Department of Basic Medicine, Fenyang College of Shanxi Medical University, Fenyang, 032200, China.
Exp Brain Res. 2024 Aug;242(8):1983-1998. doi: 10.1007/s00221-024-06858-z. Epub 2024 Jun 27.
The aim of this study was to investigate histone deacetylase 6 (HDAC6) modifies the heat shock protein 90 (HSP90) and heat shock transcription factor 1 (HSF1) affect the levels of pathological markers such as Aβ oligomers (Aβo) and Tau phosphorylation (p-Tau) in APP/PS1 double transgenic mice hippocampal tissues or HT22 neurons as well as the changes in cognitive behavioral functions of mice. (1) APP/PS1 transgenic mice (6 months old, 25 ~ 30 g) were randomly assigned to 5 experimental groups, C57BL/6J mice (6 months old, 25 ~ 30 g) were used as 4 control groups, with 8 mice in each group. All mice underwent intracerebroventricular (i.c.v.) cannulation, and the experimental groups were administered with normal saline (APP + NS group), HDAC6 agonist tubastatin A hydrochloride (TSA) (APP + TSA group) or HDAC6 agonist theophylline (Theo) (APP + Theo group), HSP90 inhibitor Ganetespib (Gane) (APP + Gane group), or a combination of pre-injected Gane by TSA (APP + Gane + TSA group); the control group received i.c.v. injections of Gane (Gane group), TSA (TSA group), Theo (Theo group) or NS (NS group), respectively. (2) Mouse hippocampal neurons HT22 were randomly divided into a control group (Control) and an Aβ intervention group (Aβ). Within the Aβ group, further divisions were made for knockdown HSP90 (Aβ + siHSP90 group), overexpression HSP90 (Aβ + OE-HSP90 group), knockdown HSF1(Aβ + siHSF1 group) and knockdown HSF1 followed by overexpression HSP90 (Aβ + siHSF1 + OE-HSP90 group), resulting in a total of 6 groups. Morris water maze test was used to evaluate the cognitive behavior of the mice. Western blot and immunohistochemistry or immunofluorescence were performed to detect the levels of HDAC6, HSP90, HSF1, Aβ, Tau protein, and p-Tau in the hippocampal tissue or HT22 cells. qRT-PCR was used to measure the levels of hdac6, hsp90, and hsf1 mRNA in the hippocampus or nerve cells. (1) The levels of HDAC6, Aβ and p-Tau were elevated, while HSP90 and HSF1 were decreased in the hippocampal tissue of APP/PS1 transgenic mice (all P < 0.01). Inhibiting HDAC6 upregulated the expressions of HSP90 and HSF1 in the hippocampal tissue of APP/PS1 mice, while decreasing the levels of Aβ and p-Tau as well as improving the spatial cognitive behavior in mice (P < 0.05 or P < 0.01). The opposite effects were observed upon HDAC6 activation. However, inhibiting HSP90 reduced the expression of HSF1 (P < 0.01) and increased the levels of Aβ and p-Tau (P < 0.05 or P < 0.01) but did not significantly affect the expression of HDAC6 (P > 0.05). No significant changes were observed in the aforementioned indicators in the 4 control groups (P > 0.05). (2) In the Aβ intervention group, HDAC6 and Aβ, p-Tau expression levels were elevated, while HSP90 and HSF1 expressions were all decreased, and cell viability was reduced (P < 0.05 or P < 0.01). Overexpression of HSP90 upregulated HSF1 expression, decreased the levels of Aβ and p-Tau, and increased cell viability (P < 0.05 or P < 0.01). Knocking down HSP90 had the opposite effect; and knocking down HSF1 increased the levels of Aβ and p-Tau and decreased cells viability (all P < 0.01), but did not result in significant changes in the expression levels of HSP90 (P > 0.05). Inhibiting HDAC6 can upregulate the expressions of HSP90 and HSF1 but reduce the levels of Aβ and p-Tau in the hippocampus of APP/PS1 mice and improvement of cognitive behavioral function in mice; Overexpression of HSP90 can increase HSF1 but decrease Aβ and p-Tau levels in the hippocampal neurons and increase cell activity. It is suggested that HDAC6 may affect the formation of Aβ oligomers and the changes in Tau protein phosphorylation levels in the hippocampus of AD transgenic mouse as well as the alterations in cognitive behavioral functions by regulating the HSP90-HSF1 pathway.
本研究旨在探讨组蛋白去乙酰化酶 6（HDAC6）是否通过调节热休克蛋白 90（HSP90）和热休克转录因子 1（HSF1）来影响 APP/PS1 双转基因小鼠海马组织或 HT22 神经元中病理标志物（如 Aβ 寡聚物（Aβo）和 Tau 磷酸化（p-Tau）的水平，以及改变小鼠的认知行为功能。（1）将 APP/PS1 转基因小鼠（6 个月大，25-30g）随机分为 5 个实验组，C57BL/6J 小鼠（6 个月大，25-30g）作为 4 个对照组，每组 8 只。所有小鼠均进行脑室内（i.c.v.）插管，实验组给予生理盐水（APP+NS 组）、HDAC6 激动剂 tubastatin A 盐酸盐（TSA）（APP+TSA 组）或 HDAC6 激动剂茶碱（Theo）（APP+Theo 组）、HSP90 抑制剂 Ganetespib（Gane）（APP+Gane 组），或 TSA 预处理联合 Gane（APP+Gane+TSA 组）；对照组分别给予 i.c.v.注射 Gane（Gane 组）、TSA（TSA 组）、Theo（Theo 组）或 NS（NS 组）。（2）将小鼠海马神经元 HT22 随机分为对照组（Control）和 Aβ 干预组（Aβ）。在 Aβ 组中，进一步分为敲低 HSP90（Aβ+siHSP90 组）、过表达 HSP90（Aβ+OE-HSP90 组）、敲低 HSF1（Aβ+siHSF1 组）和敲低 HSF1 后过表达 HSP90（Aβ+siHSF1+OE-HSP90 组），共 6 组。采用 Morris 水迷宫试验评价小鼠的认知行为。采用 Western blot 和免疫组化或免疫荧光法检测各组小鼠海马组织或 HT22 细胞中 HDAC6、HSP90、HSF1、Aβ、Tau 蛋白和 p-Tau 的水平。采用 qRT-PCR 法检测各组小鼠海马或神经细胞中 hdac6、hsp90 和 hsf1mRNA 的水平。（1）APP/PS1 转基因小鼠海马组织中 HDAC6、Aβ 和 p-Tau 水平升高，HSP90 和 HSF1 水平降低（均 P<0.01）。抑制 HDAC6 可上调 APP/PS1 小鼠海马组织中 HSP90 和 HSF1 的表达，降低 Aβ 和 p-Tau 水平，改善小鼠的空间认知行为（P<0.05 或 P<0.01）。HDAC6 激活则产生相反的效果。然而，抑制 HSP90 降低了 HSF1 的表达（P<0.01），增加了 Aβ 和 p-Tau 的水平（P<0.05 或 P<0.01），但对 HDAC6 的表达没有显著影响（P>0.05）。4 个对照组上述指标无明显变化（P>0.05）。（2）在 Aβ 干预组中，HDAC6 和 Aβ、p-Tau 表达水平升高，HSP90 和 HSF1 表达水平均降低，细胞活力降低（P<0.05 或 P<0.01）。过表达 HSP90 可上调 HSF1 的表达，降低 Aβ 和 p-Tau 的水平，增加细胞活力（P<0.05 或 P<0.01）。敲低 HSP90 则产生相反的效果；敲低 HSF1 增加了 Aβ 和 p-Tau 的水平并降低了细胞活力（均 P<0.01），但 HSP90 的表达水平没有明显变化（P>0.05）。抑制 HDAC6 可上调 APP/PS1 小鼠海马中 HSP90 和 HSF1 的表达，降低 Aβ 和 p-Tau 的水平，改善认知行为功能；过表达 HSP90 可增加 HSF1 但降低 Aβ 和 p-Tau 水平并增加细胞活性。提示 HDAC6 可能通过调节 HSP90-HSF1 通路影响 AD 转基因小鼠海马中 Aβ 寡聚物的形成和 Tau 蛋白磷酸化水平的变化以及认知行为功能的改变。
