研究文章细胞神经科学

反Na+/千+-ATPase免疫疗法可通过激活Na改善α-突触核蛋白的病理学+/千+-ATPaseα1依赖性自噬

查看全部隐藏 作者和从属关系

科学进步 2021年年1月27日:
卷7号5,eabc5062
DOI:10.1126 / sciadv.abc5062
载入中

抽象

+/千+-ATPase (NKA) plays important roles in maintaining cellular homeostasis. Conversely, reduced 恩卡 activity has been reported in aging 和 neurodegenerative diseases. However, little is known about the function of 恩卡 in the pathogenesis of 公园inson’s disease (PD). Here, we report that reduction of 恩卡 activity in 恩卡α1+/- mice aggravates α-synuclein–induced pathology, including a reduction in tyrosine hydroxylase (TH) 和 deficits in behavioral tests for memory, learning, 和 motor function. To reverse this effect, we generated an 恩卡-stabilizing monoclonal antibody, DR5-12D, against the DR region (897DVEDSYGQQWTYEQR911) of the 恩卡α1 subunit. We demonstrate that DR5-12D can ameliorate α-synuclein–induced TH loss 和 behavioral deficits by accelerating α-synuclein degradation in neurons. The underlying mechanism for the beneficial effects of DR5-12D involves activation of 恩卡α1-dependent autophagy via increased AMPK/mTOR/ULK1 pathway signaling. Cumulatively, this work demonstrates that 恩卡 activity is neuroprotective 和 that pharmacological activation of this pathway represents a new therapeutic strategy for PD.

介绍

+/千+–腺苷三磷酸酶(NKA)是一种跨膜蛋白,由三个亚基:α,β和γ组成,具有催化亚基的四个亚型(α1至α4)(1)。 In the central nervous system, 恩卡 requires about 40% of the 恩ergy delivered by respiration 至 maintain ion 梯度s across cell membranes (2)。 Recently, it has been reported that a progressive decline of 恩卡 activity can exacerbate neurodegeneration in the aging process (38)。 Accumulating evidence also suggests a close relationship between 恩卡 和 公园inson’s disease (PD). For example, clinical studies found that 恩卡 activity was substantially reduced in erythrocytes of PD patients (9),并且发现具有遗传突变的患者出现了快速发作的肌张力障碍-帕金森病(RDP)的运动症状和脑脊液中多巴胺代谢异常。 ATP1A3 (10)。 These findings, 至 some extent, provide clinical correlations suggesting that 恩卡 may play an important role in PD pathogenesis.

1997年,α-突触核蛋白(αSyn)被确定为路易小体的主要成分,并且聚集的αSyn现在被认为是PD的病理学标志(11)。泛素-蛋白酶体系统和自噬-溶酶体系统均负责αSyn降解(12)。然而,在PD中,自噬-溶酶体系统的降解效率降低,这有助于αSyn的积累(1315)。反过来,αSyn的积累会导致细胞功能障碍,包括抑制泛素-蛋白酶体和自噬-溶酶体系统,以及诱导线粒体功能障碍和内质网应激。这些缺陷累积导致神经元死亡和神经变性(16)。 Therefore, increasing αSyn degradation through autophagy may be an attractive target for PD therapy. Because 恩卡 is a key regulator of autophagy (1719), it is compelling 至 theorize that αSyn clearance may be 加速的through activation of 恩卡-dependent autophagy.

细胞外区域 897DVEDSYGQQWTYEQR911 (DR region), which is highly conserved among various 恩卡α subunits, is the activation domain of 恩卡 (20)。 Previously, our group developed a polyclonal antibody (DR-Ab) against the DR region of 恩卡 that effectively activates 恩卡. DR-Ab protects against chronic heart failure 和 ischemic stroke injury both in vitro 和 in vivo (2123)。 Given the reduction of 恩卡 activity in PD 和 the importance of 恩卡 in regulating autophagy, we hypothesize that activation of 恩卡 by DR-Ab may be potentially protective against αSyn pathology. To improve the specificity of DR-Ab in activating 恩卡 和 至 facilitate subsequent drug development, we generated a monoclonal DR-Ab (DR5-12D) 和 studied its effect 上 αSyn pathology. Intracerebral injection of preformed fibrils (PFFs) of αSyn is a widely used rodent PD model due 至 its close replication of clinical symptoms observed in PD patients (24)。 Here, we demonstrate that reduction of 恩卡 activity in 恩卡α1+/- mice exacerbates PFF-induced pathological process, while DR5-12D 所有eviates PD-related pathology through activation of 恩卡-dependent autophagy 至 increase αSyn clearance.

结果

恩卡α1 deficiency aggravates PFF-induced pathology

To investigate the role of 恩卡α1 in αSyn-induced pathology, 恩卡α1+ / +和恩卡α1+/- mice were evaluated in the PFF model as previously described (fig. S1, A 至 E). PFF or phosphate-buffered saline (PBS) was injected into the striatum of mice 90 days before behavioral analysis (fig. S2A) with the 莫里斯水迷宫 (spatial learning 和 memory) 和 the rotarod test (neuromotor performance). After training for four consecutive days in the 莫里斯水迷宫, 恩卡α1+ / + mice treated with PFF exhibited a longer escape latency relative 至 PBS-treated controls. The same trend was also found in the 恩卡α1+/- 老鼠 (图1A和fig. S2B). These data confirm that PFF successfully induced PD-like neuronal injury.

Fig. 1 恩卡α1 deficiency aggravates PFF-induced behavioral deficits.

(A)从训练第1天到第4天的逃避潜伏时间。 n = 11 in 恩卡α1+ / +和恩卡α1+/- PBS组和 n = 10 in 恩卡α1+ / +和恩卡α1+/- PFF组。 (B)第4训练日的代表游泳路线。C)探针测试日的代表性游泳路径。 (D)持续时间以探针测试日在目标象限中60s的百分比表示。 n = 11 in 恩卡α1+ / +和恩卡α1+/- PBS组和 n = 10 in 恩卡α1+ / +和恩卡α1+/- PFF组。 (E)在探针测试当天越过平台区域的频率。 n = 11 in 恩卡α1+ / +和恩卡α1+/- PBS组和 n = 10 in 恩卡α1+ / +和恩卡α1+/- PFF组。 (F)在轮播测试中连续三个测试日的延迟时间。 n = 11 in 恩卡α1+ / +和恩卡α1+/- PBS组和 n = 10 in 恩卡α1+ / +和恩卡α1+/-PFF组。值代表平均值±SEM,方差的双向分析(ANOVA),然后是Bonferroni’的多重比较测试。

Although no significant difference was found in the escape latency between 恩卡α1+ / +和恩卡α1+/- mice receiving 上ly PBS treatment, a longer escape latency was found in the PFF-treated 恩卡α1+/- mice compared 至 PFF-treated 恩卡α1+ / + 老鼠 (图1A)。训练第4天的代表性游泳路线如下 图1B。在探针测试日卸下平台后,记录了目标象限中的游泳时间和穿过平台区域的频率(图S2C)。与PBS处理的对照组相比,PFF处理的小鼠中这两个指标显着降低(图1,C到E)。 Consistently, significant differences were found between the two genotypes of mice treated with PFF but 不 PBS. PFF-treated 恩卡α1+/- mice had a shorter swimming time in the target quadrant 和 a lower frequency 至 cross the platform zone compared 至 those in PFF-treated 恩卡α1+ / + 老鼠 (图1,C到E)。 These data suggest that reduction of 恩卡α1 exacerbates PFF-induced learning 和 memory impairment. Neuromotor performance evaluated by rotarod test confirmed that the latency 至 fall was decreased in PFF-treated mice compared 至 that in PBS-treated 老鼠 (图1F)。 Moreover, reduction of 恩卡α1 further shortened the latency 至 fall in PFF-treated 老鼠 (图1F), indicating the essential role of 恩卡α1 in neuromotor performance. Together, these results demonstrate that 恩卡α1 deficiency contributes 至 PFF-induced behavioral signs.

We next examined whether 恩卡 activity is altered in the PFF model. As shown in 图2A, 恩卡 activity was decreased in the PFF-treated wild-type (WT) mice 和 this effect was exacerbated by basal reduction of 恩卡α1 in 恩卡α1+/- mice. PFF-induced loss of dopaminergic axons in the striatum 和 dopaminergic neurons in the substantia nigra pars compacta (SNpc) was observed as determined by loss of tyrosine hydroxylase (TH) immunoreactivity, 和 these effects were exacerbated when 恩卡α1 was reduced (图2,B和C)。 In addition, the relative accumulation of soluble αSyn (TX-soluble fraction) versus insoluble αSyn (SDS-soluble fraction) was examined. Insoluble αSyn aggregates were detectable in both the striatum 和 midbrain regions upon PFF treatment, 和 this increased significantly in 恩卡α1+/- mice compared 至 恩卡α1+ / + 老鼠 (图2,D和E)。与SDS可溶级分中的αSyn水平一致,磷酸化的αSyn(p-αSyn,Ser129) was also significantly increased in the PFF-treated mice 和 this was further 恩hanced in both brain regions of 恩卡α1+/- 老鼠 (图2,F和G)。 Immunostaining of the SNpc in the midbrain further confirmed that TH labeling was reduced in the PFF treatment groups, 和 this TH loss was exacerbated in 恩卡α1+/- mice compared 至 that in 恩卡α1+ / + 老鼠 (图2H)。 Furthermore, p-αSyn in the SNpc was increased with PFF treatment, 和 this increase was even greater in 恩卡α1+/- 小鼠比野生型小鼠(图2H)。 Therefore, 恩卡α1 deficiency, resulting in the reduction of 恩卡 activity, may exacerbate PFF-induced pathological characteristics.

Fig. 2 恩卡α1 deficiency exacerbates PFF-induced pathological characteristics.

(A) 恩卡 activity was measured in the striatum sample (n = 6)。 (BC) Representative Western blots 和 quantification showing the expression of 恩卡α1 和 tyrosine hydroxylase (TH) in striatum region (B) 和 in midbrain region (C) (n 纹状体区域= 4 n = 5(在中脑区域)。 GAPDH,3-磷酸甘油醛脱氢酶。 (DE)代表性的蛋白质印迹和定量分析显示TX可溶性αSyn和SDS可溶性αSyn在纹状体区域(D)和中脑区域(E)中的表达(n 纹状体和中脑区域均为5。 TX,1%Triton X-100; SDS,2%十二烷基硫酸钠。 (FG)代表性的蛋白质印迹和定量分析显示p-αSyn在纹状体区域(F)和中脑区域(G)中的表达(n 纹状体= 3 n = 4在中脑区域)。 (H)代表性的免疫荧光图像和定量显示TH阳性细胞和磷酸化-αSyn(p-αSyn; Ser129)SNpc(n = 8). Scale bars, 250 μ米值代表平均值±扫描电镜双向方差分析,然后是Bonferroni’对(H)以外的所有数字进行多重比较检验,其中p-αSyn intensity was analyzed by unpaired t 测试,两尾。

To compare the contribution of 恩卡-dependent αSyn clearance in various brain cells, we determined αSyn levels in primary cultures of various brain cells in both WT 和 恩卡α1-deficient mice. A comparable reduction of 恩卡α1 was found in neurons (40% of WT; fig. S3A), astrocytes (38% of WT; fig. S3C), 和 microglia (35% of WT; fig. S3E). The reduction of 恩卡α1 also correlated with an increase in αSyn content that was similar in 所有 cell types (neurons, fig. S3B; astrocytes, fig. S3D; 和 microglia, fig. S3F).

单克隆DR抗体改善PFF诱导的病理

On the basis of our previous study that reported the development of an 恩卡-stabilizing polyclonal antibody (22),我们生成了针对相同DR区的单克隆DR抗体(DR-Ab)(897DVEDSYGQQWTYEQR911) of the 恩卡α subunit (图3A)。通过酶联免疫吸附测定(ELISA)和Western印迹分析分别验证了DR-Ab克隆DR5-12D的效价和特异性(图3,B和C)。为了检查DR5-12D对PFF诱导的损伤的治疗效果,在注射PFF / PBS后第7天至第90天每周一次腹膜内注射DR5-12D或对照免疫球蛋白G(IgG)。在治疗性实验开始之前,通过免疫荧光在不同大脑区域对IgG的染色来验证DR抗体的血脑屏障渗透。为了最大程度地减少由内源性小鼠IgG引起的背景标记,我们使用了兔多克隆DR抗体来测量血脑屏障渗透。 DR抗体(腹膜内)治疗后24小时,在大脑区域(包括皮质,海马,纹状体和中脑)中检测到明显的标记(图S4,B至E)。在莫里斯水迷宫测试中,在训练的第3天和第4天,与PBS对照组相比,PFF治疗组的逃避潜伏期更长,而PFF + DR5-12D治疗组的潜伏期缩短了(图3D)。训练第4天的代表性游泳路线如下 图3E。在探针测试日,与PBS对照相比,PFF治疗组的目标象限持续时间和穿过平台区域的频率显着减少,而PFF + DR5-12D治疗则增加了这些时间(图3,从F到H)。此外,在轮状试验中,与PBS对照组相比,PFF治疗组的跌倒潜伏期较短,但PFF + DR5-12D治疗组的这种下降减弱了(图3I)。这些数据表明DR5-12D对PFF诱导的行为体征的治疗作用。

Fig. 3 DR5-12D改善了PFF引起的行为缺陷。

(A) Schematic illustration of 恩卡α subunit 和 DR region. (B)ELISA检测纯化的单克隆DR5-12D的滴度(n = 3)。外径450450nm处的光密度。 (C) Specificity of purified monoclonal DR5-12D detected by 蛋白质印迹分析. Samples included purified 恩卡 protein from the kidney of pigs 和 cell lysates extracted from SH-SY5Y cells 和 human embryonic kidney (HEK) 293 cells. BSA was provided as a negative control. (D)从培训第1天到第4天的延迟时间。E)第4训练日的代表游泳路线。F)探针测试日的代表性游泳路径。 (G)持续时间以探针测试日在目标象限中60s的百分比表示。 (H)在探针测试当天越过平台区域的频率。 (I)在轮播测试中连续三个测试日的延迟时间。 n PBS组= 11 n = 10 in PFF + IgG group 和 PFF + DR group. Values represent mean ±扫描电镜两向方差分析,然后是Bonferroni’的多重比较测试用于分析(D)和(I)中的数据。单向方差分析,然后是Bonferroni’的多重比较测试用于分析其他数据。

As anticipated, DR5-12D treatment attenuated 恩卡 impairment in the PFF model (图4A)。同时,DR5-12D治疗可降低多巴胺能神经元死亡,这可通过两条纹状体中TH的恢复表达来表明(图4B)和中脑区(图4C)。与纹状体中的IgG处理组相比,DR5-12D处理组的SDS可溶级分中αSyn的积累明显减少了(图4D)和中脑(图4E)地区。关于p-αSyn水平,发现相似的结果。 DR5-12D治疗还显着减少了上述两个大脑区域中PFF诱导的p-αSyn上调(图4,F和G)。 TH的免疫染色进一步证实了DR5-12D对SNpc中PFF诱导的TH丢失的保护作用(图4H)。此外,SNpc中的DR5-12D处理也显着降低了p-αSyn水平(图4H)。 Together, we conclude that DR5-12D has the potential 至 treat αSyn pathology, presumably by preservation of 恩卡 activity.

Fig. 4 DR5-12D减轻了PFF诱导的病理特征。

(A) 恩卡 activity was measured in the striatum samples (n = 6)。 (BC) Representative Western blots 和 quantification showed the expression of 恩卡α1 和 TH in striatum region (B) 和 in midbrain region (C) (n 在纹状体区域和中脑区域均为5。 (DE)代表性的蛋白质印迹和定量分析显示TX可溶性αSyn和SDS可溶性αSyn在纹状体区域(D)和中脑区域(E)中的表达(n SDS可溶级分= 6 n 纹状体和中脑区域的TX可溶性级分= 4)。 (FG)代表性的蛋白质印迹和定量分析显示p-αSyn在纹状体区域(F)和中脑区域(G)中的表达(n 纹状体= 3 n = 4在中脑区域)。 (H)代表性的免疫荧光图像和定量显示SNpc中TH阳性细胞和p-αSyn的密度(n = 8). Scale bars, 250 μ米值代表平均值±扫描电镜单向方差分析,然后是Bonferroni’的多重比较测试用于分析除(H)以外的所有数据,其中p-αSyn intensity was analyzed by unpaired t 测试,两尾。

DR5-12D抑制摄取并加速αSyn的清除

在体外实验中,我们进一步研究了DR5-12D对αSyn病理的保护作用的机制。 PFF用绿色荧光染料ATTO 488标记,用于活细胞成像(图S5A)。此后,通过αSyn抗体检测到标记的PFF以确认成功标记,显示出合并的信号(图S5B)。这些标记的PFF对SH-SY5Y细胞具有渗透性,适用于活细胞成像(图S5C)。 DR5-12D处理一小时后,将SH-SY5Y细胞用PFF-ATTO 488处理15分钟,然后洗净。与IgG治疗组相比,在PFF–ATTO 488治疗后的多个时间点,DR5-12D治疗组的αSyn水平降低(图5A)。为了排除观察到的荧光来自粘附在细胞表面的细胞外αSyn原纤维的可能性,在随后的所有实验中均使用胰蛋白酶-EDTA(0.01%)消化细胞外原纤维(25, 26)。为了确定DR5-12D对αSyn内在化的影响,将细胞用溶酶体抑制剂[bafilomycin A1(BA),100 nM]预处理1小时以阻断αSyn降解。在对DR5-12D或对照IgG处理1小时后,将PFF添加到细胞中0.5、1和2小时。如图所示 图5B在IgG处理组中,PFF孵育0.5至2小时后,αSyn被细胞摄取。用DR5-12D治疗减弱了这种作用。为了观察DR抗体对αSyn清除的作用,通过用PFF处理细胞1小时来装载等量的αSyn,然后用含有DR5-12D的新鲜培养基替换培养基。结果发现,与IgG处理相比,DR5-12D处理24小时后αSyn明显减少,表明DR5-12D处理对αSyn的清除率增加(图5C)。此外,我们通过蛋白质印迹分析验证了这种效果。 DR5-12D(而非IgG)处理可增加SH-SY5Y细胞中SDS可溶级分中的αSyn清除率(图5D)。除了外源性PFF治疗模型外,我们还使用αSyn过表达模型检查了DR5-12D对αSyn清除的影响。将αSyn增强的绿色荧光蛋白(eGFP)质粒转染到SH-SY5Y细胞中,以产生αSyn过表达的细胞系,其中可以通过Western印迹检测到αSyn(图5E)。用DR5-12D处理24小时后,与IgG处理相比,αSyn的表达明显降低,这表明DR5-12D处理的效果与细胞外PFF模型相同(图5F)。此外,DR5-12D处理还减少了原代神经元培养物中αSyn的内源表达(图S5D)。在一起,我们的数据表明DR5-12D对αSyn病理的保护作用是由清除加速和抑制αSyn摄取共同介导的。

Fig. 5 DR5-12D增加α通过维持NKA活性和激活NKA来清除Synα1-dependent autophagy.

(A)活细胞成像显示DR5-12D处理降低了αSyn水平( n = 3;比例尺,50μm)。 (BDR5-12D对PFF内部化的影响(n = 4)。 SH-SY5Y细胞用BA预处理1小时。 (CD)免疫荧光染色和Western印迹分析显示DR5-12D处理增加了αSyn的清除率(n = 4)。比例尺,10μm。 (EF) Representative Western blots showing the increased αDR5-12D处理后的顺式清除αSyn overexpression cells (n = 3)。 (G) Effect of DR5-12D 上 恩卡 activity (n = 4)。 (HI) OB pretreatment blocked DR5-12D–accelerated αPFF处理和αSyn overexpression cells [n = 5英寸(H)和 n = 4 in(I)]。 (J)溶酶体抑制剂阻断了DR5-12D对αSyn清除的影响(n = 4)。 (K) CRISPR-Cas9 technique knockout (KO) of 恩卡α1 in Neuro2a cells. (L) mRNA levels of autophagy-related genes in 恩卡α1 WT 和 KO cells (n = 6至8)。 (M) Effect of DR5-12D 上 LC3-II expression in 恩卡α1 WT 和 KO cells (n = 5)。使用双向ANOVA分析(B)和(M)中的数据。未配对 t 测试用于分析(L)中的数据。单向方差分析用于分析其他数据。

恩卡 activity 和 恩卡α1-dependent autophagy are required for DR5-12D–induced αSyn clearance

Consistent with results obtained in the PFF mouse model, 恩卡 activity was similarly decreased in the PFF cell model. SH-SY5Y cells were pretreated with PFF for 1 hour 和 then washed out, followed by DR5-12D or control IgG treatment for 24 hours. The PFF-induced decrease in 恩卡 activity was attenuated by DR5-12D but 不 by IgG treatment (图5G)。 Intriguingly, the effect of DR5-12D 上 αSyn clearance was blocked by pretreatment with 1 μM ouabain (OB; an 恩卡 inhibitor) for 1 hour in both exogenous PFF (图5H)和αSyn过表达模型(图5I), confirming the essential role of 恩卡 in the effect of DR5-12D 上 αSyn clearance. To further understand the mechanism underlying DR5-12D–accelerated αSyn clearance, we made use of inhibitors targeting the autophagy lysosomal system [BA 和 chloroquine (CQ)] 和 ubiquitin proteasome system [MG132 (MG)]. In the PFF model, inhibition of the autophagy lysosomal system with BA 和 CQ completely blocked the effect of DR5-12D 上 αSyn clearance, while MG treatment had no effect (图5J和fig. S6A). These data suggest an autophagy-dependent effect of DR5-12D 上 αSyn clearance that is independent of the ubiquitin proteasome pathway.

To confirm the role of 恩卡α1 in autophagy-dependent clearance of αSyn, we generated 恩卡α1 knockout (KO) Neuro2a cells with CRISPR-Cas9 technique (图5K)。自噬相关基因的mRNA水平包括 MAP1LC3B, SQSTM1, ULK1, BECN1ATG12 were 所有 decreased in 恩卡α1 KO cells when compared 至 those of WT cells (图5L)。 In addition, while BA (100 nM) pretreatment significantly inhibited the degradation of LC3-II in WT cells, this effect was markedly attenuated in 恩卡α1 KO cells (图5M)。尽管DR5-12D处理增加了WT细胞中LC3-II的积累,但KO细胞中没有这种作用(图5M)。 Together, our data suggest that 恩卡α1-dependent autophagy is indispensable for αSyn clearance induced by DR5-12D treatment.

DR5-12D激活PFF模型中的AMPK / mTOR / ULK1途径

接下来,我们研究了DR5-12D对PFF模型中自噬相关信号通路的影响。首先在SH-SY5Y细胞中检测了5'腺苷单磷酸激活的蛋白激酶α(AMPKα)/雷帕霉素(mTOR)/ ULK1途径的哺乳动物靶标(一种自噬的正向调节剂)。 PFF处理1小时后,用胰蛋白酶洗涤细胞以去除细胞外PFF,然后将其替换为含有DR5-12D或对照IgG的新鲜培养基24小时。发现AMPK与ATP(三磷酸腺苷)比例的降低反映出AMPK被显着抑制(图6A)和磷酸化AMPKα(p-AMPKα; Thr172)(图6B)。但是,DR5-12D(而不是IgG)通过增加AMP与ATP的比例并上调p-AMPKα的表达来减弱PFF对AMPK的抑制作用(图6,A和B)。一致地,DR5-12D减弱了下游信号分子表达的改变,包括mTOR(p-mTOR; Ser2448)(图6C)和ULK1(p-ULK1,Ser555)(图6D )。此外,DR5-12D处理还减弱了LC3-II的表达降低(图6E)。相比之下,使用AMPK抑制剂化合物C(20μM)中断该途径消除了DR5-12D对αSyn清除的影响(图6F)。

Fig. 6 DR5-12D treatment activates the AMPK/mTOR/ULK1 pathway 和 inhibits the formation of the 恩卡α1/AMPKα/αSyn complex in the PFF model.

(A)DR5-12D在PFF模型中显着减弱了PFF抑制的AMP与ATP的比率(n = 6)。 (BD)具有代表性的Western印迹,显示AMPKα和p-AMPKα(Thr172),mTOR和p-mTOR(Ser2448),ULK1和p-ULK1(Ser555)在PFF模型[n = 4 in(B), n = 5 in(C), n = 4 in(D)]。 (E)代表性的Western印迹显示了PFF模型中LC3-II的表达(n = 4)。 (F)AMPK抑制剂阻断DR5-12D对αSyn清除的影响(n = 4)。 (GH) Co-IP analysis showing the interaction between 恩卡α1 和 AMPKα in SH-SY5Y cells in physiological state (n = 3)。 (I) DR5-12D treatment inhibited the formation of the 恩卡α1/AMPKα/αSyn complex in the PFF model (n = 4)。 (JDR5-12D处理降低了质膜组分中AMPKα和αSyn的表达(n = 4至6)。除(J)中的数据外,所有数据分析均采用单向方差分析,其中通过不成对分析αSyn水平 t 测试。

我们还研究了另一种自噬相关途径,磷脂酰肌醇3激酶(PI3K)/ AKT途径,它是自噬的负调节剂。与DR5-12D对AMPK的显着作用相反,我们未能发现DR5-12D对PI3K / AKT的任何显着调节作用(图S6B)。在一起,我们得出结论,DR5-12D治疗通过激活AMPK / mTOR / ULK1途径增加αSyn清除率。

DR5-12D inhibits the formation of 恩卡α1/AMPKα/αSyn complex

Following the findings that 恩卡α1 was an essential regulator of autophagy 和 AMPK activation, we next explored the molecular interaction between 恩卡α1 和 AMPKα. Co-immunoprecipitation (IP) was used 至 examine the interplay between 恩卡α1 和 AMPKα. In the normal physiological state, AMPKα was detected from the immunoprecipitate collected by anti-NKAα1 antibody treatment in SH-SY5Y cells (图6G)。 通过 contrast, 恩卡α1 was detected from anti-AMPKα antibody precipitated proteins (图6H), confirming a direct interaction between 恩卡α1 和 AMPKα. In the PFF model, the expression of 恩卡α1 was increased in the immunoprecipitate collected by anti-AMPKα antibody treatment, suggesting the increased formation of the 恩卡α1/AMPKα complex. Meanwhile, αSyn was also detected in the same immunoprecipitate, suggesting the formation of a large 恩卡α1/AMPKα/αSyn complex. When treated with DR5-12D, the presence of 恩卡α1 和 αSyn was decreased in the immunoprecipitate (图6I)。 Hence, we determined that addition of PFF increased the formation of the 恩卡α1/AMPKα complex 和 further contributed 至 the emergence of an 恩卡α1/AMPKα/αSyn complex, but DR5-12D treatment inhibited this complex formation. As 恩卡α1 distributes predominantly 至 the plasma membrane, while AMPKα mostly exists in cytosol, we investigated the process of complex formation by isolating plasma membrane fractions from 至tal cell lysates 至 examine the presence of an 恩卡α1/AMPKα/αSyn complex. Intriguingly, PFF-induced accumulation of AMPKα 和 αSyn was observed in the plasma membrane fractions, 和 DR5-12D treatment reduced this membrane localization (图6J)。 To some extent, the above data suggest a process by which AMPKα, with the assistance of PFF, translocates from the cytosol 至 the plasma membrane 至 form a complex with 恩卡α1, while the addition of DR5-12D inhibits this complex formation (fig. S7).

讨论

Recent studies into the pathophysiology of PD have renewed our understanding of the function of the well-studied ion pump, 恩卡. Although clinical findings, such as a decreased 恩卡 activity in erythrocytes of PD patients (9)和的基因突变 ATP1A3 在RDP患者中(10), have suggested that 恩卡 may play a role in the pathogenesis of PD, the mechanisms underlying this process are poorly documented. A previous study 上 恩卡α3 demonstrated that αSyn assemblies sequester 恩卡α3 至 the plasma membrane, which leads 至 impaired 恩卡 function (27)。 This study demonstrated the action of αSyn 上 reducing the pump function of 恩卡 和 characterized the importance of freely diffusing 恩卡α3 in maintaining 恩卡 activity. Although α3 is a neuron-specific subunit of 恩卡, 恩卡α1 is expressed ubiquitously in 所有 cells including neurons, 和 is essential for normal 恩卡 activity (1)。 In our study, beyond addressing our knowledge gaps concerning the role of 恩卡α1 in PD pathogenesis, we aimed 至 develop a new therapeutic strategy based 上 maintaining 恩卡 activity 至 treat PD.

To study the role of 恩卡α1 in αSyn pathology, 恩卡α1+ / +和恩卡α1+/- 将小鼠用于PFF模型,然后进行行为分析。与先前的发现一致,我们发现PFF引起明显的学习和记忆缺陷(28, 29)。但是,与以前的报告相比,该研究发现TH损失更高且更早。差异可能是由于不同实验室产生的PFF毒性不同而引起的。诸如不同类型的αSyn,缓冲液和实验程序(例如超声处理)等因素可能会导致不同的PFF毒性(30)。 In addition, in the current study, we quantified the density of TH-positive cells rather than the 至tal neuron count, which may also contribute 至 the discrepancy. ur study demonstrated that 恩卡α1 deficiency aggravates PFF-induced learning 和 memory impairment. ur evidence shows that PFF-induced neuromotor signs worsen due 至 the loss of 恩卡α1. In addition, PFF-induced TH loss was also markedly higher in 恩卡α1+/- mice when compared 至 that in 恩卡α1+ / + mice. This may be attributed 至 decreased 恩卡 activity 和 increased pathogenic αSyn. We found that 恩卡α1 deficiency caused about a 20% reduction in 恩卡 activity, while PFF reduced 恩卡 activity in WT mice by 45%. These two manipulations seem 至 work synergistically 至 impair 恩卡 activity. These data suggest that neurons with reduced 恩卡 expression are more susceptible 至 PFF injury. This is similar 至 our previous findings that 恩卡α1-deficient mice are more susceptible 至 ischemic damage than WT 老鼠 (22)。 On the basis of these findings, we hypothesized that maintaining 恩卡 activity may be a new therapeutic strategy for αSyn pathology. To verify this hypothesis, we generated a monoclonal DR antibody (DR5-12D) that activates 恩卡. DR5-12D treatment 所有eviated the learning 和 memory impairment 和 improved neuromotor performance in the PFF model. Meanwhile, DR5-12D treatment, as expected, maintained 恩卡 activity 和 further attenuated TH loss 和 reduced pathogenic αSyn. Hence, we confirm that DR5-12D protects against PFF-induced injuries through the preservation of 恩卡 activity 和 the decrease of pathogenic αSyn.

We studied the role of 恩卡 in αSyn regulation in the PFF model. 通用电器netic reduction of 恩卡α1 恩hanced, while DR5-12D treatment reduced, accumulation of insoluble, phosphorylated αSyn in the striatum 和 midbrain regions. These data suggest that 恩卡 may play a role in the formation or clearance of αSyn aggregates. ur data in SH-SY5Y cells SUPport the idea that 恩卡 activity plays a role in clearance. Intriguingly, we found 上e species of αSyn oligomer around 50 kDa that responded significantly 至 the reduction of 恩卡α1 和 DR treatment in the PFF model. We speculate that DR treatment preferentially elicits increased degradation efficiency for αSyn oligomers with high molecular weights relative 至 those with lower molecular weights. We also compared the role of 恩卡α1 in the regulation of αSyn among different brain cell types. No significant differences were found in αSyn accumulation in neurons, astrocytes, 和 microglia when 恩卡α1 expression was reduced in these cells. This suggests that 恩卡α1 is broadly important in the regulation of αSyn level in brain cells. Because αSyn aggregation induces dopaminergic neuronal injury, the present study therefore focused 上 studying how αSyn is cleared in neuronal cells.

It is expected that binding of DR5-12D 至 恩卡α subunit activates 恩卡 because the DR region has been reported 至 be the activation domain of 恩卡 (20, 22)。 However, the potential mechanism by which DR5-12D reduces pathogenic αSyn is still elusive. We found in the present study that, in addition 至 reducing the uptake, DR-Ab 加速的αSyn clearance both in an exogenous PFF model 和 in an αSyn overexpression model at the cellular level. Inhibition of 恩卡 activity using OB blocked the effect of DR5-12D 上 αSyn clearance, reaffirming an essential role of 恩卡 activity in αSyn clearance. The ubiquitin-proteasome system 和 the autophagy-lysosome system are the two major systems for intracellular proteolysis, including αSyn degradation (12)。 Inhibition of the autophagy-lysosome system rather than the ubiquitin-proteasome system inhibited DR5-12D–induced αSyn clearance. Moreover, the effect of DR5-12D 上 increasing autophagic flux was blocked in 恩卡α1 KO cells. These data suggest the requirement of 恩卡α1-dependent autophagy for αSyn degradation with DR5-12D treatment.

越来越多的病理生理学和遗传学证据已经确定了PD中自噬的功能障碍(1315)。自噬-溶酶体系统的受损会导致αSyn聚集,进而抑制自噬,从而形成正反馈回路(31)。 In this study, we found that autophagic flux was inhibited by PFF treatment through the regulation of AMPK/mTOR/ULK1 pathway but 不 the PI3K/AKT pathway. There have been conflicting reports regarding the regulation of AMPK 上 恩卡 activity in different tissues. Studies have described positive regulation in skeletal muscle (32)和肺部负调节(33), while little is known regarding this process in the brain. These controversial findings indicate a tissue-specific 和 complex relationship between AMPK 和 恩卡. To further study the action of DR5-12D 上 activating the AMPK/mTOR/ULK1 pathway, we explored the molecular interaction between 恩卡α1 和 AMPKα. ur data show that a direct interaction between 恩卡α1 和 AMPKα exists 和 is 恩hanced by the formation of the 恩卡α1/AMPKα/αSyn complex in the PFF model, 和 that DR5-12D treatment inhibits this complex formation. In addition, we identify the process by which the 恩卡α1/AMPKα/αSyn complex forms. ur data suggest that αSyn is taken up into cells where it binds 至 cytosolic AMPKα 和 induces its translocation 至 the plasma membrane 至 form an 恩卡α1/AMPKα/αSyn complex. However, this does 不 exclude the possibility that extracellular αSyn may also induce complex formation through binding 至 恩卡α1 at the cell surface. More studies are warranted 至 investigate how the complex is formed 和 how DR5-12D disassociates this complex through binding 至 恩卡α1.

The action of αSyn 上 恩卡 activity is different between a previous study of 恩卡α3 和 our current study of 恩卡α1. In light of the 恩卡α3 study, we understand that αSyn impairs 恩卡 activity by trapping 恩卡α3 至 form nanoclusters 上 the plasma membrane (27)。 The reduced 恩卡 activity is attributed 至 the interaction between αSyn 和 the extracellular segment of 恩卡α3. Here, we revealed the intracellular events of αSyn. We found that αSyn interacts with AMPKα 和 contributes 至 the translocation of AMPKα 至 the plasma membrane 至 form a complex with 恩卡α1. The formation of the 恩卡α1/AMPKα/αSyn complex inhibits 恩卡 activity 和 恩卡α1-dependent autophagy.

In summary, our work uncovers the role of 恩卡α1 in αSyn pathology 和 explores the action of DR5-12D 上 αSyn clearance. However, more experiments are warranted 至 provide a comprehensive view of the importance of 恩卡 in PD. Although we have demonstrated the effect of DR5-12D 上 αSyn clearance, it is possible that this effect may 不 be specific for αSyn. Future studies will address the specificity of DR5-12D treatment. In addition 至 the degradation process, a greater understanding of the mechanisms by which how 恩卡 regulates αSyn internalization is needed as well. Moreover, exploration of 恩卡 in different PD models, such as 6-hydroxydopamine–induced 至xic model or genetic models, will deepen our understanding of the importance of 恩卡 in PD. Together, our study 不 上ly broadens the potential function of 恩卡 in PD but also sheds substantial light 上 developing new strategies for PD therapy.

材料和方法

αSynPFFs的制备

如前所述表达和纯化人αSyn(30)。细菌表达质粒pRK172中的人αSyn互补DNA(cDNA)是M. 长谷川的礼物(东京都医学科学研究所)(34)。简而言之,将αSyn-pRK172质粒转化并在BL21(DE3)中扩增 大肠杆菌 应变。此后收集细菌,并将沉淀重悬于含有蛋白酶抑制剂混合物的高盐缓冲液[10 mM tris(pH 7.6),750 mM 娜Cl和1 mM EDTA]中。将细胞裂解物超声处理并煮沸以沉淀不需要的蛋白质。然后,收集上清液并用10 mM tris(pH 7.6),50 mM 娜Cl和1 mM EDTA透析。将上清液加到Hi-Trap Q HP阴离子交换柱上,并用0至0.5 M 娜Cl梯度洗脱(αSyn用0.2 M 娜Cl洗脱)。浓缩洗脱液,并通过3.5 kDa MWCO Amicon Ultra Centrifuge过滤装置(Millipore)用PBS缓冲液置换。分别通过考马斯蓝染色(图S1A)和蛋白质印迹分析(图S1B)测量蛋白质的纯度和鉴定。为了形成αSynPFF,将纯化的αSyn单体[5 mg / ml,溶于50 mM tris-HCl(pH 7.5)和150 mM KCl]在37°C下孵育7天,并在热混合器(Eppendorf)中以1000 rpm连续摇动。 ,德国)。通过硫黄素T(Th-T)结合测定法(图S1C)监测PFF的形成。每天从组装反应中取出等分试样,并与Th-T(10μM)混合以测量440 nm激发下的荧光和480 nm处的发射。通过透射电子显微镜(TEM)进一步观察到PFF的形成。超声处理前后PFF的结构用磷钨酸染色并通过TEM观察(图S1D)。 Western blotting也用于确认PFF的形成(图S1E)。所有的PFF均通过Sonic消解器系统(Thermo Fisher Scientific,目录号FB120110)以0.16英寸微尖端在60个脉冲和10%的功率(合计30和0.5 s开启和0.5 s断开)下进行超声处理。适用于实验。

PFF dissolved in PBS was administered at 5 μg per mouse in animal experiments 和 2 μg/ml in cell experiments. In cell experiments, trypsin-EDTA was used 至 digest the remaining extracellular fibrils after PFF treatment. Cells were washed three times with PBS 和 incubated with trypsin-EDTA (0.01%) for 1 min at 37°C 至 remove extracellular αSyn, followed by a wash with Dulbecco’s Modified Eagle Medium (DMEM) SUPplemented with 10% fetal calf serum 至 stop the trypsinization.

抗NKA单克隆抗体的产生

抗原DR肽(DVEDSYGQQWTYEQR)(新加坡1st Base)与匙孔血蓝蛋白(KLH)偶联。每两周用DR肽-KLH腹膜内免疫BALB / c小鼠(雌性,6至8周大)3次。 DR肽– KLH的初始剂量是在完全弗氏佐剂(Sigma-Aldrich,F5881)中乳化100μg,然后两次注射不完全的Freund佐剂(Sigma-Aldrich,F5506)而乳化的DR肽– KLH(50μg)。在最后一次免疫后3天从免疫小鼠中分离脾细胞以产生杂交瘤。简而言之,使用50%(v / v)聚乙二醇将脾细胞与SP2 / 0骨髓瘤细胞以4:1的比例融合。含有20%胎牛血清和次黄嘌呤-胸腺嘧啶核苷的完整RPMI用于杂交瘤细胞培养。通过ELISA在培养的杂交瘤的上清液中测量抗体产生。挑选阳性杂交瘤并通过有限稀释克隆。通过从BALB / c小鼠(雌性,6至8周大)收集腹水来进行单克隆抗体的进一步生产。通常,将rist烷(每只小鼠0.5ml,Sigma-Aldrich,P2870)注射到小鼠的腹膜中。 7天后,杂交瘤(5×10 6 per mouse, intraperitoneally) were injected into the mice 至 produce monoclonal antibody. Ascites was collected 10 至 14 days later for monoclonal antibody purification. Protein A/G spin columns (Thermo Fisher Scientific, #89962) were used 至 purify the monoclonal antibody from ascites according 至 the manufacturer’s instruction. The titer of the purified DR antibody was measured by ELISA, while the specificity was validated by Western blotting using 恩卡 protein purified from the kidney of pigs as described (35)和从SH-SY5Y细胞和人胚肾(HEK)293细胞提取的细胞裂解液。在细胞实验中,施用了单克隆DR抗体(40μg/ ml)和对照IgG(40μg/ ml)。在动物实验中,施用了单克隆DR抗体(30 mg / kg)和对照IgG(30 mg / kg)。

立体定向注射

恩卡α1+/- 由美国辛辛那提大学(University of Cincinnati,J.36)。 The 恩卡α1+ / +和恩卡α1+/- 小鼠与C57BL / 6回交。繁殖和住房根据美国国立卫生研究院进行 实验室动物的护理和使用指南和approved by the 娜tional University of Singapore Institutional Animal Care 和 Use Committee. Intracerebral injection of PFF was performed as previously described (37)。 Briefly, after anesthesia, 3-month-old male 恩卡α1+ / +和恩卡α1+/- 给C57BL / 6小鼠立体定向注射PFF(每只小鼠5μg),对照组小鼠注射PBS。通过汉密尔顿注射器(0.1μl/ min,2.5μl)将一根针头插入右纹状体(坐标:距前reg +0.2 mm,距中线2.0 mm,硬脑膜以下2.6 mm)。手术后,对动物进行监测,并提供术后护理。注射PFF后7天(腹膜内,每周)注射单克隆抗NKA抗体(DR)(30 mg / kg)和对照IgG(30 mg / kg)。 PFF注射后第90天终止治疗。

莫里斯水迷宫

PFF注射后90天进行莫里斯水迷宫测试。在测试之前,将小鼠游泳60 s,并在平台上站立10 s,以熟悉测试设备。从第1天到第4天的测试期间,平台的位置保持不变。未找到平台的小鼠被引导至平台,并获得60 s的等待时间评分。发现平台的小鼠被允许在平台上停留10 s。测试每只小鼠两次试验,间隔间隔为15分钟。记录找到平台的平均时间和游泳路径。在第5天,进行探针测试,测试每个小鼠在不存在平台的情况下搜索平台60秒。测试每只小鼠两次试验,间隔间隔为15分钟。记录先前拥有平台的象限中消耗的平均时间以及跨平台区域的数量。

罗塔罗德测试

在Morris水迷宫测试之后进行旋转脚架测试。在连续两天的训练阶段中,将每只小鼠以12 rpm的恒定速度放在旋转脚架上,最长时间为120 s。在连续三天的测试中,将小鼠以加速模式(在5分钟内以4至40 rpm的速度)放在旋转脚架上。给小鼠两次试验,间隔60分钟。每个阶段的最长时间为5分钟,并记录了在旋转脚架上花费的平均时间。

主要文化

Primary neurons were prepared from E17 恩卡α1+ / +和恩卡α1+/- 如先前所述的小鼠(22)。简而言之,将中脑小心地在冰冷的PBS中解剖,并用胰蛋白酶解离(0.25%,在37°C下12分钟)。将神经元接种到聚d-lysine–treated culture plates in DMEM SUPplemented with 10% fetal bovine serum (FBS) 和 1% penicillin/streptomycin. The cells were incubated in a 5% CO2 在37°C下孵育4小时,然后将培养基替换为无血清Neurobasal / B27 /谷氨酰胺培养基。每3天更换一半的培养基。所有实验均在体外培养12至14天的神经元上进行。

如前所述分离原代星形胶质细胞(38)。 Briefly, the midbrain of neonatal mice was separated from meninges 和 basal ganglia. Tissues were dissociated with 0.25% trypsin at 37°C 和 terminated by DMEM SUPplemented with 10% FBS 和 1% penicillin/streptomycin. Cells were plated 上 flasks, 和 the culture medium was replaced with fresh medium 24 hours later. Then, media were replaced every 3 days. After culturing for 14 days, microglia were detached from the astrocytes by shaking flasks at 200 rpm for 24 hours at 37°C 上 a shaker. Detached microglia were plated 和 incubated for 30 min at 37°C 和 5% CO2。通过更换培养基除去未结合的细胞。

蛋白质印迹分析

准备解剖的脑区或培养的细胞,用于顺序提取蛋白质。将样品在TX可溶性缓冲液[1%Triton X-100、150 mM 娜Cl,50 mM tris(pH 8.0)和蛋白酶抑制剂]中裂解,以提取TX可溶性级分。将不溶性沉淀物重悬于SDS可溶缓冲液[2%SDS,1%Triton X-100、150 mM 娜Cl,50 mM tris(pH 8.0)和蛋白酶抑制剂]中,并超声处理成SDS可溶级分。用BCA试剂盒(美国皮尔斯)测量蛋白质浓度。样品在SDS-聚丙烯酰胺凝胶电泳凝胶上分离(8%至15%),并转移到聚偏二氟乙烯膜上。在室温下于TBST缓冲液(10 mM tris-HCl,120 mM 娜Cl和0.1%Tween 20,pH 7.4)中封闭5%脱脂牛奶1小时后,在4°C下用各种一级抗体探测膜过夜。然后将膜在TBST中洗涤,并与适当的第二抗体在室温下孵育1小时。用TBST洗涤后,在ChemiDoc XRS系统(Bio-Rad)中用ECL底物观察印迹上的靶抗原。通过使用ImageJ对扫描的印迹进行光密度分析来定量条带密度。表S1中列出了该研究中使用的一抗和二抗。

联合IP测定

用含有蛋白酶抑制剂的裂解缓冲液(1%Triton X-100、150 mM 娜Cl,50 mM tris,pH 8.0)收集细胞样品。然后将等量的蛋白质与抗AMPKα抗体或抗NKAα1抗体在4°C孵育过夜。加入蛋白A / G PLUS-琼脂糖(Santa Cruz Biotechnology,SC-2003),与样品在室温下孵育4小时。将IP复合物用裂解缓冲液洗涤3次,并通过添加2x Laemmli样品缓冲液使其变性,然后煮沸5分钟。蛋白质印迹分析用于进一步检测IP复合物。

免疫荧光测定

将脑切片(20μm)和细胞样品固定在4%多聚甲醛中,然后用0.1%Triton X-100透化。在室温下于PBS缓冲液中封闭5%牛血清白蛋白(BSA)1小时后,在4°C下用各种一抗探测样品过夜。用PBS洗涤3次后,将样品与荧光二抗在室温下孵育2小时,然后将其与包含4',6-diamidino-2-phenylindole(DAPI)的固定介质(Invitrogen,Carlsbad,CA,USA)固定在一起。 )。使用荧光显微镜(日本尼康)拍摄照片。 ImageJ用于评估TH阳性SNpc神经元。从SNpc中收集每六个冠状冷冻切片(每切片20μm),并用抗TH抗体通过免疫荧光染色。对于每只鼠标,我们检查并分析了六个部分。将图像转换为灰度并使用ImageJ设置阈值。将相同的阈值应用于每个生物学和技术重复。通过ImageJ中的“分析粒子”功能分析了每个图像中占据的面积。用ImageJ分析αSyn,p-αSyn和溶酶体底物的强度。

质膜蛋白的生物素化

用SULFO-NHS-SS-生物素(1 mg / ml,Pierce,美国)标记质膜蛋白1小时。然后用含100 mM甘氨酸的PBS彻底冲洗细胞,以淬灭未反应的生物素,并在RIPA缓冲液[50 mM tris-HCl(pH 8.0),150 mM 娜Cl,1%Triton X-100和1%脱氧胆酸钠]中裂解。将蛋白质(150至300μg)在抗生蛋白链菌素珠(Pierce Chemical Co.)存在下于4°C孵育过夜。将珠子彻底洗涤,重悬于2x Laemmli样品缓冲液中,并通过Western印迹进行分析。

恩卡 activity assay

脑和细胞样品在缓冲液A(20 mM Hepes,250 mM蔗糖,2 mM EDTA和1 mM MgCl2,pH 7.4)。将沉淀重悬于缓冲液A中,并用BCA测定试剂盒(Pierce,USA)测定蛋白质浓度。将样品分成两等份:将一等份(50μl)与反应缓冲液1 [50μl,200 mM tris(pH 7.5),30 mM MgCl2, 200 mM 娜Cl, 60 mM KCl, 和 10 mM EGTA], 和 the other aliquot (50 μl) was incubated with reaction buffer 2 (buffer 1 + 1 mM OB; Sigma-Aldrich, O3125). ATP (1 mM) was added 至 start the reactions at 37°C for 10 min. After terminating the reactions by adding trichloroacetic acid [10 μl, 100% (w/v)], samples were placed 上 ice for 1 hour. Free phosphates were then collected in the SUPernatant of the samples after centrifuging at 20,000g for 30 min. Phosphate colorimetric kit (Sigma-Aldrich, MAK030) was used 至 measure the free phosphates. The 恩zyme activity of 恩卡 was defined as the difference of absorbance at 650 nm between the two aliquots.

活细胞成像

使用ATTO 488蛋白标记试剂盒(Sigma-Aldrich,38371)标记PFF。 Hoechst 33342(Thermo Fisher Scientific,62249)用于染色细胞核。 SH-SY5Y细胞用DR5-12D预处理1小时,然后进行PFF-ATTO 488处理。 PFF-ATTO 488处理后15分钟,将细胞替换为新鲜培养基。使用Leica全自动倒置显微镜(Leica,DMi8)每15分钟记录一次实时图像,持续1小时。

稳定细胞系的产生

为了产生αSyn过表达细胞系,使用Lipofectamine 3000转染试剂盒(Invitrogen,L3000015)将αSyn-eGFP质粒(Addgene,#40822)转染到SH-SY5Y细胞中。卡那霉素筛选成功转染的细胞,并通过蛋白质印迹法验证αSyn的表达。

恩卡α1 CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology, SC-419236) was used 至 generate 恩卡α1 KO stable cell line in Neuro2a cells. Briefly, cells were transfected with 恩卡α1 CRISPR-Cas9 KO plasmid 和 homology-directed repair plasmid, which is used 至 repair site-specific double-strand breaks (DSBs) caused by KO plasmids. After DSB 和 subsequent repairment, the cells incorporated puromycin resistance 和 red fluorescent protein (RFP) for selection. Western blotting was used 至 monitor the KO efficiency of 恩卡α1.

逆转录定量聚合酶链反应

从Neuro2a细胞提取的总RNA通过逆转录试剂盒(Promega,A5001)用于生成cDNA。进行了基于SYBR Green(Promega,A6001)的实时聚合酶链反应以定量mRNA水平。表S2列出了该研究中使用的引物。

AMP和ATP水平的测量

根据制造商的说明分别使用AMP试剂盒(Promega,V5011)和ATP试剂盒(Promega,G9242)测量细胞的AMP和ATP水平。分析了AMP与ATP的比例。

统计分析

未配对学生的 t test was used 至 compare 上e variable between two groups. One-way analysis of variance (ANOVA) was used 至 compare 上e variable in three or more groups followed by Bonferroni’的多重比较测试。 Two-way ANOVA was used 至 compare two independent variables followed by Bonferroni’的多重比较测试。 GraphPad Prism 7 software was used for analysis. Graphs showed the values by mean ± SEM, with individual datapoints. P <预定0.05作为统计学显着性的阈值。在体外实验中,所有数据都是通过三个以上的生物学复制品和两个技术复制品收集的,详细信息显示在图例中。

补充材料

有关本文的补充材料,请访问: http://advances.cqonlead.com/cgi/content/full/7/5/eabc5062/DC1

//creativecommons.org/licenses/by-nc/4.0/

这是根据以下条款分发的开放获取文章 知识共享署名-非商业许可,它允许在任何介质中使用,分发和复制,只要最终的使用是 出于商业利益,并提供了适当引用的原始作品。

参考和注释

致谢: We thank J.B. 玲格尔 for the gift of 恩卡α1+/- 老鼠和D. Ramond Herr对手稿进行了批判性阅读。 资金: This work was SUPported by the Singapore 娜tional Medical Research Council (NMRC/CIRG/1432/2015), Ministry of Education of Singapore Tier 2 Research grant (MOE2017-T2-2-029), 和 娜tional 性质 科学 Foundation of China (NSFC 81872865). 作者贡献: J.-S.B. 和 L.C. conceived this project. L.C. performed most experiments, analyzed the data, 和 wrote the manuscript. S.X. generated the monoclonal DR5-12D 和 two stable cell lines. L.D., 是的Z., Z.W., 和 H。S. helped with data analysis. M.Z., W.T.L., 和 X。N. provided critical ideas. J.-S.B. SUPervised 所有 the experimental procedures 和 revised the manuscript. 利益争夺: 作者宣称他们没有竞争利益。 数据和材料可用性: 本文和/或补充材料中提供了评估本文结论所需的所有数据。作者可能需要与本文相关的其他数据。
查看摘要

保持联系 科学进步

浏览本文