Differential skeletal muscle gene expression after upper or lower motor neuron transection

Causes of disuse atrophy include loss of upper motor neurons, which occurs in spinal cord injury (SCI) or lower motor neurons (denervation). Whereas denervation quickly results in muscle fibrillations, SCI causes delayed onset of muscle spasticity. To compare the influence of denervation or SCI on muscle atrophy and atrophy-related gene expression, male rats had transection of either the spinal cord or sciatic nerve and were sacrificed 3, 7, or 14 days later. Rates of atrophy increased gradually over the first week after denervation and then were constant. In contrast, atrophy after SCI peaked at 1 week, then declined sharply. The greater atrophy after SCI compared to denervation was preceded by high levels of ubiquitin ligase genes, MAFbx and MuRF1, which then also markedly declined. After denervation, however, expression of these genes remained elevated at lower levels throughout the 2-week time course. Interestingly, expression of the muscle growth factor, IGF-1 was increased at 3 days after denervation when fibrillation also peaks compared to SCI. Expression of IGF-1R, GADD45, myogenin, and Runx1 were also initially increased after denervation or SCI, with later declines in expression levels which correlated less well with rates of atrophy. Thus, there were significant time-dependent differences in muscle atrophy and MAFbx, MuRF1, and IGF-1 expression following SCI or denervation which may result from distinct temporal patterns of spontaneous muscle contractile activity due to injury to upper versus lower motor neurons.     

Cerebrospinal fluid profile of amyloid β42 (Aβ42), hTau and ubiquitin in North Indian Alzheimer's disease patients

Alzheimer's disease (AD) is the most common form of dementia, and is characterized by the degeneration of neurons and their synapses, and a higher number of amyloid plaques and neurofibrillary tangles (NFTs) compared with that found in non-demented individuals. Amyloid-β-peptides (Aβ) are major components of amyloid plaques in AD brain whereas NFTs are composed of Tau and associated with ubiquitin. The aim of the present study was to analyze the levels of Aβ42, hTau (total Tau) and ubiquitin in CSF of North Indian population. CSF Aβ42, Tau and ubiquitin were measured in CSF of AD patients as well as controls using ELISA assays. Here we report low Aβ42 levels in AD patients (324.24 ± 76.38 pg/ml) as compared to those in non-AD (NAD) (668.34 ± 43.13 pg/ml), neurological controls (NCs) (727.28 ± 46.49 pg/ml) and healthy controls (HCs) (976.47 ± 124.46 pg/ml). In contrast, hTau and ubiquitin levels were significantly high (568.65 ± 48.89 pg/ml and 36.82 ± 4.34 ng/ml, respectively) in AD patients compared to those in NAD, NC and HC. The hTau levels were 267.37 ± 36.64 pg/ml, 167.34 ± 44.27 pg/ml and 107.62 ± 24.27 pg/ml in NAD, NC and HC, respectively. Similarly, ubiquitin levels were 23.57 ± 2.32 ng/ml, 19.76 ± 3.64 ng/ml and 13.24 ± 4.56 ng/ml in NAD, NC and HC, respectively. In conclusion, low Aβ42 and high Tau–ubiquitin levels were found in North Indian AD patients.     

泛素连接酶LNX1促进外源PBK的泛素化和降解

 目的 研究LNX1对其相互作用蛋白PBK的泛素化和降解。方法 克隆、原核表达、纯化了一系列重组人LNX1截断体蛋白和LNX1全长蛋白;在体外泛素化体系中研究其对PBK的泛素化,哺乳动物细胞内研究其对外源PBK的泛素化和降解。结果 在体外泛素化体系中LNX1泛素化PBK,并研究了不同LNX1截断体对PBK泛素化的影响;发现在哺乳动物细胞内外源LNX1促进外源PBK的泛素化,进而导致其通过蛋白酶体降解。结论 研究发现了LNX1对外源PBK的泛素化和降解,为研究LNX1的生理功能提供了重要线索。     

反义阻断CROC-1基因表达导致细胞生长变慢

目的:建立CROC- 1 基因表达被阻断的FL-CROC- 1 ^-细胞系,研究CROC- 1 基因在细胞生长中的作用。方法: 运用反义技术将新近发现的人泛素缀合酶样蛋白基因CROC- 1 的适当长度cDNA片断克隆到本室改建的真核细胞表达载体pMAMneo-amp^-中,经限制性内切酶图谱分析筛选出反向插入的表达CROC- 1 反义RNA的重组质粒。将此反义表达重组质粒(pMAM-antiCROC- 1 )用改良的磷酸钙法转染人羊膜FL细胞并用含G418的培养基筛选,测定G418抗性的FL-CROC- 1 ^- 细胞系的生长速度。 结果:建立的FL-CROC- 1 ^- 细胞系在地塞米松诱导下CROC- 1 基因表达被反义抑制后,其生长速度较对照FL细胞及转染了载体pMAMneo-amp^- 的FL细胞(FL-MAMneo)的明显要慢(P＜0.05)。结论: 本研究成功地建立了CROC- 1 基因表达被阻断的FL-CROC- 1 ^- 细胞系,并提示CROC- 1 基因编码的泛素缀合酶样蛋白在细胞生长中起正调控作用。     

Frontotemporal lobar degeneration – tau as a pied piper?

Frontotemporal lobar degeneration (FTLD) is the second most-common form of cortical dementia in the presenium after Alzheimer disease. Clinically three disease entities can be distinguished: frontotemporal dementia, semantic dementia, and primary progressive aphasia. The underlying neuropathology can be classified into disorders with tau pathology (including Pick disease, corticobasal degeneration, progressive supranuclear palsy, and familial frontotemporal dementia with parkinsonism linked to chromosome 17 – FTDP-17), and into disorders that lack tau abnormalities (including dementia lacking distinctive histology and motor neuron disease inclusion dementia). The recent discovery of tau gene mutations in FTDP-17 brought tau to the center stage, but led to the erroneous trend of collectively grouping all forms of FTLD as tauopathies. However, clinicopathological and genetic studies strongly suggest that the majority of sporadic and familial FTLD cases are not associated with tau pathology and/or tau gene mutations. Furthermore, recent studies have linked several autosomal dominantly inherited familial frontotemporal dementia cases to a variety of gene loci on different chromosomes. Thus, this review is intended to summarize our current knowledge about the sporadic and familial FTLD disorders that lack tau pathology, and shall further strengthen the view that FTLD is heterogeneous, both in terms of clinicopathological phenotypes as well as genetic backgrounds. Electronic Publication     

阻断泛素-蛋白酶体通路诱导PC12细胞死亡和泛素阳性包涵体生成

目的探讨泛素蛋白酶体通路的功能障碍对于多巴胺能细胞的活力以及胞质内包涵体生成的影响。方法应用蛋白酶体抑制剂lactacystin(5μmol/L、10μmol/L、15μmol/L和20μmol/L)处理PC12细胞24h,MTT方法检测细胞活力,WesternBlot方法测定细胞内泛素化蛋白质水平,免疫荧光细胞化学染色观察泛素免疫阳性包涵体的生成。结果经5μmol/L、10μmol/L、15μmol/L和20μmol/Llactacystin处理24h后,PC12细胞的活力显著降低(细胞存活率分别为81.5%±3.6%、75.4%±2.4%、70.2%±2.7%和60.4%±3.9%),呈现剂量依赖性。WesternBlot证实对照组细胞内未检测到相对高分子质量的泛素化蛋白质;随着lactacystin作用浓度的增加,细胞内相对高分子质量泛素化蛋白质的含量逐渐增高。免疫荧光染色显示对照组中仅有极少数细胞内含有泛素阳性包涵体;20μmol/Llactacystin处理组中含有泛素阳性包涵体的细胞数目显著增多(P<0.01)。结论泛素蛋白酶体通路的功能缺失能诱导多巴胺能细胞死亡,造成细胞内泛素化蛋白质积聚,促进胞质内泛素阳性包涵体的生成,可能在帕金森病黑质多巴胺能神经元变性死亡和Lewy小体形成中发挥重要作用。     

Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease

Abnormal neuronal cytoplasmic inclusions (NCIs) containing aggregates of -internexin and the neurofilament (NF) subunits, NF-H, NF-M, and NF-L, are the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID). The disease has a clinically heterogeneous phenotype, including frontotemporal dementia, pyramidal and extrapyramidal signs presenting at a young age. NCIs are variably ubiquitinated and about half of cases also have neuronal intranuclear inclusions (NIIs), which are also ubiquitinated. NIIs have been described in polyglutamine-repeat expansion diseases, where they are strongly ubiquitin immunoreactive. The fine structure of NIIs of NIFID has not previously been described. Therefore, to determine the ultrastructure of NIIs, immunoelectron microscopy was undertaken on NIFID cases and normal aged control brains. Our results indicate that the NIIs of NIFID are strongly ubiquitin immunoreactive. However, unlike NCIs which contain ubiquitin, -internexin and NF epitopes, NIIs contain neither epitopes of -internexin nor NF subunits. Neither NIIs nor NCIs were recognised by antibodies to expanded polyglutamine repeats. The NII of NIFID lacks a limiting membrane and contains straight filaments of 20 nm mean width (range 11–35 nm), while NCIs contain filaments with a mean width of 10 nm (range 5–18 nm; t-test, P<0.001). Biochemistry revealed no differences in neuronal IF protein mobilities between NIFID and normal brain tissue. Therefore, NIIs of NIFID contain filaments morphologically and immunologically distinct from those of NCIs, and both types of inclusion lack expanded polyglutamine tracts of the triplet-repeat expansion diseases. These observations indicate that abnormal protein aggregation follows separate pathways in different neuronal compartments of NIFID.     

Frontotemporal lobar degeneration with motor neuron disease-type inclusions predominates in 76 cases of frontotemporal degeneration

This report presents the largest series of consecutive, neuropathologically confirmed cases of frontotemporal degeneration (FTD). Prior studies have found dementia lacking distinctive histology (DLDH) to be the most common pathology underlying the clinical diagnosis of FTD. In this series of 76 cases, 29 (38%) were found to have frontotemporal lobar degeneration with motor neuron disease-type inclusions (FTLD-MND-type) or FTLD-MND (with ALS), the most common neuropathological classification in our series. Only eight (11%) were classified as Picks disease. Several cases originally designated as DLDH could be reclassified as FTLD-MND-type based on current recommendations for classification of FTD.     

Cortical ubiquitin-positive inclusions in frontotemporal dementia without motor neuron disease: a quantitative immunocytochemical study

Ubiquitin-positive tau-negative inclusions were initially described in the rare form of frontotemporal dementia (FTD) associated with motor neuron disease. However, recent studies have indicated that these inclusions are also present in typical FTD, which is usually characterized by nonspecific histological changes. To examine the contribution of these inclusions to neuronal loss and to explore their relationship with disease duration, we performed a quantitative immunocytochemical analysis of 38 typical FTD cases. Relationships between neuron and ubiquitin inclusion densities as well as between duration of illness and neuropathological parameters was studied using linear regression in both univariate and multivariate models. Ubiquitin-positive tau-negative intracytoplasmic inclusions were present in 65.8% of cases in the dentate gyrus, 57.9% in temporal cortex and 31.6% in frontal cortex. The highest densities of ubiquitin-positive inclusions were consistently observed in the dentate gyrus, followed by the temporal and frontal cortex. There was no statistically significant relationship between neuron and ubiquitin-positive inclusion densities in any of the areas studied. In contrast, ubiquitin-positive inclusion densities in the dentate gyrus were negatively related to the duration of illness. Our data suggest that the development of ubiquitin-related pathology is the rule and not the exception in typical FTD, yet is not causally related to neuronal loss. They also reveal that the development of ubiquitin-positive inclusion densities in the dentate gyrus may be associated with a more aggressive form of the disease.