骨桥蛋白在神经退行性疾病中的作用

骨桥蛋白(OPN)是一种分子量约为60 KDa的糖基化磷蛋白,广泛分布于骨、脑、肾、肺以及肝等多种重要的脏器组织中.该蛋白通过与整合素、CD44V等受体结合,参与应激反应,癌症,骨重建,炎性反应以及感染等多种生理病理性进展.由于早期分泌OPN能够诱发细胞的激活,故OPN也被称为ETA-1(早期T淋巴细胞激活因子-1).目前发现,OPN存在两种形式:一种是分泌型骨桥蛋白(sOPN),另一种是胞内型骨桥蛋白(iOPN).在体内,二者通过不同的作用途径参与免疫调节过程.近年来,随着分子生物学的进展以及对神经退行性疾病研究的不断深入,发现OPN在神经退行性疾病中似乎发挥着双刃剑的作用,即在某些特定情况下,它能够激发神经毒性和神经元的死亡;而在其他情况下,它起到的是神经保护性作用.本文就OPN的结构特点、生物学功能以及在神经退行性病变中的作用进行简要归纳.     

蛋白质组学在神经退行性疾病中的研究进展

蛋白质组学是后基因组时代兴起的新型学科,是从整体水平对蛋白质的综合分析。阿尔茨海默病、帕金森病、肌萎缩侧索硬化症等是最常见的神经退行性疾病。应用蛋白质组学对它们进行研究,不仅可从蛋白质水平上揭示疾病的本质,还有助于全面探讨其病理机制,建立诊断标准,发现药物治疗靶点。     

High-Density Lipoprotein Aggregated by Oxidation Induces Degeneration of Neuronal Cells

Abstract: We have previously reported that high-density lipoprotein (HDL) exhibits antineuritogenic effects on chicken cerebral cells in culture. In the present study, we show the effects of HDLs, oxidized by UV irradiation or heating, on chicken cerebral neurons in culture. Both treatments produced several physical and chemical changes in the HDLs, i.e., formation of lipid peroxides, enlargement of HDL diameters, an increased exposure of the tryptophan groups of the apolipoprotein A-I to a more hydrophilic environment, formation of bityrosines, and cross-linking of apolipoprotein A-I. When these treatments were performed in the absence of EDTA, most of the modifications described above were more intense and HDLs formed a macroaggregate that displays a rosette-like structure. The aggregated HDLs produced neurodegeneration and death when added to both undifferentiated and differentiated cerebral neurons in culture. This process was accompanied by the disorganization of the cellular microtubular cytoskeleton and hyperphosphorylation of the microtubule-associated protein tau. Native HDL or HDLs treated in the presence of EDTA inhibited the neuritogenesis of undifferentiated neurons but did not show any significant effect on the differentiated neurons in culture. The effects on the cellular cytoskeleton and morphology of aggregated HDLs recall those of the fibrillar β-amyloid peptide. The present results suggest that aggregated HDLs could participate in neurodegeneration associated with oxidative stress in the CNS.     

Neuronal apoptosis in mouse trisomy 16: mediation by caspases

Hippocampal neurons from the trisomy 16 (Ts16) mouse, a potential animal model of Down's syndrome (trisomy 21) and neurodegenerative disorders such as Alzheimer's disease (AD), die at an accelerated rate in vitro. Here, we present evidence that the accelerated neuronal death in Ts16 occurs by apoptosis, as has been reported for neurons in AD. First, the nuclei of dying Ts16 neurons are pyknotic and undergo DNA fragmentation, as revealed by terminal transferase-mediated dUTP nick end-labeling. Second, the accelerated death of Ts16 neurons is prevented by inhibitors of the caspase family of proteases, which are thought to act at a late, obligatory step in the apoptosis pathway. In the presence of maximally effective concentrations of caspase inhibitors, Ts16 neuron survival was indistinguishable from that of control neurons. These results suggest that overexpression of one or more genes on mouse chromosome 16 leads to caspase-mediated apoptosis in Ts16 neurons.     

Oxidative Stress Induces Dephosphorylation of τ in Rat Brain Primary Neuronal Cultures

Abstract: Oxidative stress and free radical damage have been implicated in the neurodegenerative changes characteristic of several neurodegenerative diseases, including Alzheimer's disease. There is experimental evidence that the neurotoxicity of β-amyloid is mediated via free radicals, and as the deposition of β-amyloid apparently precedes the formation of paired helical filaments (PHF) in Alzheimer's disease, we have investigated whether subjecting primary neuronal cultures to oxidative stress induces changes in the phosphorylation state of the principal PHF protein τ that resemble those found in PHF-τ. Contrary to causing an increase in τ phosphorylation, treatment of neurones with hydrogen peroxide caused a dephosphorylation of τ and so we conclude that oxidative stress is not the direct cause of τ hyperphosphorylation and hence of PHF formation.     

Parkinson's Disease: Mechanisms of Neuronal Death

Based on the published data, the authors analyze in detail events resulting in the death of neurons in the substantia nigra (according to the apoptosis scenario) and in the development of Parkinson's disease (idiopathic parkinsonism).     

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2^Y1571H cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2^R611Q, which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.     

The role of mitochondria in inherited neurodegenerative diseases

In the past decade, the genetic causes underlying familial forms of many neurodegenerative disorders, such as Huntington's disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich ataxia, hereditary spastic paraplegia, dominant optic atrophy, Charcot-Marie-Tooth type 2A, neuropathy ataxia and retinitis pigmentosa, and Leber's hereditary optic atrophy have been elucidated. However, the common pathogenic mechanisms of neuronal death are still largely unknown. Recently, mitochondrial dysfunction has emerged as a potential 'lowest common denominator' linking these disorders. In this review, we discuss the body of evidence supporting the role of mitochondria in the pathogenesis of hereditary neurodegenerative diseases. We summarize the principal features of genetic diseases caused by abnormalities of mitochondrial proteins encoded by the mitochondrial or the nuclear genomes. We then address genetic diseases where mutant proteins are localized in multiple cell compartments, including mitochondria and where mitochondrial defects are likely to be directly caused by the mutant proteins. Finally, we describe examples of neurodegenerative disorders where mitochondrial dysfunction may be 'secondary' and probably concomitant with degenerative events in other cell organelles, but may still play an important role in the neuronal decay. Understanding the contribution of mitochondrial dysfunction to neurodegeneration and its pathophysiological basis will significantly impact our ability to develop more effective therapies for neurodegenerative diseases.     

Neural-specific Deletion of FIP200 Leads to Cerebellar Degeneration Caused by Increased Neuronal Death and Axon Degeneration

FIP200 (FAK family-interacting protein of 200 kDa) is a conserved protein recently identified as a potential mammalian counterpart of yeast autophagy protein Atg17. However, it remains unknown whether mammalian FIP200 regulates autophagy in vivo. Here we show that neural-specific deletion of FIP200 resulted in cerebellar degeneration accompanied by progressive neuronal loss, spongiosis, and neurite degeneration in the cerebellum. Furthermore, deletion of FIP200 led to increased apoptosis in cerebellum as well as accumulation of ubiquitinated protein aggregates without any deficiency in proteasome catalytic functions. We also observed an increased p62/SQSTM1 accumulation in the cerebellum and reduced autophagosome formation as well as accumulation of damaged mitochondria in the mutant mice. Lastly, analysis of cerebellar neurons in vitro showed reduced JNK activation and increased susceptibility to serum deprivation-induced apoptosis in cerebellar neurons from the mutant mice. Taken together, these results provide strong genetic evidence for a role of FIP200 in the regulation of neuronal homeostasis through its function in autophagy in vivo.     

Tuberin is a component of lipid rafts and mediates caveolin-1 localization: role of TSC2 in post-Golgi transport

Mutations of the TSC2 gene lead to the development of hamartomas in tuberous sclerosis complex. Their pathology exhibits features indicative of defects in cell growth, proliferation, differentiation, and migration. We have previously shown that tuberin, the TSC2 protein, resides in multiple subcellular compartments and as such may serve multiple functions. To further characterize the microsomal pool of tuberin, we found that it cofractionated with caveolin-1 in a low-density, Triton X-100-resistant fraction (i.e., lipid rafts) and regulated its localization. In cells lacking tuberin, most of the endogenous caveolin-1 was displaced from the plasma membrane to a Brefeldin-A-sensitive, post-Golgi compartment distinct from the endosome and lysosome. Correspondingly, there was a paucity of caveolae at the plasma membrane of Tsc2-/- cells. Reintroduction of TSC2, but not a disease-causing mutant, reversed the caveolin-1 localization to the membrane. Exogenously expressed caveolin-1-GFP and vesicular stomatitis virus G protein, VSVG-GFP in the Tsc2-/- cells failed to be transported to the plasma membrane and were retained in distinct post-Golgi vesicles. Our data suggest a role of tuberin in regulating post-Golgi transport without apparent effects on protein sorting. The presence of mislocalized proteins in Tsc2-/- cells may contribute to the abnormal signaling and cellular phenotype of tuberous sclerosis.     

The Role of Glutathione in Dopaminergic Neuronal Survival

Abstract: An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1-methyl-4-phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1-methyl-4-phenylpyridinium toxicity, although 1-methyl-4-phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1-methyl-4-phenylpyridinium to cause the selective death of dopaminergic neurons.     

The blood–brain barrier in neurodegenerative disease: a rhetorical perspective

Recent studies suggest that the function of the blood–brain barrier (BBB) is not static under normal physiologic conditions and is likely altered in neurodegenerative disease. Prevailing thinking about CNS function, and neurodegenerative disease in particular, is neurocentric excluding the impact of factors outside the CNS. This review challenges this perspective and discusses recent reports suggesting the involvement of peripheral factors including toxins and elements of adaptive immunity that may not only play a role in pathogenesis, but also progression of neurodegenerative diseases. Central to this view is neuroinflammation. Several studies indicate that the neuroinflammatory changes that accompany neurodegeneration affect the BBB or its function by altering transport systems, enhancing immune cell entry, or influencing the BBB's role as a signaling interface. Such changes impair the BBB's normal homeostatic function and affect neural activity. Moreover, recent studies reveal that alterations in BBB and its transporters affect the entry of drugs used to treat neurodegenerative diseases. Incorporating BBB compromise and dysfunction into our view of neurodegenerative disease leads to the inclusion of peripheral mediators in its pathogenesis and progression. In addition, this changing view of the BBB raises interesting new therapeutic possibilities for drug delivery as well as treatment strategies designed to reinstate normal barrier function.     

Protection of Flupirtine on β-Amyloid-Induced Apoptosis in Neuronal Cells In Vitro: Prevention of Amyloid-Induced Glutathione Depletion

Abstract: Effective drugs are not available to protect against β-amyloid peptide (Aβ)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment Aβ25-35 at 1 µ M in the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 µ M Aβ25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5 µg/ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with Aβ25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like DNA fragmentation pattern and by the TUNEL technique. Aβ25-35-induced DNA fragmentation could be abolished by preincubation of the cells with 1 µg/ml flupirtine. Incubation with Aβ25-35 reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/10⁶ cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where Aβ accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.     

Role for dopamine in malonate-induced damage in vivo in striatum and in vitro in mesencephalic cultures

Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.     

Oligodendrocytes in neurodegenerative diseases

Oligodendrocyte is a highly specialized glial cell type in the vertebrate central nervous system, which guarantees the long-distance transmission of action potential by producing myelin sheath wrapping adjacent axons. Disrupted myelin and oligodendrocytes are hallmarks of some devastating neurological diseases, such as multiple sclerosis, although their contribution to neurodegeneration in a given disease is still controversial. However, accumulating evidence from clinical studies and genetic animal models implicates oligodendrocyte dysfunction as one of major events in the processes of initiation and progression of neurodegeneration. In this article, we will review recent progress in understanding non-traditional function of oligodendrocytes in neuronal support and protection independent of myelin sheath and its possible contribution to neurodegeneration. Oligodendrocytes play a pivotal role in neurodegenerative diseases among which special emphasis is given to multiple system atrophy and Alzheimer’s disease in this review.     

Initiation of Neuronal Damage by Complex I Deficiency and Oxidative Stress in Parkinson's Disease

Oxidative stress and partial deficiencies of mitochondrial complex I appear to be key factors in the pathogenesis of Parkinson's disease. They are interconnected; complex I inhibition results in an enhanced production of reactive oxygen species (ROS), which in turn will inhibit complex I. Partial inhibition of complex I in nerve terminals is sufficient for in situ mitochondria to generate more ROS. H2O2 plays a major role in inhibiting complex I as well as a key metabolic enzyme, alpha-ketoglutarate dehydrogenase. The vicious cycle resulting from partial inhibition of complex I and/or an inherently higher ROS production in dopaminergic neurons leads over time to excessive oxidative stress and ATP deficit that eventually will result in cell death in the nigro-striatal pathway.     

Inflammatory Mediator Stimulation of Astrocytes and Meningeal Fibroblasts Induces Neuronal Degeneration via the Nitridergic Pathway

Abstract: The role of inflammatory cytokines in the pathogenesis of neurological disorders is not entirely clear. The neurotoxic effects of cytokines, and perhaps indirectly bacterial endotoxins, could be mediated by the stimulation of immunocompetent cells in the brain to produce toxic concentrations of nitric oxide (NO) and reactive nitrogen oxides. NO is a short-lived, diffusible molecule that has a variety of biological activities including vasorelaxation, neurotransmission, and cytotoxicity. Both constitutive and inducible NO synthase has been described in astrocytes in vitro. Here we demonstrate that newborn mouse cortical astrocytes, when coincubated with neonatal mouse cerebellar granule cells or hippocampal neurons, induced neurotoxicity upon stimulation with endotoxin (lipopolysaccharide) (ED₅₀ 30 ng/ml). Astrocytes were unresponsive to the cytokines tumor necrosis factor-α or interleukin-1β individually, but exhibited a marked synergistic stimulation in their combined presence. Moreover, meningeal fibroblasts treated with tumor necrosis factor-α, but not interleukin-1β or lipopolysaccharide, elaborated neurotoxicity for cocultured granule cells (ED₅₀ 30 U/ml). In cocultures of immunostimulated astrocytes or meningeal fibroblasts, neurotoxicity was blocked by the NO synthase inhibitors N^ω-nitro-l -arginine and N^ω-nitro-d -arginine methyl ester, and by oxyhemoglobin, which inactivates NO. Astroglial-induced neurotoxicity was not affected by N-methyl-d -aspartate receptor antagonists. Superoxide dismutase, which degrades superoxide anion, attenuated astrocyte- and fibroblast-mediated neurotoxicity, indicating that endogenous superoxide anion may react with NO to form toxic peroxynitrite and its breakdown products. These findings suggest a potentially important role for glial- and meningeal fibroblast-induced NO synthase in the pathophysiology of CNS disease states of immune or inflammatory origin.     

结节性硬化症伴癫痫患儿临床特征和基因型分析

摘要 目的：探讨结节性硬化症（TSC）伴癫痫患儿的临床特征和基因型特点,旨在了解 TSC伴癫痫患儿的临床表现,以及表型与基因型的相关性,为临床诊治提供更有效的方案。方法：回顾性分析 2019年 12月至 2021年 1月安徽省儿童医院神经内科收治的 10例 TSC伴癫痫患儿的临床表现,采用芯片捕获高通量测序以及 Sanger测序验证,对 TSC伴癫痫患儿及父母进行基因检测,分析其临床及遗传变异的特征。结果：10例中男性 4例,女性 6例,首次发作痉挛 3例,局灶性 7例,70.00%首发年龄小于 1岁。临床表现：90.00%皮肤病变,80.00%心脏横纹肌瘤,20.00%眼底异常,未见肾脏、肝脏、肺脏病变。视频脑电图显示 60.00%痫样波位于额颞区,100.00%伴神经影像学皮层 /皮层下异常,90.00%双侧室管膜 /室管膜下异常,10.00%室管膜下巨细胞星形细胞瘤。研究患儿均完善基因检测,3例为 TSC1基因突变,7例为 TSC2突变,包括错义、移码及剪接位点突变,2例检测出家族变异,7例均未检测出家族变异。结论：TSC伴癫痫患儿男女发病无明显差异性,散发病例多见,发病年龄多为 1岁内,首次发作常为局灶性。视频脑电图痫样波额颞区为主,头颅影像学多为皮层 /皮层下及双侧室管膜 /室管膜下异常。TSC2突变较 TSC1突变常见,临床表现严重,痉挛发生率高,发病年龄小,多控制不佳。早期基因检测,对于发病年龄小,疗效欠佳患儿尤为重要。