Grant Philip, Pant Harish C
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
J Alzheimers Dis. 2002 Aug;4(4):269-81. doi: 10.3233/jad-2002-4402.
At autopsy, a most distinctive pathology seen in Alzheimer's disease (AD) brains is numerous abnormal neurons filled with neurofibrillary tangles (NFTs) containing stable complexes of hyperphosphorylated tau (PHF), neurofilaments and various kinases, among other proteins. Though these neuronal aggregates have been actively studied, their nature and origin are still poorly understood. Our studies of regulation of phosphorylation in neurons of the squid giant fiber system, using P13(suc1) affinity chromatography, suggest that neuronal phosphorylation of cytoskeletal proteins is compartmentalized into active axonal and inactive cell body-specific multimeric complexes of kinases, substrates and phosphatases. To determine whether such compartment-specific phosphorylation complexes are present in human brains, we separated gray matter (enriched in cell bodies) and white matter (enriched in axons) from normal and AD brains and studied the total kinase activities in lysates, pellets and P13(suc1) complexes. In addition, Western blot analysis was used to characterize the proteins associated with P13(suc1) multimeric complexes extracted from gray and white matter. We tested the hypothesis that P13 phosphorylation complexes were abnormally compartmentalized in AD neurons with the more active complexes shifted to cell bodies (gray matter) instead of axons (white matter). We found that (1) endogenous and exogenous substrate-dependent kinase activities of AD and control brain extracts were similar in both gray and white matter. (2) Long post mortem times tend to erase any differences in kinase activity between control and AD extracts. In contrast to shorter post mortem times (4.5-10 hrs), long post mortem times (13-34 hrs) significantly minimize the variances in kinase activities between control and AD brain extracts suggesting that cell death and proteolysis may eliminate any intrinsic differences in enzyme activities. (3) Except for the significantly higher level of histone phosphorylation in control white extracts, the kinase activities of P13(suc1)-derived multimeric complexes from gray and white matter were also similar in control and AD brains. Here, too, variances between control and AD distributions were significantly different (p < 0.001-0.02) suggesting that the P13 complexes were different. We also found differences in the Western blot profiles of P13suc1-associated kinases and cytoskeletal proteins; higher expression of phosphorylated NF-H and PHF-tau in gray matter of AD brains was detected. We believe that such differences in P13 complexes from human control and AD brain samples displaying extensive heterogeneity in age, post mortem time and clinical history, may be important.
在尸检中，阿尔茨海默病（AD）大脑中最显著的病理特征是大量异常神经元，这些神经元充满了神经原纤维缠结（NFTs），其中包含高度磷酸化tau蛋白（PHF）、神经丝和各种激酶以及其他蛋白质的稳定复合物。尽管对这些神经元聚集体进行了积极研究，但其性质和起源仍知之甚少。我们利用P13（suc1）亲和色谱法对鱿鱼巨纤维系统神经元中的磷酸化调节进行研究，结果表明，细胞骨架蛋白的神经元磷酸化被分隔到激酶、底物和磷酸酶的活性轴突特异性和非活性细胞体特异性多聚体复合物中。为了确定人类大脑中是否存在这种特定区域的磷酸化复合物，我们从正常和AD大脑中分离出灰质（富含细胞体）和白质（富含轴突），并研究了裂解物、沉淀和P13（suc1）复合物中的总激酶活性。此外，蛋白质印迹分析用于表征从灰质和白质中提取的与P13（suc1）多聚体复合物相关的蛋白质。我们检验了这样一个假设，即AD神经元中P13磷酸化复合物的区域分隔异常，活性更高的复合物转移到了细胞体（灰质）而非轴突（白质）。我们发现：（1）AD和对照脑提取物的内源性和外源性底物依赖性激酶活性在灰质和白质中相似。（2）较长的死后时间往往会消除对照和AD提取物之间激酶活性的任何差异。与较短的死后时间（4.5 - 10小时）相比，较长的死后时间（13 - 34小时）显著减小了对照和AD脑提取物之间激酶活性的差异，这表明细胞死亡和蛋白水解可能消除了酶活性的任何内在差异。（3）除了对照白质提取物中组蛋白磷酸化水平显著较高外，来自灰质和白质的P13（suc1）衍生多聚体复合物的激酶活性在对照和AD大脑中也相似。同样，对照和AD分布之间的差异也显著不同（p < 0.001 - 0.02），这表明P13复合物是不同的。我们还发现了与P13suc1相关的激酶和细胞骨架蛋白在蛋白质印迹图谱上的差异；在AD大脑灰质中检测到磷酸化NF - H和PHF - tau的表达更高。我们认为，来自人类对照和AD脑样本的P13复合物存在这些差异，这些样本在年龄、死后时间和临床病史方面表现出广泛的异质性，可能具有重要意义
