Divalent transition-metal ions (Cu and Zn) in the brains of epileptogenic and normal mice

The concentrations of Cu²⁺ and Zn²⁺ in 3 strains of mice were determined spectrophotometrically. The brain of the inborn audiogenic mouse (DBA/2J) contains higher levels of Zn²⁺ and Cu²⁺ than those found in the normal mouse (CBA/Ca or Parkes). Small differences in the metallic content in the whole brains of audiogenic and normal mice are accentuated in the hippocampus and the colliculus.     

丙泊酚可调节缺血再灌注损伤脊髓Ca2+, Mg2+, Cu2+，Zn2+的平衡

检测血清和脊髓匀浆中Ca2+、Mg2+、Cu2+、Zn2+的变化规律,揭示丙泊酚对缺血再灌注损伤脊髓保护作用的可能机制。结果发现,随着缺血再灌注损伤时间的延长,兔血清Ca2+,Cu2+浓度逐渐升高,Mg2+,Zn2+浓度逐渐下降,至脊髓损伤后7d,以上离子变化最明显;缺血再灌注损伤7 d,兔缺血脊髓匀浆中的各离子浓度变化与血清中相一致。给予丙泊酚干预后,缺血再灌注期间兔血清和脊髓匀浆中Ca2+、Mg2+、Cu2+、Zn2+浓度均无显著的波动。提示丙泊酚可通过稳定或恢复脊髓缺血再灌注损伤区金属离子的平衡发挥对脊髓缺血再灌注损伤的保护作用。     

Modification of hippocampal excitability in brain slices pretreated with a low nanomolar concentration of Zn2+

Synaptic Zn²⁺ homeostasis may be changed during brain slice preparation. However, much less attention has been paid to Zn²⁺ in artificial cerebrospinal fluid (ACSF) used for slice experiments than has been paid to Ca²⁺. The present study assesses addition of Zn²⁺ to ACSF, focused on hippocampal excitability after acute brain slice preparation. When the static levels of intracellular Zn²⁺ and Ca²⁺ were compared between brain slices prepared with conventional ACSF without Zn²⁺ and those pretreated with ACSF containing 20 nM ZnCl₂ for 1 hr, both levels were almost the same. On the other hand, intracellular Ca²⁺ levels were significantly increased in the stratum lucidum of the control brain slices after stimulation with high K⁺, although the increase was significantly suppressed by the pretreatment with ACSF containing Zn²⁺, suggesting that neuronal excitation is enhanced in brain slices prepared with ACSF without Zn²⁺. The increase in extracellular Zn²⁺ level, an index of glutamate release, after stimulation with high K⁺ was also significantly suppressed by pretreatment with ACSF containing Zn²⁺. When mossy fiber excitation was assessed in brain slices with FM4‐64, an indicator of presynaptic activity, attenuation of FM 4‐64 fluorescence based on presynaptic activity was suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn²⁺. The present study indicates that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn²⁺. It is likely that a low nanomolar concentration of Zn²⁺ is necessary for ACSF. © 2015 Wiley Periodicals, Inc.     

A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis

About 15–20% of patients with familial amyotrophic lateral sclerosis (ALS) carry one of several missense mutations in the gene for Cu,Zn superoxide dismutase (SOD1). We have previously reported on an animal model of this disease produced by the transgenic expression of a mutant form of human SOD1 containing a Gly⁹³→Ala amino acid substitution. Several lines of transgenic mice were produced, characterized by a differing tempo and severity of disease that generally correlated with the number of mutant gene copies that these lines expressed. We reported that mice expressing high copy numbers (18–25) developed a disease with a relatively short course and with a pathology mainly characterized by severe vacuolar degeneration of motor neurons and their processes. Lewy-like bodies and swollen axons were also present. The exquisite localization to motor neurons was the feature that made the pathology in these overexpressors germane to the human disease. Severe vacuolar degeneration, however, was considered to be at variance with human ALS, in which similar changes have not been described. In the present study, we have made a temporal characterization of microscopic and immunohistochemical changes in a line of transgenic mice expressing lower copy numbers of the mutant gene. These mice, designated G5/G5, survive more than 400 days and present pathological changes which are virtually identical to those in the human disease. In fact, in these animals, anterior horn cell depletion, atrophy, astrocytosis, and the presence of numerous ubiquitinated Lewy-like bodies and axonal swellings are the main pathological features, while vacuolar pathology is minimal. This study underscores the importance of the level of expression of the mutant enzyme in the resulting clinical and pathological disease, and supports the value of this transgenic model as an excellent tool for investigating both pathogenesis of human ALS and possible therapeutic avenues. Received: 11 September 1996 / Revised, accepted: 18 October 1996     

Stabilization of mutant Cu/Zn superoxide dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the disease progression in familial amyotrophic lateral sclerosis mice

Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)-linked mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A-mice) and G85R (G85R-mice) genes develop limb paresis. Introduction of human wild type SOD1 (hWT-SOD1) gene, which does not cause motor impairment by itself, into different FALS mice resulted in different effects on their clinical courses, from no effect in G85R-mice to acceleration of disease progression in G93A-mice. However, the molecular mechanism which causes the observed difference, has not been clarified. We hypothesized that the difference might be caused by the stability of mutant SOD1 proteins. Using a combination of mass spectrometry and enzyme-linked immunosorbent assay, we found that the concentration of G93A-SOD1 protein was markedly elevated in tissues of transgenic mice carrying both G93A- and hWT-SOD1 genes (G93A/hWT-mice) compared to that in G93A-mice, and also found that the concentration of G93A-SOD1 protein had a close relation to the disease duration. The concentration of metallothionein-I/II in the spinal cord, reflecting the degree of copper-mediated oxidative stress, was highest in G93A/hWT-mice, second in G93A-mice, and normal in the mice carrying hWT-SOD1 gene. These results indicated that the increase of G93A-SOD1 protein was responsible for the increase of oxidative stress and disease acceleration in G93A/hWT-mice. We speculate that coexpression of hWT-SOD1 protein is deleterious to transgenic mice carrying a stable mutant such as G93A-SOD1, because this mutant protein is stabilized by hWT-SOD1 protein, but not to transgenic mice carrying an unstable mutant such as G85R-SOD1, because this mutant protein is not stabilized by hWT-SOD1.     

Intracellular Zn2+ signaling in cognition

Brain zinc homeostasis is strictly controlled under healthy conditions, indicating the importance of zinc for physiological function in the brain. A part of zinc in the brain exists in the synaptic vesicles, is released from a subclass of glutamatergic neurons (i.e., zincergic neurons), and serves as a signal factor (Zn²⁺ signal) in the intracellular (cytosol) compartment as well as in the extracellular compartment. Zn²⁺ signaling is dynamically linked to glutamate signaling and may be involved in synaptic plasticity, such as long‐term potentiaion and cognitive activity. In zincergic synapses, intracellular Zn²⁺ signaling in the postsynaptic neurons, which is linked to Zn²⁺ release from zincergic neuron terminals, plays a role in cognitive activity. When nonzincergic synapses participate in cognition, on the other hand, it is possible that intracellular Zn²⁺ signaling, which is due mainly to Zn²⁺ release from the internal stores and/or metallothioneins, also is involved in cognitive activity, because zinc‐dependent system such as zinc‐binding proteins is usually required for cognitive process. Intracellular Zn²⁺ dynamics may be modified via an endocrine system activity, glucocorticoid secretion in both zincergic and nonzincergic neurons, which is linked to a long‐lasting change in synaptic efficacy. On the basis of the evidence of cognitive decline caused by the lack and/or the blockade of synaptic Zn²⁺ signaling, this article summarizes the involvement of intracellular Zn²⁺ signaling in zincergic synapses in cognition and a hypothetical involvement of that in nonzincergic synapses. © 2014 Wiley Periodicals, Inc.     

Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS)

Two different lines of mice, G1 and G20, carrying a transgene for a mutant form of Cu,Zn SOD, found in a family with familial amyotrophic lateral sclerosis (FALS), develop clinical and pathological changes which are, in their late stages, strikingly similar to those in human disease. We have analyzed the distribution and characteristics of lesions in the central and peripheral nervous systems of such mice. The most affected structure was the spinal cord, followed by the medulla, pons and midbrain. The early stages of the disease were characterized by vacuolar degeneration of anterior horn neurons and their processes, while, in the late stages, the main changes consisted of neuronal loss and atrophy of the anterior horns and the deposition in these areas of multiple filamentous inclusions resembling Lewy bodies. In the late stages of the disease, the white matter of the spinal cord was also involved, particularly in the anterior and lateral columns. Posterior columns were also involved, but to a much lesser degree. The brainstem structures also showed vacuolar degeneration of several motor nuclei and of several groups neurons in the reticular formation. Anterior roots and peripheral nerves showed the classical features of Wallerian degeneration. The dorsal root ganglia, with rare exceptions, were unremarkable. The posterior roots showed mild changes in the most severely affected mice. Changes in these two affected lines were compared to changes in mice overexpressing wild type, rather than mutant human Cu,Zn SOD. These mice never developed clinical disease, although, pathologically, they developed very mild vacuolar changes in the anterior horns of the spinal cord and in motor axons. This study shows that although simple overexpression of SOD may be injurious to motor neurons, albeit very mildly, the mutant form is necessary to produce both clinical disease and severe pathological changes which, in the chronic stage of the disease, have striking similarities to human familial ALS. A dominant gain of function, therefore, is the most likely pathogenesis of tissue injury induced by mutations in Cu,Zn SOD.     

Cu, Zn superoxide dismutase in patients with dementia of the Alzheimer type

Cu, Zn superoxide dismutase (SOD) levels in the serum, cerebrospinal fluid (CSF) and skin fibroblasts of patients with dementia of the Alzheimer type (DAT) were estimated using enzyme immunoassay. The SOD mRNA level in the skin fibroblasts was also determined by the Northern blot analysis. As compared with the age-matched control groups of neurological patients without dementia, the AD group consisting of patients with DAT at ages under 65 years of age as well as the SDAT group of patients with DAT at ages over 65 years of age showed no significant changes in serum or CSF SOD levels. However, the skin fibroblast SOD- and SOD mRNA levels, which were correlate each other, were significantly higher in the AD group (p<0.05), while lower in the SDAT group (p<0.05). These results suggest that the determination of the SOD level of skin fibroblast may be useful for diagnosis of DAT and that the abnormality of SOD may play an important role in developing DAT.     

Sporadic ALS associated with the D90A Cu,Zn superoxide dismutase mutation in Russia

Twenty blood samples from Russian patients (Moscow) with idiopathic motor neurone disease were analysed for mutations in the Cu,Zn superoxide dismutase (Cu,Zn SOD) gene. Two patients (10%) with the amyotrophic lateral sclerosis (ALS) form of the disease were found to have a disease-related mutation. One patient appears to have autosomal recessive adult-onset ALS associated with homozygosity for D90A and presents the characteristic phenotype of very slowly ascending paresis with both lower and upper motor neurone signs. Another patient, heterozygous for D90A, presents ALS with lumbar onset and rapid progression. This is the first report of a Cu,Zn SOD mutation in ALS in Russia.     

Molecular and synaptic changes in the hippocampus underlying superior spatial abilities in pre-symptomatic G93A+/+ mice overexpressing the human Cu/Zn superoxide dismutase (Gly93 --> ALA) mutation

Although amyotrophic lateral sclerosis (ALS) is mainly considered as a motor disease, extramotor neural and cognitive alterations have also been reported in ALS patients. There is evidence that mutations in the Cu/Zn superoxide dismutase (SOD1) gene are implicated in about 20% of familiar ALS and transgenic mice overexpressing the human Cu/Zn superoxide dismutase (GLY(93) --> ALA) mutation show an ALS-like phenotype. However, while motor behavior has been extensively analyzed in these mutants, little is known on their cognitive abilities. To characterize the pre-symptomatic cognitive profile of G93A+/+ mice, we estimated their capability to detect spatial novelty and examined several indexes of their hippocampal function. We found an enhancement of spatial abilities in mutant mice associated with (1) a higher expression of hippocampal AMPA subunit GluR1 mRNA and of GluR1 protein levels, and (2) an increased induction and maintenance of long-term potentiation (LTP) at Schaffer collateral-CA1 synapses. Thus, before leading to extensive neuronal excitotoxicity, the high endogenous levels of glutamate present in the brain of pre-symptomatic G93A+/+ mice could mediate site-specific molecular and synaptic changes providing favorable conditions to spatial information processing. These findings suggest that identification of pre-symptomatic behavioral changes in murine models of ALS may point to early neural abnormalities selectively associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene.     

Altered neurochemical parameters in the brains of mice (Frings) susceptible to audiogenic seizures

In this study, several biochemical aspects of serotonergic neurons were compared in mice susceptible to audiogenic seizures (Frings) and in mice not susceptible to audiogenic seizures (CF 1). Tryptophan hydroxylase (TPH) activity was significantly lower in brains of Frings mice compared to CF1 mice; however, brain levels of 5-HT were similar in both strains. The significantly lower TPH activity in Frings mice was due to the altered kinetic characteristics of TPH activity in this strain. TPH from Frings mice had a significantly lower apparent maximal velocity and a significantly higher affinity for the substrate, as indicated by a lower apparent Km for tryptophan. The uptake of tryptophan and accumulation of 5-HT (following pargyline) were similar in Frings and CF 1 mice. The levels of 5-HT in selected brain regions of Frings and CF 1 mice were also similar, but NE levels were higher in the cerebral cortex and DA levels were higher in the neostriatum of Frings mice. It is unclear at this point what role, if any, 5-HT plays in susceptibility to audiogenic seizures in brains of Frings mice.     

Immunohistochemical study on the distribution of Bcl-2 and Bax in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we performed immunohistochemical studies to investigate the changes of Bcl-2 and Bax in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation. In contrast to the controls, a high density of Bcl-2-IR astrocytes were detected all around the gray matter of the spinal cord of the mutant transgenic mice. Bcl-2-IR astrocytes were also detected in the cerebellum and brainstem of transgenic mice. Specific immunoreactivity for Bax was seen in the spinal cord and brainstem of transgenic mice. Immunostaining for Bax was identified only in neurons and not in glial cells. Our present study demonstrated the distribution of Bcl-2 and Bax in detail using immunohistochemical methods through the central nervous system of the transgenic mice, for the first time.     

Audiogenic seizure susceptibility of C57BL/6 ↔ DBA/2 allophenic mice

Allophenic mice composed of cells from a strain (DBA/2) susceptible to sound-induced seizures and cells from a strain (C57BL/6) resistant to them were produced by embryo aggregation techniques. Twenty-eight allophenic mice were tested for audiogenic seizure susceptibility. The results were compared with the genotypic composition of the coat melanocytes. For those animals with a predominance of one genotype or the other in the coat, their seizure phenotype was the same as that of the strain most represented in the coat. In contrast, those animals with major contributions ofboth genotypes in the coat demonstrated the entire spectrum of susceptibility phenotypes. Such results are likely to be a manifestation of a relatively small target tissue for the genes influencing the development of audiogenic seizure susceptibility.     

老年和成年APP转基因小鼠脑蛋白质组2-DE图谱比较

目的:比较老年和成年APP转基因小鼠脑蛋白质组双向电泳(2-DE)图谱差异,从蛋白质水平初步探索老年痴呆发病机制。方法:以固相pH梯度等电聚焦(IPG)为第一向,SDS-PAGE垂直电泳为第二向进行2-DE。图象分析软件Imagemaster 2D Elite分析电泳图谱。结果:老年和成年转基因小鼠脑组织2-DE图谱分别检出964和947个蛋白点。对两张电泳图进行匹配后,发现有17个蛋白点仅在老年转基因小鼠脑蛋白2-DE图谱检测到表达,而有5个蛋白点只在成年转基因小鼠检测到。部分蛋白在2组小鼠脑组织中含量发生了明显变化。结论:初步建立了AD动物模型差异表达蛋白质组学的技术方法;差异点的发现为研究AD机理及治疗AD提供了有益的线索。     

Calcineurin A and calbindin immunoreactivity in the spinal cord of G93A superoxide dismutase transgenic mice

A qualitative immunohistochemical study was performed on calcineurin A- and calbindin-positive neurons in the spinal cord of transgenic mice, an animal model of amyotrophic lateral sclerosis, carrying the G93A mutation of the Cu/Zn-superoxide dismutase gene. The results show that calcineurin A-immunoreactive motoneurons are affected by the neurodegenerative process; in contrast, calbindin-positive cells are selectively spared. The findings suggest that calcineurin plays a role as an accessory factor responsible for selective vulnerability in the neurodegenerative process of amyotrophic lateral sclerosis.     

突变SOD1 cDNA在大鼠皮层神经元定位和作用

目的:分析一个ALS家系突变铜、锌超氧化物歧化酶(Cu/Zn superoxide dismutase,SOD1)基因在原代培养的大鼠皮层神经元中的定位及作用。方法:将构建好的含有SOD1正常基因与突变基因在内的增强型绿色荧光蛋白真核表达载体(pEGFP-hSOD1和pEGFP-mSOD1)分别转染大鼠皮层神经元,转染后观察绿色荧光在细胞内的分布情况,同时设对照组,检测神经元的SOD1活性、MDA含量及细胞活性。结果:转染后荧光物质表达于胞浆内,与转染pEGFP-mSOD1质粒的神经元相比,转染pEGFP-hSOD1质粒神经元的SOD1活性、MTT值显著增高,而MDA、LDH水平显著降低。结论:突变SOD1蛋白的酶活性下降,使脂质过氧化物相对增多,导致神经元损伤,可能是该家系发病的主要原因之一。     

Protective effects of extracellular glutathione against Zn2+-induced cell death in vitro and in vivo

The central nervous system reserves high concentrations of free Zn(2+) in certain excitatory synaptic vesicles. In pathological conditions such as transient cerebral ischemia, traumatic brain injury, and kainic acid (KA)-induced seizure, free Zn(2+) is released in excess at synapses, which causes neuronal and glial death. We report here that glutathione (GSH) can be used as an effective means for protection of neural cells from Zn(2+)-induced cell death in vitro and in vivo. Chronic treatment with 35 microM Zn(2+) led to death of primary cortical neurons and primary astrocytes. The Zn(2+) toxicity of cortical neurons was partially protected by 1 mM of GSH, whereas the Zn(2+) toxicity of primary astrocyte cultures was blocked completely by 100 microM of GSH. To evaluate the beneficial effects of GSH in vivo, an excitotoxin-induced neural cell death model was established by intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 microg) KA, which produced selective neuronal death, especially in CA1 and CA3 hippocampal regions. The i.c.v. co-injection of 200 pmol of GSH significantly attenuated KA-induced neuronal cell death and reactive gliosis in hippocampus. The results of this study suggest the contribution of Zn(2+) in the excitotoxin-induced neural cell death model and a potential value of GSH as a therapeutic means against Zn(2+)-induced pathogenesis in brain.     

Autoradiographic distribution of Mu opioid receptors in the brains of Cu/Zn-superoxide dismutase mice

Superoxide dismutase (SOD) is an important free radical scavenging enzyme which dismutates the superoxide anion radical. We have evaluated the role of SOD in the regulation of opioid receptors by comparing the concentration of μ opioid receptors labeled with [³H]DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol) in SOD-transgenic (SOD-Tg) mice and their non-transgenic (Non-Tg) littermates. SOD-Tg mice had higher maximal binding capacity (Bmax) in the shell division of the nucleus accumbens (NAcshell) in comparison to Non-Tg littermates. There were no differences in Bmax in μ receptors in the core subdivision of the nucleus accumbens (NAc-core). There were no significant differences in receptor affinity (Kd) in either the NAc-shell or in the NAc-core. Moreover, there were no significant differences in either Bmax or Kd in the matrices nor in the patches of any of the striatal subdivisions. However, in a fashion similar to the situation in the NAc-shell, [³H]DAGO binding in the substantia nigra pars compacta (SNpc), the ventral tegmental area (VTA), and the ventral part of the central grey was significantly higher in the SOD-Tg mice in comparison to Non-Tg mice. The present results are discussed in terms of their support for a possible involvement of free radicals in the differences observed in various regions of the SOD-Tg and control mice, which differ in their ability to scavenge the superoxide anion. Moreover, because of the differences in β-opioid receptors in the NAc-shell, these transgenic mice might provide an excellent model in which to evaluate the role of the two accumbal subdivisions in paradigms of drug addiction, which appears to depend on the functional integrity of the mesocorticolimbic system. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Global view of transcriptome in the brains of aged NR2B transgenic mice

NR2B subunits are involved in regulating aging, in particular, age-related learning and memory deficits. We examined 19-month-old NR2B transgenic mice and their littermate controls. First, we detected ...