磁共振扩散张量成像（diffusion tensor imaging，DTI）技术，研究了Wilson疾病模型TX（toxic milk）小鼠的脑组织微观结构改变和结构连接情况.基于感兴趣区域（region of interest，ROI）的分析发现，与对照组相比，TX模型组的各向异性比值（fractional anisotropy，FA）在海马、尾状核和苍白球显著下降；平均扩散率（mean diffusivity，MD）则呈现上升趋势，但无统计学意义.纤维束追踪法结果表明TX模型组小鼠的脑结构连接并未受到严重破坏，证明了铜累积对脑组织的损伤具有区域性.     

基于多输出的 3D 卷积神经网络诊断阿尔兹海默病

随着人口老龄化的加深，阿尔兹海默疾病更加大众化地出现在我们生活中，而早期精准诊断阿尔兹海默疾病并进行正向干预可有效延缓阿尔兹海默疾病的进程．基于磁共振图像的阿尔兹海默疾病的精准诊断需要综合利用多个感兴趣区域（ROIs）的信息，而单个ROI无法体现不同ROIs之间存在的联系与影响．本文首先提出三输入3D卷积神经网络（CNN），综合利用大脑3D磁共振图像中海马体、灰质（无海马体）和白质3个ROIs的信息．此外，随着神经网络的加深，原始图像的重要特征信息会部分丢失，因此我们又提出一种多输出3D CNN，通过增加中间层的连接和输出，缩短输入和输出之间的距离，增强特征传播，减少特征信息的丢失．结果显示采用多输出3DCNN模型实现整个测试集三分类的准确率为90.5%、精确率为91.0%、灵敏度为90.4%、特异性为95.2%、F1-score为90.5%，诊断性能优于单输出3D CNN模型．     

Structure-based design and analysis of MAO-B inhibitors for Parkinson’s disease: using in silico approaches

Parkinson’s disease (PD) is a degenerative disorder of the CNS, characterized by cerebral depletion of dopamine (DA), hence one of the approaches to delay the depletion of DA is to inhibit its oxidative deamination. Monoamine oxidases (MAO) carry out the oxidative deamination of monoamines like DA. These are intracellular enzymes, located on the outer mitochondrial membrane. MAO-A and MAO-B are the two subtypes of which MAO-B is the most predominant enzyme and is commonly found in the brain. Inhibition of the MAO-B enzyme boosts the effect of both endogenous and exogenous DA. In this study, we have carried out crystal structure analysis and structure-based design of MAO-B inhibitors. We also performed molecular dynamics, flexible docking, induced-fit docking and ADME prediction of the newly designed compounds.     

Synthesis and evaluation of novel arylisoxazoles linked to tacrine moiety: in vitro and in vivo biological activities against Alzheimer’s disease

Molecular Diversity - Alzheimer’s disease (AD) is now ranked as the third leading cause of death after heart disease and cancer. There is no definite cure for AD due to the multi-factorial...     

Neurotoxicity of low-dose repeatedly intranasal instillation of nano- and submicron-sized ferric oxide particles in mice

Olfactory tract has been demonstrated to be an important portal for inhaled solid nanoparticle transportation into the central nervous system (CNS). We have previously demonstrated that intranasally instilled Fe₂O₃ nanoparticles could transport into the CNS via olfactory pathway. In this study, we investigated the neurotoxicity and size effect of repeatedly low-dose (130 μg) intranasal exposure of nano- and submicron-sized Fe₂O₃ particles (21 nm and 280 nm) to mice. The biomarkers of oxidative stress, activity of nitric oxide synthases and release of monoamine neurotransmitter in the brain were studied. Our results showed that significant oxidative stress was induced by the two sizes of Fe₂O₃ particles. The activities of GSH-Px, Cu,Zn-SOD, and cNOS significantly elevated and the total GSH and GSH/GSSG ratio significantly decreased in the olfactory bulb and hippocampus after the nano- and submicron-sized Fe₂O₃ particle treatment (p < 0.05). The nano-sized Fe₂O₃ generally induced greater alteration and more significant dose–effect response than the submicron-sized particle did. Some slight perturbation of monoamine neurotransmitters were found in the hippocampus after exposure to the two sizes of Fe₂O₃ particle. The TEM image showed that some ultrastructural alterations in nerve cells, including neurodendron degeneration, membranous structure disruption and lysosome increase in the olfactory bulb, slight dilation in the rough endoplasmic reticulum and lysosome increase in the hippocampus were induced by the nano-sized Fe₂O₃ treatment. In contrast, in the submicron-sized Fe₂O₃ treated mice, slightly swollen mitochondria and some vacuoles were observed in the olfactory bulb and hippocampus, respectively. These results indicate that intranasal exposure of Fe₂O₃ nanoparticles could induce more severe oxidative stress and nerve cell damage in the brain than the larger particle did. This is the first study to compare the neurotoxicity of nano- and submicron-sized Fe₂O₃ particles in the central nervous system after long-term and low-dose intranasal exposure.     

Neurovascular dysfunction in vascular dementia,Alzheimer’s and atherosclerosis

Efficient blood supply to the brain is of paramount importance to its normal functioning and improper blood flow can result in potentially devastating neurological consequences. Cerebral blood flow in response to neural activity is intrinsically regulated by a complex interplay between various cell types within the brain in a relationship termed neurovascular coupling. The breakdown of neurovascular coupling is evident across a wide variety of both neurological and psychiatric disorders including Alzheimer’s disease. Atherosclerosis is a chronic syndrome affecting the integrity and function of major blood vessels including those that supply the brain, and it is therefore hypothesised that atherosclerosis impairs cerebral blood flow and neurovascular coupling leading to cerebrovascular dysfunction. This review will discuss the mechanisms of neurovascular coupling in health and disease and how atherosclerosis can potentially cause cerebrovascular dysfunction that may lead to cognitive decline as well as stroke. Understanding the mechanisms of neurovascular coupling in health and disease may enable us to develop potential therapies to prevent the breakdown of neurovascular coupling in the treatment of vascular brain diseases including vascular dementia, Alzheimer’s disease and stroke.     

Ultrasonic stimulation of the brain to enhance the release of dopamine – A potential novel treatment for Parkinson’s disease

Parkinson’s disease (PD) is characterized by the decrease of dopamine (DA) production and release in the substantia nigra and striatum regions of the brain. Transcranial ultrasound has been exploited recently for neuromodulation of the brain in a number of fields. We have stimulated DA release in PC12 cells using low-intensity continuous ultrasound (0.1 W/cm² − 0.3 W/cm², 1 MHz), 12 h after exposure at 0.2 W/cm², 40 s, the amount of DA content eventually increased 78.5% (p = 0.004). After 10-day ultrasonic treatment (0.3 W/cm², 5 min/d), the DA content in the striatum of PD mice model restored to 81.07% of the control (vs 43.42% in the untreated PD mice model). In addition to this the locomotion activity was restored to the normal level after treatment. We suggest that the low intensity ultrasound-induced DA release can be attributed to a combination of neuron regeneration and improved membrane permeability produced by the mechanical force of ultrasound. Our study indicates that the application of transcranial ultrasound applied below FDA limits, could provide a candidate for relatively safe and noninvasive PD therapy through an amplification of DA levels and the stimulation of dopaminergic neuron regeneration without contrast agents.     

Dopamine mediated iron release from ferritin is enhanced at higher temperatures: Possible implications for fever-induced Parkinson's disease

A new molecular mechanism is proposed to explain the pathogenesis of fever-induced Parkinson's disease. This proposal is based on dopamine and 6-hydroxydopamine-mediated free iron release from ferritin magnetic nanoparticles, which is enhanced at higher temperatures, and which may lead to substantial peroxidation and injury of lipid biomembranes of the substantia nigra in the brain.     

On the use of information theory for detecting upper limb motor dysfunction: An application to Parkinson’s disease

Parkinson’s disease (PD) is a chronic neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra, decreased striatal dopamine levels, and consequent extrapyramidal motor dysfunctions. Several potential early diagnostic markers of PD have been proposed. Since they have not been validated in presymptomatic PD, the diagnosis and monitoring of the disease is based on subjective clinical assessment of cognitive and motor symptoms. In this study, we investigated interjoint coordination synergies in the upper limb of healthy and parkinsonian subjects during the performance of unconstrained linear-periodic movements in a horizontal plane using the mutual information (MI). We found that the MI is a sensitive metric in detecting upper limb motor dysfunction, thus suggesting that this method might be applicable to quantitatively evaluating the effects of the antiparkinsonian medication and to monitor the disease progression.     

Time course and progression of wild type α-Synuclein accumulation in a transgenic mouse model

Background

Progressive accumulation of α-synuclein (α-Syn) protein in different brain regions is a hallmark of synucleinopathic diseases, such as Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy. α-Syn transgenic mouse models have been developed to investigate the effects of α-Syn accumulation on behavioral deficits and neuropathology. However, the onset and progression of pathology in α-Syn transgenic mice have not been fully characterized. For this purpose we investigated the time course of behavioral deficits and neuropathology in PDGF-β human wild type α-Syn transgenic mice (D-Line) between 3 and 12 months of age.

Results

These mice showed progressive impairment of motor coordination of the limbs that resulted in significant differences compared to non-transgenic littermates at 9 and 12 months of age. Biochemical and immunohistological analyses revealed constantly increasing levels of human α-Syn in different brain areas. Human α-Syn was expressed particularly in somata and neurites of a subset of neocortical and limbic system neurons. Most of these neurons showed immunoreactivity for phosphorylated human α-Syn confined to nuclei and perinuclear cytoplasm. Analyses of the phenotype of α-Syn expressing cells revealed strong expression in dopaminergic olfactory bulb neurons, subsets of GABAergic interneurons and glutamatergic principal cells throughout the telencephalon. We also found human α-Syn expression in immature neurons of both the ventricular zone and the rostral migratory stream, but not in the dentate gyrus.

Conclusion

The present study demonstrates that the PDGF-β α-Syn transgenic mouse model presents with early and progressive accumulation of human α-Syn that is accompanied by motor deficits. This information is essential for the design of therapeutical studies of synucleinopathies.