Center for Translational Research in Neurodegenerative Disease and Department of Neuroscience, University of Florida, Gainesville, FL 32610, USA.
J Alzheimers Dis. 2013;33(1):249-63. doi: 10.3233/JAD-2012-121093.
Conditional overexpression of four-repeat human tau containing the P301L missense mutation in the rTg4510 mouse model of tauopathy leads to progressive accumulation of neurofibrillary tangles and hyperphosphorylated, sarkosyl-insoluble tau species, which are biochemically comparable to abnormal tau characteristic of hereditary tauopathies termed FTDP-17. To fully understand the impact of tau species at different stages of self-assembly on neurodegeneration, we fractionated rTg4510 brain representing several stages of tauopathy to obtain TBS-extractable (S1), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble (P3) fractions. Under reducing condition, the S1 fraction was demonstrated by western blotting to contain both 50-60 kDa normally-sized and 64 kDa tau. Both are thermo-stable, but the 64 kDa tau showed a higher degree of phosphorylation. Under non-reducing condition, nearly all TBS-extractable 64 kDa tau were detected as ∼130 kDa species consistent with the size of dimer. Quantitative analysis showed ∼80 times more 64 kDa tau in S1 than P3 fraction. Immunoelectron microscopy revealed tau-positive granules/short filaments in S1 fraction. These structures displayed MC1 immunoreactivities indicative of conformational/pathological change of tau. MC1 immunoreactivity was detected by dot blotting in samples from 2.5 month-old mice, whereas Ab39 immunoreactivity indicative of late stages of tau assembly was detected only in P3 fraction. Quantitative analysis also demonstrated a significant inverse correlation between brain weight and 64 kDa tau, but the level of TBS-extractable 64 kDa tau reflects neurodegeneration better than that of sarkosyl-insoluble 64 kDa tau. Together, the findings suggest that TBS-extractable 64 kDa tau production is a potential target for therapeutic intervention of tauopathies.
条件性过表达四重复人类 tau，其中包含 P301L 错义突变，在 rTg4510 tau 病模型中导致神经原纤维缠结和过度磷酸化、 Sarkosyl 不溶性 tau 物种的进行性积累，这些物质在生化上与称为 FTDP-17 的遗传性 tau 病的异常 tau 相当。为了充分了解不同自组装阶段 tau 物种对神经退行性变的影响，我们对 rTg4510 大脑进行了分级，代表 tau 病的几个阶段，以获得 TBS 可提取（S1）、高盐/Sarkosyl 可提取（S3）和 Sarkosyl 不溶性（P3）级分。在还原条件下，Western blot 证实 S1 级分既含有 50-60 kDa 正常大小的 tau，也含有 64 kDa tau。两者都是热稳定的，但 64 kDa tau 的磷酸化程度更高。在非还原条件下，几乎所有 TBS 可提取的 64 kDa tau 都被检测为约 130 kDa 的二聚体大小的物质。定量分析显示，S1 级分中的 64 kDa tau 比 P3 级分多约 80 倍。免疫电镜显示 S1 级分中存在 tau 阳性颗粒/短丝。这些结构显示出 MC1 免疫反应性，表明 tau 的构象/病理变化。Dot blot 检测到来自 2.5 月龄小鼠的样品中的 MC1 免疫反应性，而 Ab39 免疫反应性表明 tau 组装的晚期阶段仅在 P3 级分中检测到。定量分析还表明，脑重与 64 kDa tau 之间存在显著的负相关，但 TBS 可提取的 64 kDa tau 水平比 Sarkosyl 不溶性 64 kDa tau 更能反映神经退行性变。总之，这些发现表明，TBS 可提取的 64 kDa tau 的产生是 tau 病治疗干预的潜在靶点。
