调节 AMPK 的催化活性对 α- 突触核蛋白毒性具有神经保护作用

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Modulating the catalytic activity of AMPK has neuroprotective effects against α-synuclein toxicity

Abstract

摘要

Background

背景

Metabolic perturbations and slower renewal of cellular components associated with aging increase the risk of Parkinson’s disease (PD). Declining activity of AMPK, a critical cellular energy sensor, may therefore contribute to neurodegeneration.

代谢紊乱和与衰老相关的细胞组分更新缓慢增加了患帕金森病(PD)的风险。细胞能量传感器 AMPK 的活性下降可能是神经退行性疾病的原因之一。

Methods

方法

Here, we overexpress various genetic variants of the catalytic AMPKα subunit to determine how AMPK activity affects the survival and function of neurons overexpressing human α-synuclein in vivo.

在这里,我们过度表达了 AMPKα 催化亚基的各种遗传变异,以确定 AMPK 活性如何影响在体内过度表达人 α- 突触核蛋白的神经元的存活和功能。

Results

结果

Both AMPKα1 and α2 subunits have neuroprotective effects against human α-synuclein toxicity in nigral dopaminergic neurons. Remarkably, a modified variant of AMPKα1 (T172Dα1) with constitutive low activity most effectively prevents the loss of dopamine neurons, as well as the motor impairments caused by α-synuclein accumulation. In the striatum, T172Dα1 decreases the formation of dystrophic axons, which contain aggregated α-synuclein. In primary cortical neurons, overexpression of human α-synuclein perturbs mitochondrial and lysosomal activities. Co-expressing AMPKα with α-synuclein induces compensatory changes, which limit the accumulation of lysosomal material and increase the mitochondrial mass.

AMPKα1和 α2亚基对人 α- 突触核蛋白毒性具有神经保护作用。值得注意的是,AMPKα1(T172Dα1)的一个修饰变异体(T172Dα1)具有组成性低活性,能够最有效地防止多巴胺神经元的丢失,以及 α- 突触核蛋白积累引起的运动损伤。在纹状体中,T172Dα1减少了包含聚集 α- 突触核蛋白的营养不良性轴突的形成。在原代皮质神经元中,人 α- 突触核蛋白的过度表达扰乱了线粒体和溶酶体的活性。Ampkα 与 α- 突触核蛋白共表达可诱导补偿性改变,限制溶酶体物质积累,增加线粒体质量。

Conclusions

结论

Together, these results indicate that modulating AMPK activity can mitigate α-synuclein toxicity in nigral dopamine neurons, which may have implications for the development of neuroprotective treatments against PD.

这些结果表明,调节 AMPK 活性可以减轻 α- 突触核蛋白对黑质多巴胺神经元的毒性作用,这可能对帕金森病神经保护治疗的发展具有启示意义。

Background

背景

Parkinson’s disease (PD) is a debilitating neurodegenerative disease mainly characterized by motor symptoms, which result from a dysfunction of basal ganglia caused by degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc). Alpha-synuclein (α-syn), a small presynaptic protein highly expressed in the dopamine neurons of the ventral midbrain, plays an important role in PD etiology [12], via mechanisms involving its misfolding and aggregation [34].

帕金森病(Parkinson’ s disease,PD)是一种神经退行性疾病衰弱性疾病,主要表现为拥有属性运动症状,由黑质致密部多巴胺神经元退化引起的基底神经节功能障碍。α- 突触核蛋白(α-syn)是一种在腹侧中脑多巴胺神经元中高度表达的突触前小蛋白,通过其错误折叠和聚集机制在 PD 病因[1,2]中发挥重要作用[3,4]。

Apart from rare genetic forms with early onset [5], incidence of the disease depends on age and therefore, aging is considered a major risk factor for PD. The exact nature of the interplay between aging and pathogenic mechanisms remains however poorly explored. Nevertheless, it has been shown that α-syn protein levels increase significantly with age [6]. Aging may also impair capacity of cells to cope with metabolic stress. This may affect the survival and function of neurons exposed to high energy demand, such as nigral dopamine neurons [7]. Decreased turnover rates of organelles and proteins, including α-syn, may further contribute to metabolic defects and prompt neurodegeneration.

除了具有早发型的罕见遗传形式外,该病的发病率取决于年龄,因此,老化被认为是帕金森病的主要危险因素。然而,衰老和致病机制之间相互作用的确切性质仍然没有得到很好的探讨。然而,随着年龄的增长,α- 同位素蛋白水平显著增加。衰老也可能削弱细胞应对代谢应激的能力。这可能影响神经元的生存和功能暴露于高能量需求,如黑质多巴胺神经元[7]。细胞器和蛋白质,包括 α-syn 的周转率降低,可能进一步导致代谢缺陷和神经退行性疾病。

AMP-activated protein kinase (AMPK) is a molecular gauge of energy status, at both cellular and whole-body levels [89]. Mammalian AMPK is a heterotrimeric complex consisting of α, β and γ subunits, which carry out enzymatic, scaffolding and regulatory functions, respectively [8,9,10,11]. In mammals, there exist two isoforms of the α and β subunits, and three isoforms of the γ subunit, all encoded by separate genes [8912]. AMPK becomes catalytically active when cellular levels of AMP rise substantially, as a consequence of metabolic stress. Binding of AMP to the CBS domains in γ subunit, triggers allosteric activation of the complex, favors T172 phosphorylation by AMPK kinases, and protects this residue from potential dephosphorylation. As a consequence of AMP binding, the catalytic activity of the AMPK complex is increased by nearly 1000-fold [8913,14,15]. Once activated, AMPK induces catabolic processes and inhibits energy consumption, in order to maintain metabolic homeostasis [891116].

AMP活化蛋白激酶是细胞和整个身体水平上能量状态的分子标准[8,9]。哺乳动物 AMPK 是一种由 α、 β 和 γ 亚基组成的异三聚体复合物,分别具有酶、支架和调节功能[8,9,10,11]。在哺乳动物中,存在着 α 亚基和 β 亚基的两种亚型,以及 γ 亚基的三种亚型,它们都由不同的基因编码[8,9,12]。作为代谢应激的结果,当 AMP 的细胞水平大幅度上升时,AMPK 变得具有催化活性。AMP 与 CBS 结合,激活复合物的变构激活,有利于 AMPK 激酶的 T172磷酸化,并保护这种残基不受潜在的去磷酸化作用的影响。作为 AMP 结合的结果,AMPK 配合物的催化活性增加了近1000倍[8,9,13,14,15]。一旦激活,AMPK 诱导分解代谢过程和抑制能量消耗,以维持代谢稳态[8,9,11,16]。

A growing body of evidence indicates a role for AMPK in aging, which might possibly be linked to the risk of developing neurodegenerative diseases, including PD. AMPK signaling gradually declines with age [17,18,19,20], whilst activation of AMPK and its downstream targets has been shown to increase longevity in model organisms like Drosophila [21] and C. elegans [22]. Remarkably, low activity of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), one of the key downstream effectors of AMPK, has been linked to sporadic PD cases [23]. Furthermore, metformin and glitazone, anti-diabetic drugs acting via AMPK and PGC-1α, respectively, have been shown to significantly decrease the risk of PD in large cohort clinical trials [2425].

越来越多的证据表明腺苷酸活化蛋白激酶在衰老中起作用,这可能与患上神经退行性疾病的风险有关,包括帕金森病。AMPK 信号随着年龄(17,18,19,20)逐渐衰退,而 AMPK 及其下游目标的激活已被证明能延长果蝇和秀丽隐杆线虫等模式生物的寿命。值得注意的是,AMPK 下游关键效应因子之一的过氧化物酶体增殖物活化受体 γ 辅激活因子 -1α (pgc-1α)的低活性与散发性帕金森病有关[23]。此外,二甲双胍和格列酮这两种分别通过 AMPK 和 pgc-1α 作用的抗糖尿病药物,在大型队列临床试验中已被证明能显著降低帕金森病的风险[24,25]。

Only a few studies have explored the effect of AMPK on α-syn toxicity. In vitro, overexpression of α-syn lowers AMPK activity, which can be compensated by exposure to metformin or 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). Conversely, reduced expression of AMPK lowers cellular resistance to α-syn [26]. However, inducing chronic high AMPK activity was also found to be detrimental in cell-based assays [27]. It is well recognized that α-syn may also perturb key cellular processes including mitochondrial activity, which may indirectly impinge on AMPK signaling. Recently, α-syn has been found to bind PIKE-L, an inhibitor of AMPK, as a function of S129 phosphorylation [28]. PIKE sequestration in Lewy bodies may hence contribute to neuronal degeneration via AMPK overactivation. Overall, it is however not known how AMPK may control the survival of nigral dopamine neurons exposed to the α-syn pathology in vivo.

目前,关于 AMPK 对 α- 合成氨基酸毒性作用的研究还很少。在体外,过量表达 α- 合成氨基酸降低 AMPK 活性,这可以通过暴露于二甲双胍或5- 氨基咪唑 -4- 甲酰胺 -1-β-d- 呋喃核苷(AICAR)来补偿。相反,AMPK 表达的减少降低了细胞对 α-syn 的抗性[26]。然而,在以细胞为基础的实验中也发现诱导慢性高 AMPK 活性是有害的。众所周知,α-syn 还可能干扰包括线粒体活性在内的关键细胞过程,这可能间接影响 AMPK 信号传导。最近,人们发现 α-syn 能结合 AMPK 的抑制剂 PIKE-L,作为 S129磷酸化的功能[28]。派克在路易体可能因此有助于神经元退化通过 AMPK 过度激活。总的来说,目前还不清楚 AMPK 是如何控制体内 α- 合成酶病理作用下的黑质多巴胺神经元的存活。

Here, we explore with a genetic approach how the activity of the AMPK complex determines vulnerability of neurons overexpressing α-syn. In order to modulate the energy-sensing ability of the complex, we overexpress variants of the AMPKα subunit, which differ in their activity pattern, and test their neuroprotective effects in neurons overexpressing human α-syn. In vivo, our results provide evidence that the catalytic AMPKα subunit is neuroprotective at early stages of the developing α-syn pathology. In particular, the AMPK T172Dα1 variant, which provides mild chronic catalytic activity, leads to the most efficient neuroprotection by reducing the accumulation of misfolded α-syn within dopamine axons projecting to the striatum (STR). In vitro, we show that the overexpression of AMPKα subunits in primary neurons, modifies the effects of α-syn on autophagic and mitochondrial activities. These results suggest that pre-emptive measures to chronically activate AMPK or enhance the catalytic response of this metabolic sensor, can mitigate the effects of α-syn toxicity in a genetic rodent model of PD pathology.

在这里,我们用遗传学的方法探讨 AMPK 复合物的活性如何决定过度表达 α-syn 的神经元的脆弱性。为了调节复合体的能量感受能力,我们过量表达 ampkα 亚单位的不同活性模式的变体,并测试它们在过量表达人 α-syn 的神经元中的神经保护作用。在体内,我们的结果提供了证据,催化 ampkα 亚基是神经保护的早期阶段发展的 α- 同位素病理学。特别是 AMPK T172Dα1变异体具有温和的慢性催化活性,通过减少投射到纹状体(STR)的多巴胺轴突内错折叠的 α- 同位素的积累,从而达到最有效的神经保护作用。在体外实验中,我们发现 ampkα 亚基在原代神经元中的过度表达,调节了 α-syn 对自噬和线粒体活性的影响。这些结果表明,先发制人的措施,慢性激活 AMPK 或增强这一代谢传感器的催化反应,可以减轻 α- 突变毒性的影响,在一个遗传啮齿动物模型的 PD 病理。

Methods

方法

Vector construction

矢量结构

Plasmid constructs encoding the K45Rα2 and 1-310α2 variants were generated from human wild-type AMPKα2 (nucleotides 72–1730, NM_006252) via site-directed mutagenesis and PCR amplification. These sequences, as well as the α-syn cDNA (nucleotides 46–520, NM_000345), were introduced into the pAAV-pgk-MCS-WPRE backbone using standard cloning procedures. Similar pAAV-pgk vector constructs encoding wild-type human AMPKα1 and the T172Dα1 variant were kindly provided by Dr. K. Sakamoto. To assess construct functionality, each of the pAAV-AMPKα plasmid was co-transfected in HEK293T cells, either alone or in combination with plasmids encoding the β1 (pECE-HA-AMPKbeta1, Addgene #31666) and γ3 subunits of AMPK (pDONR223-PRKAG3, Addgene #23549). The pAAV constructs encoding the mCherry-GFP-LC3 reporter of autophagy and mitoDsRed have been previously described [2930].

质粒构建编码 K45Rα2和1-310α2变异体的质粒构建是从人类野生型 AMPKα2(核苷酸72-1730,NM _ 006252)通过定点突变扩增和 PCR 扩增产生的。这些序列以及 α-syn 基因(核苷酸46-520,NM _ 000345)通过标准的克隆程序被引入 paav-pgk-mcs-w pre 骨干中。类似的 pAAV-pgk 载体构建编码人类野生型 AMPKα1和 T172Dα1变异体,已由 k. Sakamoto 博士提供。为了评价质粒的构建功能,将 paav-AMPKα 质粒单独或与编码 ampk1(pee-ha-ampkbeta1,Addgene # 31666)和 γ3亚基(pDONR223-PRKAG3,Addgene # 23549)的质粒共转染 HEK293T 细胞。pAAV 构建编码 mCherry-GFP-LC3自噬和 mitoDsRed 的报告已经在前面描述过[29,30]。

Production of AAV2/6 vector particles and vector titration

AAV2/6载体颗粒的制备及载体滴定

Viral vectors were produced and titrated as previously described [31]. Briefly, relative AAV infectivity was determined by real-time PCR (rtPCR) quantification of double-stranded vector genomes present in total DNA isolated from HEK293 cells, 48 h post-infection. The infectivity rate expressed in ‘transducing units’ (TU) was calculated according to a known infectivity of a standard virus encoding GFP (AAV2/6-cmv-eGFP), whose titer was estimated via flow cytometry.

病毒载体的产生和滴定以前描述[31]。简要地说,通过实时荧光定量 PCR (rtPCR)定量分析从 HEK293细胞中分离的双链载体基因组,在感染48h 后测定了相对的腺相关病毒感染性。根据编码绿色荧光蛋白(GFP)的标准病毒(AAV2/6-cmv-eGFP)的已知感染性,通过流式细胞仪测定其滴度,计算其转导单位(TU)表达的感染率。

Cultures of mouse primary cortical neurons

小鼠原代皮层神经元的培养

Primary cortical neurons were derived from C57BL6/J mouse embryos, at day E16.5. Unless stated otherwise, cells were cultured in the Neurobasal medium (Thermofisher Scientific #21103–049), supplemented with 2% B27 (Thermofisher Scientific #17504–044), 1% GlutaMax (Thermofisher Scientific #35050–061) and 1% mix of Penicillin/Streptomycin (Thermofisher Scientific #10378–016) at 37 °C and 5% CO2. For all biochemical purposes, neurons were grown on adequate culture plates, pre-coated overnight at 37 °C, with 100 μg/ml poly-DL-ornithine (Sigma #P8638). For immunocytochemistry assays, neurons were grown in the 24-well plate formats, on glass cover slips pre-coated with 0.2 mg/ml poly-D-lysine (Sigma #P6407) and 33.2 μg/ml laminin (Thermofisher Scientific #23017015).

从 C57BL6/J 小鼠胚胎中分离出初级皮质神经元,于第16.5天培养。除非另有说明,细胞培养基为 neurobasis 培养基(Thermofisher Scientific # 21103-049) ,辅以2% B27(Thermofisher Scientific # 17504-044)、1% GlutaMax (Thermofisher Scientific # 35050-061)和1% 青霉素/链霉素混合培养基(Thermofisher Scientific # 10378-016) ,温度为37 ° c,CO2为5% 。对于所有生化目的,神经元生长在适当的培养板上,在37 ° c 的条件下预涂一夜,用100μg/ml 的聚 dl-ornithine (Sigma # p8638)。免疫细胞化学实验表明,神经元生长呈24孔平板形式,在玻璃盖片上分别涂有0.2 mg/ml 多聚赖氨酸(Sigma # p6407)和33.2 μg/ml 层粘连蛋白(Thermofisher Scientific # 23017015)。

Mitochondrial DNA and mitochondrial mass estimation

线粒体脱氧核糖核酸和线粒体质量评估

Neurons were plated at a density of 200,000 per well in a 24-well plate format, in medium without phenol red (Thermofisher Scientific #12348–017). On the fifth day, they were infected with AAV2/6-mitoDsRed vector in combination with the other vectors mentioned in the text. All vectors were used at a dose of 0.8E6 TU. Seven days later, neurons were collected by gentle dissociation. The arithmetic mean of mitoDsRed red fluorescence intensity was determined using Accuri C6 Flow Cytometer, recording events in the gated population of living cells.

神经元以200,000个单位的密度在24孔板的形式被镀上,在不含酚红的培养基中(Thermofisher Scientific # 12348-017)。在第五天,他们被感染 AAV2/6-mitoDsRed 载体结合其他载体在文中提到。所有载体均在0.8 E6 TU 剂量下使用。7天后,采用轻度分离法收集神经元。用 Accuri C6流式细胞仪测定了 mitoDsRed 荧光强度的算术平均值,记录了活细胞门控种群中的事件。

To estimate the amount of mitochondrial DNA (mtDNA) using rtPCR, primary neurons were infected with AAV2/6 vectors at a dose of 1E6 TU for each vector on day 5 after plating. On day 12, cells were lysed and total DNA was extracted, following Maxwell 16 Viral Total Nucleic Acid Purification Kit protocol (Promega). SYBR-green rtPCR (Qiagen) was used to determine the relative amounts of mtDNA and gDNA in each sample, using two sets of primers: 16S rRNA for mtDNA and Hexokinase-2 (Hk2) for gDNA. The relative amounts of mtDNA/gDNA were determined using the ΔΔCt method. For the primer sequences please refer to the Supplementary Methods (Additional file 1).

为了利用 rtPCR 估计线粒体 dna (mtDNA)的数量,在每个载体上,AAV2/6载体在第5天被感染,每个载体剂量为1E6 TU。第12天,细胞裂解和总 DNA 提取,遵循麦克斯韦16病毒总核酸纯化试剂盒方案(Promega)。采用 SYBR-green rtPCR (Qiagen)方法,以线粒体 dna (mtDNA)16S rRNA 和 gDNA (Hexokinase-2,Hk2)为引物,测定了每份样品中线粒体 dna 和 gDNA 的相对含量。用 δδct 方法测定了线粒体 dna/gdna 的相对含量。关于入门序列,请参阅补充方法(附加文件1)。

Estimation of the autophagic activity using LC3B-mCherry-EGFP reporter

LC3B-mCherry-EGFP 报告基因在自噬活性测定中的应用

Neurons were plated at a density of 200,000 cells on glass cover slips, pre-coated with laminin and poly-D-lysine, and cultured in medium without phenol red. On day 5 after plating, neurons were infected with 1E6 TU of each AAV2/6 vector. On day 12, cultures were fixed for 20 min with ice-cold 4% paraformaldehyde (PFA). Following staining with DAPI, coverslips were mounted on glass slides using Mowiol medium (Fluka).

神经细胞以20万个细胞密度在玻璃片上被包裹,预先包裹层粘连蛋白和多聚赖氨酸,培养在无酚红的培养基中。电镀后第5天,用 AAV2/6载体的1E6 TU 感染神经元。第12天,用4% 冰冻多聚甲醛(PFA)固定培养20分钟。采用 DAPI 染色后,用 Mowiol 培养基(Fluka)将柯氏唇安装在玻片上。

Neurons expressing the AAV-encoded LC3B-mCherry-EGFP probe were analyzed using Zeiss LSM 700 confocal microscope at 63× magnification. Pictures were taken in random fields. Each condition was run in triplicates, for a total number of 22–30 neurons per condition. Yellow or red dots, corresponding to autophagosomes and autolysosomes, respectively, were counted manually, in a blind manner, after subtracting mean values of fluorescent signal in each channel. Numbers of autophagic vesicles were normalized to cytosol area. The analysis was performed using Fiji software plugins.

采用蔡司 lsm700共聚焦显微镜,放大63倍,对表达 aav 编码的 LC3B-mCherry-EGFP 探针的神经元进行了分析。照片是在随机场所拍摄的。每个条件运行在三倍,总数为22-30个神经元每个条件。在每个通道减去荧光信号的平均值后,盲法人工计数分别对应于自噬体和自溶体的黄点和红点。自噬囊泡数量归一化为胞质溶胶区。分析是使用斐济软件插件完成的。

Biochemical analysis of protein expression

蛋白质表达的生化分析

Neurons were plated at a density of 650,000 per well in 12-well plate format. On day 5 after plating, neurons were infected with 3.9E7 TU of each AAV2/6 vector. At day 12, neurons were harvested in lysis buffer containing 0.5% NP40 with protease and phosphatase inhibitors (Roche), and incubated on ice for 10 min. Following cell lysis, protein extracts were centrifuged for 20 min at 14,000 rpm at 4 °C and the supernatant was collected. Protein concentration was evaluated using BCA Protein Kit Assay (Thermofisher Scientific #23225). For detailed description of the protein analysis, refer to Supplementary Methods (Additional file 1).

神经元以12孔板形式每个孔650,000个密度被镀。第5天,每个 AAV2/6载体的3.9 E7 TU 感染神经元。第12天,用蛋白酶和磷酸酶抑制剂(Roche)在含有0.5% NP40的溶解液中收集神经元,并在冰箱中孵育10min。细胞裂解后,蛋白质提取物在4 ° c 下14000rpm 离心20min,收集上清液。用 BCA 蛋白试剂盒法(Thermofisher Scientific # 23225)测定蛋白质浓度。有关蛋白质分析的详细描述,请参阅补充方法(附加文件1)。

Primary antibodies: AMPK alpha (Cell Signaling #2532) 1:1000; pAMPK alpha (Thr 172) (40H9; Cell Signaling #2535) 1:1000; pACC (Millipore #07–303) 1:1000; Actin (I-19; Santa Cruz #sc-1616) 1:5000; anti-α-syn (Becton Dickinson Biosciences #610787) 1:8000; anti-MAP1LC3B (Lifespan BioSciences) 1:1000. Secondary antibodies: Goat Anti-Rabbit IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401353), Goat Anti-Mouse IgG H&L Chain Specific Peroxidase Conjugate (Calbiochem #401215).

主要抗体: AMPK alpha (细胞信号 # 2532)1:1000; pAMPK alpha (Thr 172)(40H9; 细胞信号 # 2535)1:1000; pACC (Millipore # 07-303)1:1000; Actin (I-19; Santa Cruz # sc-1616)1:5000; 抗 -α-syn (美国BD公司生物科学 # 610787)1:8000; anti-1lc3b (生物科学)1:1000。次级抗体: 山羊抗兔 IgG h 和 l 链特异性过氧化物酶结合物(Calbiochem # 401353) ,山羊抗鼠 IgG h 和 l 链特异性过氧化物酶结合物(Calbiochem # 401215)。

Stereotaxic injection of viral vectors

病毒载体的立体定向注射

All in vivo experiments were performed using adult female Sprague-Dawley rats (Janvier), weighing around 200 g at the time of surgical procedure. Animals were housed in standard 12 h light/dark cycles, with ad libitum access to water and food. All procedures were approved by a local ethics committee and performed in accordance with the Swiss legislation and the European Community Council directive (86/609/EEC) regulating care and use of laboratory animals. In order to minimize stress, animals were accustomed for at least one week prior to behavioral test and surgical manipulation. For detailed description of the injection procedure, refer to Supplementary Methods (Additional file 1).

所有的活体实验都是用成年雌性 Sprague-Dawley 大鼠(Janvier)进行的,在手术过程中大约重200克。动物被按照标准的12小时光/暗周期饲养,可随意获得水和食物。所有程序都由一个地方道德委员会批准,并根据瑞士立法和欧洲共同体理事会关于实验室动物护理和使用的指令(86/609/EEC)进行。为了尽量减少压力,动物在行为测试和手术操作之前至少有一周习惯了。有关注入过程的详细描述,请参阅补充方法(附加文件1)。

Coordinates used to target the SNpc: −5.2 mm (anteroposterior), −2 mm (mediolateral), −7.8 mm (dorsoventral, relative to skull surface), −3.3 mm (tooth bar). Vectors were used at a total injected dose of 1.5E7 TU (AAV2/6-α-syn) and 1.2E6 TU (AAV2/6-AMPKα and AAV2/6-non-coding vector).

用于瞄准 SNpc 的坐标:-5.2毫米(前后) ,-2毫米(中外侧) ,-7.8毫米(背腹侧,相对于颅骨表面) ,-3.3毫米(牙条)。载体的总注射剂量为1.5 E7 TU (AAV2/6-α-syn)和1.2 E6 TU (aav2/6-ampkα 和 AAV2/6-non-coding vector)。

Animal behavior

动物行为

During the time course of the study, spontaneous forelimb activity was estimated periodically using the cylinder test. Animal’s performance was evaluated during 5 min. The time was extended in case an animal was poorly active, until it cumulatively used both of its forepaws a minimum of 20 times.

在研究过程中,使用圆柱试验周期性地估计自发前肢活动。动物生产性能评价时间为5分钟。如果一只动物不太活跃,这个时间就会延长,直到它累积使用两只前爪至少20次。

Right forepaw preference was calculated according to the following formula:

右前爪偏好是根据以下公式计算出来的:RPx(%)=(RxRx+Lx−RoRo+Lo)×100RPx(%)=(RxRx+Lx−RoRo+Lo)×100

Where: RPx-right forepaw preference at a given time point; Rx-total number of the right forepaw use at a given time point; R0– total number of the right forepaw use before surgery; Lx– total number of the left forepaw use at a given time point; L0– total number of the left forepaw use before surgery.

其中: rpx-右前爪在特定时间点的偏好; rx-在特定时间点右前爪使用的总数; R0-手术前右前爪使用的总数; Lx-在特定时间点左前爪使用的总数; L0-手术前左前爪使用的总数。

Immunohistochemistry

免疫组织化学

Procedures for the preparation of brain tissues and immunohistochemistry are described in the Supplementary Methods (Additional file 1).

补充方法(附加文件1)描述了脑组织和免疫组织化学的制备过程。

Primary antibodies (immunofluorescence): anti-TH (Millipore #AB152) 1:1000; anti-α-syn (Millipore #AB5334P) 1:1000; anti-α-syn clone 5G4 (AJ Roboscreen) 1:1000; anti-phospho S129 α-syn (Abcam ab59264) 1:1000. Secondary antibodies: Cy2-conjugated donkey anti-rabbit IgG (H + L) (Jackson ImmunoResearch Inc. #711–225-152) 1:1000; Cy3-conjugated F(ab’)2 fragment donkey anti-sheep IgG (H + L) (Jackson ImmunoResearch Inc. #713–166-147) 1:1000.

主要抗体(免疫荧光) : 抗 th (Millipore # ab152)1:1000; 抗 -α-syn (Millipore # ab5334p)1:1000; 抗 -α-syn 克隆5G4(AJ Roboscreen)1:1000; 抗磷酸化 S129 α-syn (Abcam ab59264)1:1000。次级抗体: cy2结合的驴抗兔 IgG (h + l)(Jackson 免疫研究公司 # 711-225-152)1:1000; cy3结合的 f (ab’)2片段驴抗羊 IgG (h + l)(Jackson 免疫研究公司 # 713-166-147)1:1000。

Primary antibodies (DAB): anti-TH (Millipore #AB152 1:1000); anti-DAT (Millipore #MAB369) 1:4000; anti-HA-tag (Covance clone 16B12 #MMS-101P) 1:1000. Secondary antibodies: peroxidase Goat anti-rabbit IgG (Vector Laboratories #PI-1000) 1:200; peroxidase goat anti-mouse IgG (Vector Laboratories #BA-9200) 1:200; biotinylated rabbit anti-rat IgG (Vector Laboratories #BA-4001).

一级抗体(DAB) : 抗 th (Millipore # ab1521:1000) ; 抗 dat (Millipore # mab369)1:4000; 抗 ha-tag (Covance clone 16B12 # mms-101p)1:1000。二级抗体: 过氧化物酶山羊抗兔 IgG (载体实验室 # pi-1000)1:200; 过氧化物酶山羊抗小鼠 IgG (载体实验室 # ba-9200)1:200; 生物素化兔抗大鼠 IgG (载体实验室 # ba-4001)。

Optical densitometry and stereological evaluation of neuron loss

神经元丢失的光学密度测量和体视学评价

On average, a total number of 16 sections (one in six interval) covering the whole STR were DAB-stained for tyrosine hydroxylase (TH) or dopamine transporter (DAT) in order to visualize dopamine fibers. Stained sections were scanned using Epson Perfection V750 Pro scanner. For each STR section, optical density, defined as integrated density of grey pixel values corrected for background noise and striatal surface, was measured in each hemisphere using the ImageJ software. For each animal, results are expressed as a percent loss of total optical density on the injected, compared to non-injected side.

为了观察多巴胺纤维的形态结构,平均每6个 STR 位点共有16个区段(1个区段)对酪氨酸羟化酶(TH)或多巴胺转运蛋白(DAT)进行染色。染色部分用 Epson Perfection V750 Pro 扫描仪进行扫描。对于每个 STR 切片,用 ImageJ 软件测量每个半球的光密度,定义为经背景噪声和纹状体表面校正的灰色像素值的综合密度。对于每个动物,结果表示为总光密度的百分比损失注射,相比未注射侧。

Total α-syn overexpression in the midbrain was evaluated by fluorescence immunohistochemistry (anti-α-syn, Millipore #AB5334P). Three sections near the site of vector injection were imaged using a slide scanner (Olympus VS120-L100). Integrated fluorescence intensity was determined on three sections in the entire midbrain of the right hemisphere using Fiji software, after subtraction of the background intensity measured on the same sections in the contralateral hemisphere.

采用荧光染色法(anti-α-syn,Millipore # ab5334p)检测 α-syn 在中脑的过度表达情况。使用一台幻灯扫描仪(Olympus VS120-L100)对矢量注入点附近的三个部分进行了成像。在减去对侧大脑半球同一部分测量的背景强度后,用斐济软件测定了右半球整个中脑的三个部分的综合荧光强度。

Aggregated forms of α-syn were visualized in the rat STR using immunostaining with 5G4 anti-α-syn monoclonal antibody. For the analysis, we used 5–6 sections per animal. Fluorescent pictures of the injected side were taken in the dorsal part of medial STR using a Leica DM5500 microscope, and assembled into a mosaic image. Optical densitometry of a given area was calculated using the ImageJ software. Data represent the mean ± SEM of the grey value of pixels, averaged from 5 to 6 sections of the medial STR per animal.

用5G4抗 -α-syn 单克隆抗体免疫组织化学染色法观察了 α-syn 在大鼠 STR 中的聚集形态。为了进行分析,我们使用了每只动物5-6个切片。用徕卡 DM5500显微镜在内侧 STR 背侧部分拍摄注射侧的荧光图像,并组装成镶嵌图像。用 ImageJ 软件计算给定面积的光学密度。数据代表像素灰度值的平均值 ± 扫描电镜,平均为每只动物内侧 STR 的5ー6个断面。

Detailed description of the procedures for stereological assessment of neuron counts are provided in the Supplementary Methods (Additional file 1).

对神经元计数的体视学评估程序的详细描述见补充方法(附加文件1)。

Transmission electron microscopy

透射电子显微术

For detailed description of sample preparation, refer to Supplementary Methods (Additional file 1). Each condition consisted of neurons analyzed from two animals. Thin sections in the SNpc region were viewed in the electron microscope and images taken of every neuronal cell body containing profiles of nuclei. Only large neuronal cell bodies were imaged. All the mitochondria and the cytosol were annotated in the TrakEM2 software running in the Fiji software. Relative comparisons of mitochondrial size, density, and their volume fraction in the cytosol were based on the 2D images.

有关样品制备的详细说明,请参阅补充方法(附加文件1)。每个条件包括神经元分析从两个动物。在每个神经元细胞体包含细胞核的剖面图中,可以看到 SNpc 区域的薄切片电子显微镜。只有大型神经元胞体成像。在 Fiji 软件上运行的 TrakEM2软件中注释了所有的线粒体和胞浆。线粒体的大小、密度和它们在胞质中的体积分数的相对比较是基于2D 图像的。

Statistical analysis

统计分析

All data are expressed as arithmetic mean with a standard error of the mean (SEM). If not mentioned otherwise, statistical analysis of the data was performed using one- or two-way analysis of variance (ANOVA), with a subsequent Tukey’s honest significant difference (HSD) post hoc test, using Statistica software (Statsoft). The alpha level of significance was set at 0.05. For each experiment, the number of replicates is indicated in the figure legend. The experimenter acquiring data was blinded to the experimental protocol.

所有数据表示为算术平均值与标准误差的平均值(SEM)。如果没有提到另外,统计分析的数据进行了单向或双向方差分析变异数(ANOVA) ,随后的 Tukey 的诚实显着性差异(HSD)事后测试,使用 stattica 软件(Statsoft)。显著性的 alpha 水平被设置为0.05。对于每个实验,复制的数量在图例中指出。获取数据的实验者对实验方案视而不见。

Results

结果

AAV-based constructs for overexpression of AMPKα modulate AMPK activity in neuronal cells

基于 aav 的 AMPKα 过表达载体对神经细胞 AMPK 活性的调控

We generated AAV vector constructs encoding different variants of AMPKα to modulate activity of the AMPK complex. These constructs are described in Fig. 1a. They comprise wild-type forms of the AMPKα1 and α2 subunits, as well as the catalytically inactive K45R mutant of the α2 subunit (K45Rα2). In addition, two previously characterized constitutively active forms of AMPKα were included in our experiments: T172Dα1 encodes a full-length AMPKα1 mutant, which carries an aspartate residue on the critical position 172 (T172D) to mimic phosphorylation. This variant has low but constitutive activity [3233]; 1-310α2 encodes a constitutively active truncated version of AMPKα2 (amino acids 1–310), which does not integrate into the AMPK complex because it is devoid of both the C-terminal auto-inhibitory domain (AID) and the β subunit-binding domain (α-CTD) [34]. To assess the activity of these constructs as a function of the amount of AMPK complex, we used an assay based on AMPK overexpression in HEK293T cells and measured the level of phospho-acetyl-CoA carboxylase (pACC), a product of AMPK activity (Additional file 2: Fig. S1). To determine if the activity of each of these variants was dependent on the availability of other AMPK subunits, we compared a condition in which only the α subunit was overexpressed, with a condition in which the β1 and γ3 subunits were co-overexpressed with the α subunit. In cells expressing either the AMPKα1 or the α2 subunit, pACC levels appeared to increase when the β and γ subunits were co-overexpressed (Additional file 2: Fig. S1), indicating that AMPK activity is controlled by complex formation. However, in cells overexpressing the 1-310α2, T172Dα1 and K45Rα2 subunits, the level of pACC remained very similar in both conditions, which shows that the activity of these variants is to a large extent independent from the AMPK complex. Furthermore, the level of pACC was found to be higher with the 1-310α2 subunit, as compared to the T172Dα1 and K45Rα2 subunits.

我们构建了编码 AMPKα 不同变体的 AAV 载体来调节 AMPK 复合物的活性。这些结构如图1a 所示。它们包括 AMPKα1和 α2亚基的野生型,以及催化活性非活性的 α2亚基 K45R 突变体(K45Rα2)。另外,我们的实验中还发现了两种 ampkα 的组成活性形式: T172Dα1编码一个 AMPKα1全长突变体,该突变体在临界位置172(T172D)携带一个天冬氨酸残基来模拟磷酸化。该突变体具有较低的组成型活性[32,33] ; 1-310α2编码 AMPKα2(氨基酸1-310)的组成型活性缺失,不能整合到 AMPK 复合物中,因为它缺乏 c 末端自抑制结构域(AID)和 β 结合结构域(α-ctd)[34]。为了评估这些构建的活性作为 AMPK 复合物数量的功能,我们使用了一个基于 AMPK 过表达的分析在 HEK293T 细胞和测量磷酸-乙酰辅酶 a 羧化酶(pACC)的水平,AMPK 活性的产物(附加文件2: Fig。中一)。为了确定这些变异的活性是否依赖于其他 AMPK 亚基的可用性,我们比较了一个只有 α 亚基过表达的条件和一个 β1和 γ3亚基与 α 亚基共表达的条件。在表达 AMPKα1或 α2亚基的细胞中,当 β 亚基和 γ 亚基共同过表达时,pACC 水平升高。表明 AMPK 活性受复合物形成控制。然而,在过度表达1-310α2、 T172Dα1和 K45Rα2亚基的细胞中,pACC 的水平在两种条件下保持极为相似,这表明这些变异体的活性在很大程度上独立于 AMPK 复合体。此外,与 T172Dα1和 K45Rα2亚基相比,1-310α2亚基的 pACC 水平较高。

figure1
Fig. 1 图一

To assess the effects of AMPKα overexpression in neuronal cells, we next transduced primary neurons from the mouse E16.5 cortex with AAV2/6 particles encoding each of these forms of AMPKα. Of note, we measured by real-time PCR (rt-PCR) the endogenous expression of the α1 and α2 isoforms of the catalytic AMPKα subunit. Cortical neurons were found to predominantly express the α1 subunit, as the mRNA level of AMPKα1 was found to be 9.2 ± 1.2 fold higher than that of α2 (Additional file 3: Fig. S2a). In the adult rat SN, the transcript of the α1 subunit was 85.0 ± 11.6 fold more abundant than α2 (Additional file 3: Fig. S2b). Hence, the α1 subunit is likely to be the main catalytic component of the AMPK complex both in cortical neurons and in the rat ventral midbrain.

为了评估 ampkα 过度表达在神经细胞中的作用,我们接下来用 AAV2/6编码 ampkα 的每一种形式的颗粒转导来自小鼠 E16.5皮层的初级神经元。值得注意的是,我们通过实时 PCR (rt-PCR)检测了催化 ampkα 亚基 α1和 α2亚型的内源性表达。AMPKα1的 mRNA 表达水平比 α2高9.2 ± 1.2倍(附图3)。S2a).在成年大鼠 SN 中,α1亚单位的转录本比 α2丰度高85.0 ± 11.6倍。第2b 条)。因此,α1亚基可能是大鼠皮层神经元和腹侧中脑 AMPK 复合物的主要催化成分。

To determine the effect of human α-syn accumulation on AMPKα activity, primary neurons were co-transduced with an AAV2/6 vector encoding human α-syn (AAV-α-syn, 3.9E7 TU). In each control condition, a similar non-coding AAV2/6 vector was added to the primary neurons to reach the same total dose of vector.

为了探讨人 α-syn 的积累对 ampkα 活性的影响,本研究用编码人 α-syn 的 AAV2/6载体(AAV-α-syn,3.9 E7 TU)共转导原代神经元。在每个控制条件下,在初级神经元中加入类似的非编码 AAV2/6载体,使其达到相同的总剂量。

To assess the overall AMPK activity, we analyzed via western blotting the levels of total AMPKα, T172 phospho-AMPKα (pAMPK), pACC and total α-syn (Fig. 1b-d). Using a pan-AMPKα antibody, we could detect a clear increase in the total level of AMPKα for all conditions in which a variant of AMPKα was overexpressed (Fig. 1b, c). Subsequently, we analyzed the levels of T172 phosphorylation (Fig. 1b, d). An increase in T172 pAMPK was mainly observed in neurons overexpressing AMPKα1, indicating that α1 is the subunit which is the most efficiently phosphorylated in these neurons. Of note, pAMPK was nearly totally suppressed in neurons transduced with the T172Dα1 vector, suggesting that endogenous phosphorylated AMPKα was no more part of the complex when T172Dα1 was overexpressed (Fig. 1b, d).

为了评估 AMPK 的总体活性,我们分析了 AMPKα、 T172磷酸化 AMPKα (pAMPK)、 pACC 和 α-syn 的总西方墨点法(图1b-d)。使用泛 ampkα 抗体,我们可以检测到在所有条件下 ampkα 变异过度表达时 ampkα 总水平的明显增加(图1b,c)。随后,我们分析了 T172磷酸化水平(图1b,d)。结果表明,过量表达 AMPKα1的神经元 t172pampk 增加,提示 α1是这些神经元中最有效的磷酸化亚基。值得注意的是,pAMPK 在 T172Dα1转导的神经元中几乎完全被抑制,这表明当 T172Dα1过度表达时,内源性磷酸化 ampkα 不再是复合体的一部分(图1b,d)。

To determine how each of these variants affects AMPK activity, we analyzed the level of pACC, a product of AMPK kinase activity. The level of pACC was decreased in neurons expressing the T172Dα1 and K45Rα2 variants, which is consistent with the lower (T172Dα1) and abolished (K45Rα2) catalytic activity of these mutants (Fig. 1b and Additional file 4: Fig. S3). Therefore, both the T172Dα1 and K45Rα2 variants act as dominant negative regulators of AMPK activity, although the T172Dα1 variant is expected to maintain low constitutive activity. There was no increase in the level of pACC level in neurons chronically overexpressing the active AMPKα subunits.

为了确定这些变异体是如何影响 AMPK 活性的,我们分析了 AMPK 活性的产物 pACC 的水平。表达 T172Dα1和 K45Rα2突变体的神经元 pACC 水平降低,这与这些突变体较低的(T172Dα1)和消失的(K45Rα2)催化活性一致(图1b 和附加文件4:。S3).因此,T172Dα1和 K45Rα2变异体都是 AMPK 活性的显性负调节因子,虽然 T172Dα1变异体可能维持较低的组成型活性。慢性过度表达 ampkα 亚基的神经元 pACC 水平无明显升高。

Co-infection with AAV-α-syn led to overexpression of the human α-syn protein. Total α-syn protein levels were similar in each of the conditions co-overexpressing AMPKα subunits, indicating that AMPK activity does not have any major effects on the total level of overexpressed α-syn in vitro (Fig. 1b). Next, we assessed if the overexpression of human α-syn led to any changes in the AMPKα levels in primary neurons. Remarkably, neurons accumulating human α-syn had a significant reduction in the level of total AMPKα in all conditions in which the α subunit was overexpressed (F6,14 = 5.754) (Fig. 1b, c). The level of endogenous AMPKα was not affected by α-syn overexpression in the control and 1-310α2 conditions. We did not observe any overall change in the level of pAMPK when α-syn was overexpressed, indicating that α-syn did not have any major effect on T172 phosphorylation despite lower levels of total AMPKα.

共感染 AAV-α-syn 导致人 α-syn 蛋白过表达。在共表达 AMPKα 亚基的各条件下,α-syn 总蛋白水平相似,表明 AMPK 活性对体外过表达 α-syn 的总水平没有显著影响(图1b)。接下来,我们评估了人 α-syn 的过度表达是否导致了初级神经元 ampkα 水平的变化。显著地,在 α 亚基过表达的所有条件下,累积人 α- 氨基酸的神经元的 ampkα 总水平均明显降低(F6,14 = 5.754)(图1b,c)。在对照组和1-310α2条件下,内源性 ampkα 水平不受 α-syn 过表达的影响。我们没有观察到过量表达时 pAMPK 水平的整体变化,表明尽管 ampk 总水平较低,但 α-syn 对 T172磷酸化没有重大影响。

Overall, these results show that the accumulation of human α-syn in neuronal cells leads to a general reduction in the total level of the overexpressed AMPKα subunit, which may have implications on the activity of the complex in specific subcellular locations. However, the global level of pAMPK in basal conditions does not seem to be affected by α-syn. By over-expressing various forms of AMPKα in neuronal cells, we can modulate the formation of pAMPK and thereby the activity of the complex.

总的来说,这些结果表明,人类 α-syn 在神经元细胞中的积累导致 ampkα 亚单位过度表达的总水平普遍下降,这可能意味着复合物在特定亚细胞位置的活性。在基础条件下,pAMPK 的整体水平似乎不受 α-syn 的影响。通过在神经细胞中过量表达各种形式的 ampk,我们可以调节 pAMPK 的形成,从而调节复合物的活性。

Overexpression of the AMPKα2 subunit protects dopamine neurons from α-syn toxicity in vivo, in an AMPK complex dependent manner

AMPKα2亚基的过表达对多巴胺能神经元 α- 突触毒性的保护作用

We sought to establish if overexpression of wild-type form of AMPKα subunit could be neuroprotective against α-syn toxicity in vivo. First, AAV2/6 vectors encoding either the wild-type α2 subunit or the truncated 1-310α2 form, which does not integrate in the AMPK complex and carries constitutive catalytic activity, were tested for expression of AMPKα following injection in the ventral midbrain (1.2E6 TU). Expression of both AMPKα2 variants (HA-tag staining) was detectable in the SNpc at one month post-vector injection (Fig. 2a). Furthermore, tyrosine hydroxylase (TH) immunostaining showed that there was no evident loss of dopaminergic neurons in the SNpc following overexpression of AMPKα2 alone (Fig. 2b).

本研究旨在探讨 ampkα 野生型亚基的过表达是否具有抗 α- 合成酶毒性的神经保护作用。首先,在腹侧中脑注射 AMPKα (1.2 E6 TU)后,检测了 AAV2/6载体对 AMPKα 表达的影响。两种 AMPKα2变体(HA-tag 染色)在注射后一个月的 SNpc 中均可检测到(图2a)。此外,酪氨酸羟化酶(TH)免疫组织化学染色显示,AMPKα2单独过度表达后,SNpc 中的多巴胺能神经元没有明显丢失(图2b)。

figure2
Fig. 2 图二

To assess the potential neuroprotective effects of AMPKα2 overexpression, rats were unilaterally co-injected in the SNpc with the AAV-α-syn vector (1.5E7 TU) as previously described [35], together with either a control non-coding vector (1.2E6 TU), or with each of the two vectors overexpressing the AMPKα2 subunit at the same vector dose. Immunostaining for α-syn, 4 months after vector injection, confirmed overexpression of human α-syn in the SN in all conditions (Fig. 2c). To assess α-syn abundance, we quantified its level of expression in the midbrain by immunofluorescence. Compared to the animals co-injected with the AAV-α-syn and control vectors, we found a significant reduction in the α-syn level when either AMPKα2 or the truncated 1-310α2 form were co-expressed (F2,13 = 15.64) (Fig. 2c, d).

为了评估 AMPKα2过表达的潜在神经保护作用,将大鼠单方面注射到 SNpc 中,与先前描述的 AAV-α-syn 载体(1.5 E7 TU)共同注射,并与一个对照非编码载体(1.2 E6 TU)或两个载体在同一剂量下共同注射。免疫组织化学染色显示载体注射后4个月,在所有条件下均可见人 α-syn 在 SN 中过表达(图2c)。为了评估 α- 合成酶丰度,我们用免疫荧光定量了它在中脑的表达水平。与 AAV-α-syn 和对照载体共注射相比,AMPKα2和截短的1-310α2共表达时,α-syn 水平显著降低(F2,13 = 15.64)(图2c,d)。

The survival of the nigral neurons was analyzed four months after injection, by stereological counting of neurons positive for TH in the SNpc. Compared to the non-injected hemisphere, overexpression of human α-syn led to a 49.4 ± 3.9% loss of nigral TH-positive neurons (Fig. 2e, f). Remarkably, overexpression of the AMPKα2 subunit showed a significant neuroprotective effect on TH-positive neurons, as the α-syn-induced loss was decreased to an average value of 30.7 ± 4.9% in this group (F2,24 = 5.187). In contrast, the truncated 1-310α2 variant, which has constitutive activity but does not integrate into the AMPK complex, did not induce any significant effect on TH-positive neuron loss (Fig. 2e, f).

采用体视学方法对注射后4个月黑质神经元的存活情况进行分析。与未注射的大脑半球相比,过度表达的人 α-syn 导致黑质 th 阳性神经元损失49.4 ± 3.9% (图2e,f)。AMPKα2亚单位的过表达对 th 阳性神经元具有明显的神经保护作用,α-syn 诱导的损失率平均为30.7 ± 4.9% (F2,24 = 5.187)。相比之下,截短的1-310α2基因具有组成活性,但不能整合到 AMPK 复合体中,对 th 阳性神经元丢失没有任何显著影响(图2e,f)。

Overall, overexpressing AMPKα reduces the α-syn level in the midbrain, and AMPKα2 has neuroprotective effects on dopamine neurons accumulating human α-syn. The observed neuroprotection depends on the incorporation of the active subunit into the heterotrimeric AMPK complex, suggesting that the catalytic activity of AMPK may have to be coupled with local sensing of the energy status in order to grant protection.

总的来说,过量表达 ampkα 降低了中脑的 α- 同位素水平,AMPKα2对累积人 α- 同位素的多巴胺神经元具有神经保护作用。观察到的神经保护取决于纳入异三聚体 AMPK 复合物的活性亚单位,表明 AMPK 的催化活性可能必须与能量状态的局部感知相结合,以便提供保护。

Overexpression of AMPKα2 subunit increases mitochondrial size and mitochondrial mass in vivo

AMPKα2亚基过表达增加了体内线粒体体积和质量

To further explore the effects of AMPKα2 overexpression in vivo, we examined mitochondrial morphology in nigral dopamine neurons. Using transmission electron microscopy (TEM), we investigated the status of mitochondria after one month of AMPKα2 and α-syn co-overexpression (Fig. 3a, b), when nigral neurodegeneration is still mild. Neurons in the SNpc injected with the α-syn-encoding and non-coding vectors were compared with the non-injected contralateral side and with the SNpc in rats co-injected with the α-syn- and AMPKα2-encoding vectors. We analyzed the relative number of mitochondria per μm2 of cytosol (mitochondrial density) for each group. For this parameter, there was no significant difference across conditions (Fig. 3c). However, we noticed that only in the SNpc injected with the vector encoding human α-syn, some neurons (27% of the neurons analyzed) displayed abnormal mitochondrial morphology, mainly characterized by concentric circles of cristae membranes (Fig. 3b). Compared to the SNpc injected with AAV-α-syn, mitochondrial morphology was improved following co-injection of the AMPKα2-expressing vector, with a decrease in the overall proportion of neurons that displayed abnormal cristae (9% of the neurons analyzed). Furthermore, we observed a significant increase in the size of mitochondria (Fig. 3d) (F2,5437 = 22.5), as well as an increase of the mitochondrial area fraction per neuron (Fig. 3e) (F2,69 = 4.23). The effects related to mitochondrial size and area fraction were even more pronounced when compared to neurons in the non-injected SNpc.

为了进一步探讨 AMPKα2在体内过度表达的影响,我们检测了黑质多巴胺神经元的线粒体形态。利用透射电镜观察了 AMPKα2和 α-syn 过表达一个月后,当黑质神经退行性疾病仍然较轻时线粒体的状态。将 α-syn 编码和非编码载体注射的 SNpc 神经元与对侧未注射的 SNpc 神经元以及与 α-syn 和 ampkα2编码载体共注射的 SNpc 神经元进行比较。分析各组细胞质(线粒体密度)线粒体相对数量。对于这个参数,不同条件之间没有显著差异(图3c)。然而,我们注意到只有注射编码人 α- 同工酶载体的 SNpc,一些神经元(分析的神经元的27%)呈现异常的线粒体形态,主要是拥有属性膜的同心圆(图3 b)。与注射 AAV-α-syn 的 SNpc 相比,共注射 ampkα2表达载体后,线粒体形态得到改善,表现嵴异常的神经元总比例下降(9%)。此外,我们观察到线粒体大小显著增加(图3d)(F2,5437 = 22.5) ,以及增加线粒体每神经元面积分数(图3e)(F2,69 = 4.23)。与未注射 SNpc 的神经元相比,线粒体大小和面积分数的影响更加明显。

figure3
Fig. 3 图3

All in all, in vivo overexpression of α-syn has clear effects on mitochondrial morphology and causes a significant increase in average mitochondrial size. Co-injection with the AMPKα2-encoding vector improves mitochondrial morphology and increases mitochondrial mass in nigral neurons.

总之,在体内过度表达 α-syn 对线粒体形态有明显的影响,使线粒体平均大小显著增大。与 ampkα2编码载体共注射改善黑质神经元线粒体形态,增加线粒体质量。

Overexpression of the T172Dα1 AMPK variant provides the most effective neuroprotection against the toxic effects of α-syn accumulation

过量表达 T172Dα1 AMPK 变异体可提供最有效的神经保护,抵抗 α- 同位素积累的毒性作用

The possibility to provide neuroprotective effects against α-syn by increasing the capability of nigral dopamine neurons to sense energy status via AMPKα overexpression was further tested by comparing the α1 and α2 subunits. We also included the T172D variant of the α1 subunit, which is expected to provide a low but constitutive AMPK activity. The paradigm was similar to the previous experiment, based on the unilateral injection of AAV-AMPKα vectors at the same time as the pathogenic AAV-α-syn vector in the rat SNpc.

通过对 α1亚基和 α2亚基的比较,进一步验证了通过 ampkα 过表达提高黑质多巴胺神经元感受能量状态能力的可能性,从而提供对 α-syn 的神经保护作用。我们还包括了 α1亚基的 T172D 变异体,它有望提供一个低但组成型 AMPK 活性。这个实验与之前的实验相似,基于大鼠单侧注射 aav-ampkα 载体同时注射致病的 AAV-α-syn 载体。

The extent of α-syn-induced toxicity was evaluated at four months after vector injection. The abundance of α-syn in the midbrain was assessed by fluorescence immunohistochemistry (Fig. 4a), and quantification of the α-syn level near the site of vector injection again revealed a significant decrease in the groups co-injected with AAV encoding active forms of AMPKα, including the T172Dα1 variant (F3,16 = 59.6) (Fig. 4b). The total number of neurons with dopamine morphology present in the SNpc was estimated using TH and Nissl co-staining (Fig. 4d). Remarkably, the protective effect on the number of Nissl-positive neurons was significant in all AMPKα overexpressing conditions (F3,31 = 9.955), and no major difference was observed between the α1 and α2 subunits. The effect was most evident for the T172Dα1 variant, with an average loss of Nissl-positive neurons of only 14.2 ± 2.9%, compared to 31.9 ± 2.5% for the control group co-injected with AAV-α-syn (Fig. 4d). Next, we quantified the loss of TH-positive neurons in the SNpc (Fig. 4e). There was a trend towards a protective effect with the α1 and α2 subunits. However, only overexpression of the T172Dα1 variant induced a statistically significant protection. The loss of TH-positive nigral neurons was decreased to 38.1 ± 3.2%, with respect to 49.1 ± 3.5% for the control non-coding vector (Fig. 4e). The reduced neuroprotective effects observed when measuring the number of TH-positive neurons, as compared to the total number of Nissl-positive neurons in the SNpc, indicates a likely down-regulation of TH expression in neurons overexpressing AMPKα. This was further confirmed by measuring TH immunoreactivity in the STR (Fig. 4f, g). Indeed, no protection was observed at the level of TH-positive fiber loss between the groups (Fig. 4g). Down-regulation of the dopamine markers was further confirmed using immunohistochemistry for the dopamine transporter (DAT) in the STR (Additional file 5: Fig. S4a). Optical density of the DAT signal was decreased to a similar extent as compared to TH (Additional file 5: Fig. S4b).

在载体注射后4个月评价 α- 同位素的毒性程度。中脑 α-syn 丰度采用荧光免疫组织化学(图4a)测定,在载体注射部位附近的 α-syn 水平再次定量显示,与腺相关病毒(AAV)共注射 ampkα 编码活性形式的组明显减少,包括 T172Dα1变异体(F3,16 = 59.6)(图4b)。用 TH 和 Nissl 联合染色法测定多巴胺形态的神经元总数(图4d)。值得注意的是,在所有 ampkα 过表达条件下(F3,31 = 9.955) ,对 nissl 阳性神经元的数量均有明显的保护作用,α1亚基与 α2亚基之间无明显差异。对 T172Dα1基因变异的影响最为明显,平均 nissl 阳性神经元损失率仅为14.2 ± 2.9% ,对照组为31.9 ± 2.5% (图4d)。接下来,我们量化了 SNpc 中 th 阳性神经元的损失(图4e)。α1和 α2亚基有保护作用的趋势。然而,只有 T172Dα1基因的过表达才能产生显著的保护作用。Th 阳性黑质神经元的丢失率为38.1 ± 3.2% ,对照组为49.1 ± 3.5% (图4e)。与尼氏阳性神经元的总数相比,在测量 TH 阳性神经元数量时观察到的神经保护作用减弱,表明过表达 ampkα 的神经元中 TH 的表达可能下调。通过在 STR 中测量 TH 免疫反应性进一步证实了这一点(图4f,g)。事实上,没有观察到保护水平之间的 th- 阳性纤维损失组(图4g)。多巴胺标志物的下调通过使用免疫组织化学的多巴胺转运蛋白(DAT)在 STR 中进一步得到证实。S4a).与 TH (附加文件5: 图5)相比,DAT 信号的光密度降低到了相似的程度。4b 条)。

figure4
Fig. 4 图4

The development of asymmetric spontaneous motor behavior was assessed using the cylinder test (Fig. 4h). Animals from the control group injected with the AAV-α-syn vector showed a progressive increase in preferential right forepaw use, reaching 17.2 ± 5.3% at week 4 and 19.6 ± 4.9% at week 16. The gradual motor impairment due to α-syn overexpression was statistically significant (time effect: F2,40 = 22.440). In contrast, the rats in the T172Dα1 group did not show any significant progression of motor asymmetry over time, reaching only 6.0 ± 2.4% of preference for the non-affected side at week 16 (Fig. 4h). Although the difference between these two groups did not reach significance, the lack of progression of motor asymmetry in the T172Dα1 animals indicates a preserved motor function following unilateral injection of the α-syn-encoding vector.

非对称自发运动行为的发展用圆柱体试验评估(图4h)。对照组动物注射 AAV-α-syn 载体后,右前爪优先使用率逐渐增加,第4周为17.2 ± 5.3% ,第16周为19.6 ± 4.9% 。α-syn 过表达引起的运动功能逐渐减退具有统计学意义(时间效应: F2,40 = 22.440)。与此相反,T172Dα1组大鼠随着时间的推移没有显示任何明显的运动不对称性进展,在第16周只达到非受影响侧偏好的6.0 ± 2.4% (图4h)。虽然两组之间的差异没有达到显著性,但 T172Dα1动物的运动不对称性缺乏进展表明单侧注射 α- 同位素编码载体后运动功能保持不变。

Summarizing, overexpression of AMPKα subunits provides a significant neuroprotection of dopamine neurons in SNpc against α-syn-driven toxicity. This effect is not dependent on the isoform of the catalytic subunit being overexpressed, since both AMPKα1 and α2 showed a similar level of protection. Nevertheless, the most prominent neuroprotective effect was obtained via overexpression of AMPK T172Dα1 subunit. Surprisingly however, it did not coincide with a protection of TH-positive neurons or striatal fibers, which hints to a possible down-regulation of dopamine marker expression in the surviving neurons. Nevertheless, overexpression of the T172Dα1 subunit showed an effective rescue of motor behavior, which suggests a functional preservation of the dopamine nigrostriatal function.

Ampkα 亚基的过表达为 SNpc 多巴胺神经元提供了抗 α- 突变毒性的神经保护作用。这种效应并不依赖于过度表达的催化亚基的亚型,因为 AMPKα1和 α2表现出相似的保护水平。AMPK T172Dα1亚单位的过表达可以获得最显著的神经保护作用。然而令人惊讶的是,它并没有与 th 阳性神经元或纹状体纤维的保护相吻合,这暗示着存活的神经元中多巴胺标记物的表达可能下调。然而,T172Dα1亚单位的过度表达对运动行为有明显的拯救作用,提示多巴胺可以保护黑质纹状体的功能。

T172Dα1 AMPK variant decreases the presence of dystrophic axons in the STR

T172Dα1腺苷酸活化蛋白激酶(AMPK)基因突变可减少 STR 中营养不良性轴突的存在

Next, we explored how different forms of AMPKα affect the formation of dystrophic dopaminergic fibers projecting to the STR, characterized by the pathologic accumulation of α-syn. We performed an immunofluorescent staining using the 5G4 anti-α-syn monoclonal antibody, which specifically recognizes pathological species of aggregated α-syn [36]. Within the STR, the signal for 5G4 was observed in axonal fibers, which often appeared dystrophic (Fig. 5a). These dystrophic fibers were also evident in TH immunostaining (Fig. 5b).

接下来,我们探讨了不同形式的腺苷酸活化蛋白 α 如何影响肌营养不良的多巴胺能纤维投射到 STR 的形成,以及 α-syn 的病理积累拥有属性。我们使用5G4抗 -α-syn 单克隆抗体进行了免疫荧光染色,这种染色能够特异识别聚集的 α-syn 病理种。在 STR 中,轴突纤维中可见5G4信号,常表现为营养不良(图5a)。这些肌营养不良纤维在 TH 免疫染色中也很明显(图5b)。

figure5
Fig. 5 图5

Phosphorylation of α-syn is considered as a marker for the pathological deposition of this protein, as the majority of Lewy bodies contain α-syn phosphorylated on residue S129 (pS129-α-syn) [37]. Immunostaining of STR sections revealed the presence of dystrophic fibers positive for pS129-α-syn, with a morphology closely resembling that of 5G4-positive dystrophic fibers. Despite being detected in all conditions, they appeared however to be less abundant than their 5G4 counterparts (Fig. 5c).

由于大多数路易体在 S129(pS129-α-syn)[37]的残基上都含有 α-syn 的磷酸化,因此 α-syn 的磷酸化被认为是该蛋白病理性沉积的标志。免疫组织化学染色显示 pS129-α-syn 阳性肌营养不良纤维,形态与5g4阳性肌营养不良纤维极为相似。尽管在所有条件下都能检测到它们,但是它们的含量似乎比5G4的同类要少(图5c)。

The abundance of dystrophic fibers was particularly high in the AMPKα1 and α2 injected rats, and appeared to be reduced in the injected hemisphere of the T172Dα1 rats (Fig. 5d). To quantify the effects of AMPK on dystrophic axonal fibers, we performed an optical densitometry (OD) analysis of the 5G4 signal in the dorsal part of the STR. Remarkably, the mean 5G4 fluorescence intensity was significantly decreased in rats overexpressing the T172Dα1 variant, compared to all three other groups (F3,32 = 6.4) (Fig. 5e). By contrast, in the AMPKα1 condition, the mean intensity appeared slightly higher than in the control group, although the difference was not significant. Overall, these results indicate that AMPK T172Dα1 overexpression, which leads to constitutive low AMPK activity, significantly reduces the density of dystrophic dopamine fibers containing aggregated α-syn, as compared to all other conditions.

肌营养不良纤维的丰度在注射 AMPKα1和 α2的大鼠中特别高,在注射 T172Dα1大鼠的大脑半球中似乎减少(图5d)。为了定量研究 AMPK 对肌营养不良轴突纤维的影响,我们对5G4信号进行了光学密度测定(OD)分析。高表达 T172Dα1基因的大鼠平均5G4荧光强度显著低于其他三组(F3,32 = 6.4)(图5e)。与此相反,AMPKα1组的平均强度略高于对照组,但差异不显著。结果表明,与其他条件相比,AMPK T172Dα1过表达导致组成型 AMPK 活性降低,显著降低了含聚集 α-syn 的营养不良多巴胺纤维的密度。

Overall, AMPK T172Dα1 overexpression leads to a significant reduction in the abundance of dystrophic fibers that contain aggregated (5G4-positive) α-syn, as compared to all other conditions. These results indicate that the constitutive low AMPK activity conferred by overexpressing the T172Dα1 variant has protective effect on the development of α-syn pathological features in the STR. However, other mechanisms are likely to account for the protective effects of AMPKα1 and α2.

与其他条件相比,AMPK T172Dα1的过度表达导致含有聚集的(5g4阳性) α-syn 的营养不良纤维数量显著减少。这些结果表明,过表达 T172Dα1基因的组成型低 AMPK 活性对短串联重复序列中 α-syn 病理特征的发展具有保护作用。AMPKα1和 α2的保护作用可能与其他机制有关。

In cortical neurons, AMPKα overexpression limits the accumulation of autolysosomes caused by human α-syn

在大脑皮质神经元中,ampkα 的过表达限制了人 α- 合成氨基酸引起的自溶体的积累

To further explore the effects of AMPKα on autophagic and mitochondrial activity, we next conducted in vitro experiments using mouse primary cortical neurons induced to overexpress human α-syn. As it is well established that AMPK controls autophagic activity [3839], we explored the effect of the different forms of AMPKα on autophagic and lysosomal markers at steady state, either in the absence or presence of human α-syn.

为了进一步探讨 ampkα 对自噬和线粒体活性的影响,我们接下来进行了体外实验,利用小鼠皮层原代神经元诱导过表达人 α-syn。由于 AMPK 控制自噬活性[38,39] ,我们探讨了不同形式的 AMPKα 在稳定状态下对自噬和溶酶体标记物的影响。

As a first indicator, we assessed by western blotting the level of the microtubule-associated protein 1B light chain 3 (LC3-I), and LC3-II, its lipidated form associated to autophagosome formation (Fig. 6a). Overexpression of human α-syn did not have any significant effect on the levels of LC3-I and LC3-II in the control condition (Fig. 6b, c). However, we noticed that α-syn accumulation modified the expression of these autophagic markers in neurons overexpressing AMPKα. In neurons that do not overexpress α-syn, LC3-I expression remained very similar across conditions, regardless of the overexpression of the AMPKα subunits (Fig. 6b). In contrast, the level of LC3-II was significantly reduced in neurons overexpressing either AMPKα1 or α2 (F3,12 = 3.98) (Fig. 6c). In contrast, when human α-syn was present, we observed a significant reduction in the LC3-I level in neurons overexpressing AMPKα, which was most pronounced with the constitutively active T172Dα1 variant (F3,12 = 4.0) (Fig. 6c). The level of LC3-II however remained similar across conditions. These results indicated an adaptive response to α-syn in neurons overexpressing AMPKα.

作为第一个指标,我们通过西方墨点法评估微管组织蛋白1 b 轻链3(LC3-I)和 LC3-II 的水平,它的脂肪化形式与自噬体的形成有关(图6 a)。在对照条件下,人 α-syn 的过表达对 LC3-I 和 LC3-II 水平没有显著影响(图6b,c)。然而,我们注意到 α-syn 的积累改变了 ampkα 过表达的神经元自噬标记的表达。在不过度表达 α-syn 的神经元中,不管 ampkα 亚基的过度表达如何,LC3-I 的表达在各种条件下都保持非常相似(图6b)。与此相反,过表达 AMPKα1或 α2的神经元 LC3-II 水平明显降低(F3,12 = 3.98)(图6c)。与此相反,我们观察到,当人类的 α-syn 存在时,过表达 ampkα 的神经元 LC3-I 水平显著降低,其中最明显的是组成性活跃的 T172Dα1变异(F3,12 = 4.0)(图6c)。然而,LC3-II 的水平在各种条件下保持相似。这些结果提示过表达 ampkα 的神经元对 α-syn 有适应性反应。

figure6
Fig. 6 图6

Accumulation of α-syn can affect autophagic activity and is associated with perturbations of the lysosomal function [40,41,42,43]. To determine the effects of α-syn on the distribution of LC3-II between autophagosome and lysosome vesicles, primary cortical neurons were co-transduced with a reporter construct encoding LC3B fused with mCherry and EGFP (LC3B-mCherry-EGFP described in [29]). Yellow puncta positive for both mCherry and EGFP indicate autophagosomes, which have not yet fused with lysosomes, whereas red puncta are characteristic for autolysosomes (Fig. 6d). Consistent with the effect observed for LC3-II (Fig. 6c), the total number of puncta was found to be lower in the conditions in which the AMPKα1 and α2 subunits were overexpressed, as compared to the control condition. This effect was statistically significant for the α2 subunit (α-syn x AMPK effect: F3,208 = 2.4) (Fig. 6e).

α-syn 的积累可影响自噬活性,并与溶酶体功能紊乱有关[40,41,42,43]。为了研究 α- 同位素对自噬体和溶酶体囊泡间 LC3-II 分布的影响,将原代皮层神经元与报告基因编码的 LC3B 融合于 mCherry 和 EGFP (LC3B-mCherry-EGFP,见文献[29])。mCherry 和 EGFP 阳性的黄色斑点表明自噬体尚未与溶酶体融合,而红色斑点则是自溶体的特征(图6d)。与 LC3-II 的影响相一致(图6c) ,AMPKα1和 α2亚基过表达的条件下,斑点总数低于对照条件。α2亚基(α-syn x AMPK 效应: F3,208 = 2.4)具有统计学意义(图6e)。

Remarkably, human α-syn overexpression was found to increase the number of red puncta, which indicates an abnormal accumulation of autolysosomes (α-syn effect: F1,208 = 22.08) (Fig. 6e). Although the effect of α-syn on the number of autolysosomes was observed in all conditions, the number of red puncta remained lower in neurons overexpressing AMPKα1 or α2 (Fig. 6e). In neurons overexpressing AMPKα2, the number of autolysosomes was still reduced by 48% when compared to the control α-syn condition.

显著地,人 α-syn 过表达增加了红色斑点的数量,表明自溶素的异常积累(α-syn 效应: F1,208 = 22.08)(图6e)。虽然在所有条件下都可观察到 α- 同位素对自溶体数目的影响,但过表达 AMPKα1或 α2的神经元中的红点数目仍较低(图6e)。在过表达 AMPKα2的神经元中,自溶体的数量比对照的 α- 同步酶条件下仍减少48% 。

Importantly, there was no evidence that the effect of AMPKα was associated to any downregulation of autophagic activity in neurons overexpressing α-syn. Indeed, both the total number of autophagic vesicles (Fig. 6e), as well as the number of autophagosomes positive for both mCherry and EGFP (Additional file 6: Fig. S5), remained similar in neurons co-expressing α-syn and AMPKα as compared to the control α-syn condition.

重要的是,没有证据表明 ampkα 的作用与过度表达 α-syn 的神经元自噬活性的下调有关。事实上,mCherry 和 EGFP 的自噬囊泡总数(图6e)和自噬体阳性数(附加文件6: 图6)。共表达 α-syn 和 ampkα 的神经元与对照的 α-syn 条件相似。

These results indicate that human α-syn causes an accumulation of autolysosomal markers, which may reflect defective lysosomal enzymatic activity, as previously reported [4144]. However, in neurons overexpressing either AMPKα2 or AMPKα1, albeit to a lesser extent, the number of autophagic vesicles was increased when human α-syn was overexpressed (Fig. 6e). This effect coincides with a significant reduction in the number of autolysosomes, as compared to neurons overexpressing α-syn only. In contrast, α-syn did not induce any change in the total number of autophagic vesicles in neurons overexpressing the constitutively active T172Dα1 variant (Fig. 6e). Overall, AMPKα contributes to controlling the accumulation of autolysosomes in neurons overexpressing α-syn.

这些结果表明,人类 α- 同位素引起自溶酶体标记物的积累,这可能反映了有缺陷的溶酶体酶活性,正如先前报道的[41,44]。然而,在过表达 AMPKα2或 AMPKα1的神经元中,当人 α-syn 过表达时,自噬泡的数量增加,虽然程度较轻(图6e)。与过度表达 α-syn 的神经元相比,这种效应与自溶酶体数量的显著减少相吻合。与此相反,过量表达组成性活性 T172Dα1变异体的神经元,其自噬小泡的总数并未发生任何变化(图6e)。Ampkα 对过表达 α-syn 的神经元内自溶体的积累具有调控作用。

AMPKα increases mitochondrial mass in neurons overexpressing human α-syn

Ampkα 增加过表达人 α-syn 神经元的线粒体质量

AMPKα2 overexpression has significant effects on the mitochondrial network in vivo (see Fig. 3). To further characterize the combined effects of AMPKα and α-syn on mitochondria, we determined the mitochondrial mass and mitochondrial DNA (mtDNA) quantity in cortical neurons overexpressing each of the AMPKα variants in vitro. In parallel, the neurons were challenged with α-syn overexpression, which has been reported to affect the turnover as well as the dynamics of mitochondria [4546].

AMPKα2过表达对体内线粒体网络有显著影响(见图3)。为了进一步研究 ampkα 和 α-syn 对线粒体的联合作用,我们测定了大脑皮层神经元中线粒体质量和线粒体线粒体脱氧核糖核酸(mtDNA)数量。同时,这些神经元受到 α-syn 过表达的挑战,据报道这种过表达会影响线粒体的更替和动态。

First, we determined the mitochondrial mass by overexpressing the mitoDsRed probe in neuronal cultures and measuring fluorescence intensity using flow cytometry (Fig. 7a). In control neurons, we found that α-syn overexpression did not induce any change in the total mitochondrial load. However, neurons overexpressing AMPKα1 and α2 responded to α-syn by increasing the average mitoDsRed fluorescence per cell (F1,86 = 4.87) (Fig. 7a). The increase in mitoDsRed fluorescence reached statistical significance in neurons overexpressing the AMPKα2 variant (Fig. 7a), which is in line with the aforementioned increase in mitochondrial mass observed in vivo (see Fig. 3f). Remarkably, this effect was not observed in neurons expressing the constitutively active T172Dα1 variant (Fig. 7a), which further supports the role of fully active and responsive AMPKα subunits in sensing α-syn-induced changes.

首先,我们通过在神经细胞培养中过度表达 mitoDsRed 探针并用流式细胞术测量荧光强度来确定线粒体质量(图7a)。在对照神经元中,我们发现 α-syn 过表达并不引起线粒体总负荷的改变。而过表达 AMPKα1和 α2的神经元对 α-syn 的反应是增加平均 mitoDsRed 荧光(F1,86 = 4.87)(图7a)。在过度表达 AMPKα2变异的神经元中,mitoDsRed 荧光的增加达到了统计学意义上的显著性(图7a) ,这与上述在体内观察到的线粒体质量的增加相一致(图3f)。值得注意的是,这种效应在表达组成性活跃 T172Dα1变异的神经元中没有观察到(图7a) ,这进一步支持了 ampkα 亚单位在感知 α- 同位素诱导的变化中的作用。

figure7
Fig. 7 图7

Next, we measured the relative quantity of mtDNA per cell by rt-PCR, seven days after vector transduction (Fig. 7b). Overexpression of α-syn induced a significant decrease in the cellular quantity of mtDNA (F1,23 = 25.367). Remarkably however, the α-syn-induced difference was attenuated in neurons overexpressing AMPK T172Dα1, mainly because the amount of mtDNA was also decreased in the absence of α-syn overexpression (F3,23 = 3.8) (Fig. 7b).

然后,在载体转导后7天,用 rt-PCR 方法测定每个细胞线粒体 dna 的相对数量。过量表达 α-syn 使线粒体 dna 的细胞数量显著减少(F1,23 = 25.367)。而在过表达 AMPK T172Dα1的神经元中,α-syn 诱导的差异明显减弱,这主要是因为在没有 α-syn 过表达的情况下,线粒体 dna 的数量也减少了(F3,23 = 3.8)(图7b)。

To determine the overall effects on metabolic activity, we sought to investigate mitochondrial respiration in primary cortical neurons co-overexpressing AMPKα and α-syn. We measured both respiration in basal conditions and following exposure to CCCP, a mitochondrial uncoupling agent used to assess maximal respiration (Additional file 7: Fig. S6). Overexpression of the AMPKα variants per se did not have any major effects on basal neuronal respiration, except for a slight reduction in oxygen consumption rate (OCR) in neurons overexpressing AMPKα1. Even though overexpression of human α-syn had only mild effects on OCR, we did notice an overall statistically significant increase of basal OCR and reduced reserve respiratory capacity. The effect on the reserve capacity was particularly evident in neurons overexpressing AMPKα. However, in neurons expressing the constitutively active T172Dα1 variant, the reserve capacity was similarly decreased both in the presence and absence of α-syn. AMPKα does not induce any major increase in mitochondrial activity and reduces the reserve respiratory capacity in neurons expressing human α-syn, an effect consistent with the measured mtDNA content. Overall, these results show that in primary neurons overexpressing human α-syn, AMPKα has an effect mainly on the mitochondrial mass, without increasing the mtDNA content and the respiratory capacity.

为了确定对代谢活动的整体影响,我们研究了共表达 ampkα 和 α-syn 的原代皮层神经元的线粒体呼吸。我们测量了基础条件下的呼吸和接触 ccp 后的呼吸,ccp 是一种用于评估最大呼吸的线粒体解偶联剂(附加文件7: 图。中六)。Ampkα 变异体本身的过度表达对基底神经元的呼吸没有明显影响,除了 AMPKα1过度表达的神经元的耗氧率(OCR)有轻微下降。尽管人 α-syn 的过度表达对 OCR 只有轻微的影响,但我们确实注意到基础 OCR 总体上有统计学意义上的显著增加和储备呼吸能力的降低。过表达 ampkα 的神经元对储备能力的影响尤为明显。而在表达组成性活跃 T172Dα1变体的神经元中,无论是否存在 α-syn,其储备能力也同样下降。Ampkα 不引起线粒体活性的显著增加,并降低表达人 α-syn 的神经元的储备呼吸能力,这与线粒体 dna 含量的测定结果一致。结果表明,在过度表达人 α-syn 的原代神经元中,ampkα 主要影响线粒体质量,而不增加线粒体 dna 含量和呼吸容量。

Discussion

讨论

Overexpression of AMPKα1 or α2 subunits, which integrate into the AMPK complex, can protect dopamine neurons against the toxic effects of human α-syn accumulation in vivo. In particular, the T172Dα1 variant, which is characterized by low chronic catalytic activity, provides the most effective neuroprotection of dopamine neurons following unilateral injection of the α-syn-expressing AAV vector. Furthermore, T172Dα1 significantly decreases the number of dystrophic axons containing aggregated forms of α-syn in the STR.

AMPK 复合物中过表达 AMPKα1或 α2亚基可以保护多巴胺能神经元免受体内 α- 同位素积累的毒性作用。特别是,T172Dα1变异体,这是一个拥有属性低的慢性催化活性,提供了最有效的神经保护多巴胺神经元单方面注射 α- 同位素表达的 AAV 载体。此外,T172Dα1能显著减少短串联重复序列中含有聚集形式 α-syn 的营养不良性轴突的数量。

We used the same genetic approach to control AMPK activity in primary neurons and assess effects on mitochondria and autophagy markers in vitro. Overexpressing AMPKα subunits increases the mitochondrial mass and limits the accumulation of lysosomal material in neurons co-expressing human α-syn. By contrast, α-syn has only limited effects on parameters related to lysosomal and mitochondrial activity in neurons expressing the constitutively active T172Dα1 variant.

我们使用相同的遗传学方法来控制原代神经元的 AMPK 活性,并在体外评估线粒体和自噬标记的影响。Ampkα 亚基的过表达增加了线粒体质量,限制了共表达人 α-syn 的神经元中溶酶体物质的积累。相比之下,α-syn 对表达组成活性 T172Dα1变异的神经元中溶酶体和线粒体活性相关参数的影响有限。

Alpha-synuclein and AMPK activity in neuronal cells

神经元细胞的 α- 突触核蛋白和 AMPK 活性

As α-syn can perturb mitochondria [4547], it may lead to metabolic stress in neurons and affect AMPK signaling. To address this question, α-syn was co-expressed with different forms of human AMPKα. It has been previously reported that α-syn reduces AMPK phosphorylation in neuronal cells [26]. Although we did not observe any significant change in pAMPK level, we noticed a significant reduction of total AMPKα in mouse cortical neurons overexpressing α-syn. This effect was evident on overexpressed AMPKα, and not on endogenous AMPKα, which may indicate that the overabundance of the subunit facilitates α-syn-induced reduction of its expression level. It is therefore possible that α-syn mainly affects the fraction of the protein which is not associated to the AMPK complex, and may therefore not cause any change in pAMPK level. Although α-syn accumulation may affect AMPK levels, the absence of any increase in AMPK or ACC phosphorylation in neurons overexpressing α-syn, confirms that α-syn is a rather mild metabolic stressor, at least in vitro. Nevertheless, the effects of α-syn on neuronal metabolism may be more evident at later stages of the pathology [47].

α-syn 能扰乱线粒体[45,47] ,可能导致神经元代谢应激,影响 AMPK 信号转导。为了解决这个问题,人类不同形式的 ampkα 共同表达了 α-syn。先前有报道说 α- 同位素减少神经元细胞中 AMPK 的磷酸化[26]。虽然我们没有观察到 pAMPK 水平的任何显著变化,但我们注意到在过度表达 α-syn 的小鼠皮层神经元中,ampk 总量显著减少。这种作用在过量表达的 ampkα 上表现得尤为明显,而在内源性 ampkα 上表现得不明显,这可能表明过量的 ampkα 促进了 α-syn 诱导的 ampkα 表达水平的降低。因此,可能 α-同位素主要影响与 AMPK 复合物无关的蛋白质部分,因此可能不会引起 pAMPK 水平的任何变化。虽然 α-syn 的积累可能影响 AMPK 的水平,但是在过度表达 α-syn 的神经元中,AMPK 和 ACC 的磷酸化水平没有增加,这证实了 α-syn 是一个相当温和的代谢应激源,至少在体外是这样。尽管如此,α-syn 对神经元代谢的影响可能在病理的后期更为明显[47]。

Although we have not observed any effect of AMPKα on the level of α-syn in primary neuronal cultures, the level of α-syn is significantly reduced in the ventral midbrain when active forms of AMPKα are co-expressed with α-syn. Although one cannot rule out that AMPKα affects α-syn translation [48], it appears more likely that the down-regulation of α-syn expression is caused by increased degradation of the protein. AMPK has been shown to enhance protein degradation in various tissues, via mechanisms that involve either the ubiquitin proteasome pathway or autophagy [49,50,51]. Although both systems are implicated in α-syn turnover, autophagic activity is mainly recruited when the α-syn burden is increased [52]. The effects of AMPK on α-syn turnover will need to be further explored.

虽然我们还没有观察到 ampkα 对原代培养神经元 α-syn 水平的影响,但是当 ampkα 的活性形式与 α-syn 共表达时,中脑腹侧区的 α-syn 水平显著降低。虽然我们不能排除 ampkα 影响 α-syn 翻译[48]的可能性,但是看起来 α-syn 表达的下调更可能是由于蛋白质降解的增加所致。AMPK 已经被证明可以通过泛素蛋白酶体途径或自噬途径增强蛋白质在各种组织中的降解。虽然这两个系统都涉及到 α- 同位素周转,但自噬活性主要是在 α- 同位素负荷增加时产生的[52]。AMPK 对 α- 同位素周转率的影响有待进一步研究。

Overexpression of AMPKα is neuroprotective against α-syn

腺苷酸活化蛋白激酶 α 的过表达对 α- 合成酶的神经保护作用

In normal circumstances, AMPK activity is adjusted as a function of time and location at which metabolic stress occurs [53]. In order to identify the optimal pattern of AMPK activity to counteract α-syn toxicity, we chose to overexpress four variants of the α subunit in a model of PD based on the chronic overexpression of human α-syn [35]. We show that overexpression of either the AMPKα1 or α2 subunit is able to equally protect nigral dopamine neurons against α-syn toxicity. This indicates that, at least regarding neuroprotection, there is no major difference between these isoforms, when overexpressed. Moreover, the neuroprotective effect of AMPKα is dependent on integration into the AMPK complex, as overexpression of the truncated 1-310α2 form of AMPK does not provide any protection, despite its constitutive catalytic activity.

在正常情况下,AMPK 活性会随着代谢应激发生的时间和地点的变化而调整。为了确定 AMPK 活性的最佳模式以抵消 α- 突变毒性,我们选择在人 α- 突变体[35]慢性过表达的 PD 模型中过表达4个 α 亚基变体。我们发现 AMPKα1或 α2亚单位的过表达均能同样保护黑质多巴胺神经元免受 α-syn 毒性。这表明,至少在神经保护方面,当过度表达时,这些亚型之间没有重大区别。此外,AMPK 的神经保护作用依赖于 AMPK 复合物的整合,因为截短型1-310α2 AMPK 的过度表达不能提供任何保护,尽管它具有组成性的催化活性。

Here, we show that in vivo overexpression of α-syn has dramatic effects on mitochondrial morphology (Fig. 3). It has been previously shown that neurons deprived of PGC-1α, exhibit significant alterations in mitochondrial cristae morphology and reduced respiratory chain complex activity following in vitro or in vivo overexpression of α-syn [5455]. Alpha-syn can bind to the promoter sequence of PGC-1α and cause promoter methylation, a phenomenon associated with sporadic PD cases and which can lead to decreased PGC-1α expression [5556]. Remarkably, co-expression of α-syn with the AMPKα2 subunit increases both mitochondrial size and mass, with a partial rescue of the morphological alterations observed in mitochondria. Whether these effects are due to activation of PGC-1α and increased mitochondrial biogenesis remains to be further explored.

在这里,我们表明在体内过度表达的 α- 合成酶对线粒体形态有巨大的影响(图3)。先前的研究表明,在体内或体外过度表达 α-syn [54,55]后,去除 pgc-1α 的神经元在线粒体嵴形态学上表现出明显的改变,并降低呼吸链复合物的活性。α-syn 可与 pgc-1α 启动子序列结合,引起启动子甲基化,这种现象与散发性 PD 病例有关,可导致 pgc-1α 表达下降[55,56]。结果表明,α-syn 与 AMPKα2亚单位共表达后,线粒体大小和质量均增加,线粒体形态学改变得到部分修复。这些影响是否是由于 pgc-1α 的激活和线粒体生物发生的增加有待进一步探讨。

The neuroprotective effect of AMPKα is particularly evident when assessing the number of nigral neuronal cell bodies using Nissl staining. However, the AMPKα-induced protection of TH-positive neuronal cell bodies is less consistent, reaching statistical significance with AMPKα2 only in the first experiment (Fig. 2f), whereas being less effective in the second study (Fig. 4e). In addition, AMPKα expression has failed to protect TH- and DAT-positive axonal fibers in the STR. These results indicate that AMPKα-induced neuroprotection is most likely concomitant with an eventual decrease in the expression of dopaminergic markers. This effect is reminiscent of the phenotype of dopamine marker loss observed after chronic overexpression of PGC-1α with a postulated mechanism involving Pitx3 downregulation [3057].

用尼氏染色法检测黑质神经元胞体数目时,ampkα 的神经保护作用尤为明显。然而,ampkα 对 th- 阳性神经元细胞体的保护作用不太一致,仅在第一个实验中与 AMPKα2有统计学意义(图2f) ,而在第二个实验中效果较差(图4e)。此外,ampkα 的表达对 TH- 和 dat- 阳性轴突纤维的保护作用不明显。这些结果表明,ampkα 诱导的神经保护最有可能伴随着多巴胺能标记物表达的最终减少。这种效应使人想起在慢性 pgc-1α 过度表达后观察到的多巴胺标记物丢失的表型,并假定了 Pitx3下调的机制[30,57]。

Most effective neuroprotection against α-syn is achieved with constitutive low AMPK activity

最有效的神经保护是通过组成型低 AMPK 活性来实现的

Remarkably, the T172Dα1 variant provides the most substantial neuroprotective effect against α-syn toxicity, mitigating the loss of Nissl-positive neurons in the SNpc by more than 50%. This form of AMPKα, which integrates into the AMPK complex, has a constitutive catalytic activity, which is independent from T172 phosphorylation. However, T172Dα1 has also a dominant negative effect on AMPK, as the activity of this variant is clearly lower than wild-type forms of AMPKα (Fig. 1b) [3233]. The chronic mode of low activity induced by T172Dα1 overexpression, which is likely to prevent fluctuations in AMPK activity, appears to be particularly neuroprotective against α-syn-induced toxicity.

值得注意的是,T172Dα1变异体具有最实质性的神经保护作用,可减轻 SNpc 中 nissl 阳性神经元50% 以上的损失。这种形式的 AMPKα 整合到 AMPK 复合物中,具有独立于 T172磷酸化的本构型催化活性。然而,T172Dα1对 AMPK 也有显著的负作用,因为这个变异的活性明显低于 AMPKα 的野生型(图1b)[32,33]。T172Dα1过表达引起的慢性低活性模式可能阻止 AMPK 活性的波动,特别是对 α-syn 诱导的毒性具有保护作用。

Regarding the possible mechanism of neuroprotection, T172Dα1 variant prevents development of the α-syn pathology. Using 5G4 antibody to detect α-syn aggregates [36], we show a significant reduction of α-syn inclusions in the medial STR, only in the animals overexpressing T172Dα1 AMPK (Fig. 5e). In contrast, AMPKα1 and α2 tend to increase the deposition of aggregated α-syn and formation of dystrophic TH-positive axonal fibers. These effects are in line with previous observations that enhanced AMPK activity increases α-syn oligomers’ formation in vitro [27], although the activation of AMPK via AICAR or resveratrol was also been reported to mitigate α-syn oligomerization in a neuroglioma cell line [58]. While it remains to be determined if chronic low AMPK activity reduces the formation of α-syn oligomers in vivo, the lower level of α-syn deposition may account for the observed neuroprotective effects of the T172Dα1 variant. By preventing α-syn accumulation and aggregation, T172Dα1 overexpression could prevent the formation of dystrophic fibers in the STR and facilitate vesicular dopamine release. This might explain the improved symmetry of these animals in the cylinder test, despite the observed decrease in striatal TH immunoreactivity. However, other mechanisms are likely to account for the neuroprotective effects observed following overexpression of AMPKα1 and α2, as there is no apparent decrease in α-syn deposition in these conditions.

关于可能的神经保护机制,T172Dα1变异体阻止了 α-syn 病理的发展。用5G4抗体检测 α-syn 聚集体[36] ,我们发现只有在 T172Dα1 AMPK 过表达的动物中 α-syn 包涵体明显减少(图5e)。与此相反,AMPKα1和 α2则有助于增加 α 聚合体的沉积和形成 th- 阳性轴突纤维。这些效应与先前的观察结果一致,即增强 AMPK 活性可以增加体外 α- 寡聚体的形成[27] ,虽然也有报道说,通过 AICAR 或白藜芦醇激活 AMPK 可以减轻神经胶质瘤细胞系中 α- 寡聚体的形成[58]。虽然慢性低 AMPK 活性是否会减少体内 α- 同位素寡聚体的形成仍有待确定,但较低水平的 α- 同位素沉积可能解释 T172Dα1变异体的神经保护作用。T172Dα1过表达可以阻止 α-syn 的积累和聚集,从而阻止 STR 中营养不良纤维的形成,促进多巴胺囊泡的释放。这可能解释了圆柱试验中这些动物的对称性得到改善的原因,尽管观察到纹状体 TH 免疫反应性下降。然而,其他机制可能解释 AMPKα1和 α2过度表达后观察到的神经保护作用,因为在这些条件下没有明显减少 α-syn 沉积。

The effects of AMPKα on mitochondrial and lysosomal activities

Ampkα 对线粒体和溶酶体活性的影响

Using primary neuronal cultures, we have analyzed changes in mitochondrial and lysosomal parameters following α-syn overexpression. Cortical neurons show a significant increase in the number of autolysosomes loaded with the LC3B-mCherry-EGFP reporter when overexpressing human α-syn. This effect might reflect the accumulation of lysosomal material, possibly caused by defects in the lysosomal enzymatic activity associated with impaired trafficking of proteins [4144], or by perturbed lysosome recycling, as observed in neurons with defective glucocerebrosidase activity [4059]. At the level of mitochondria, α-syn mainly causes a decrease in the amount of mtDNA per cell, without any major effect on mitochondrial mass or respiration.

利用原代神经细胞培养,我们分析了 α-syn 过表达后线粒体和溶酶体参数的变化。当过量表达人 α- 同位素时,携带 LC3B-mCherry-EGFP 报告基因的大脑皮层神经元自溶体数量显著增加。这种效应可能反映了溶酶体物质的积累,可能是由于与蛋白质运输受损有关的溶酶体酶活性缺陷[41,44] ,或者是由于溶酶体破坏了某些再循环,正如在具有缺陷性葡萄糖脑苷脂酶活性的神经元中所观察到的[40,59]。在线粒体水平,α-syn 主要引起细胞线粒体 dna 含量下降,对线粒体质量和呼吸作用无明显影响。

Neurons overexpressing AMPKα1 and α2 appear to be more responsive to changes induced by the overexpression of human α-syn. The observed response may not represent a rescue effect of the alterations caused by α-syn. Rather, AMPKα-overexpressing neurons may promptly undergo adaptive changes to cope with the α-syn-induced stress. AMPKα-expressing neurons respond to α-syn overexpression by increasing the total number of autophagosomes, while limiting the accumulation of autolysosomes (see Fig. 6e), which may indicate an improved autophagic process. These effects are consistent with the role of AMPK in regulating lysosome activity [6061]. However, further studies are warranted to determine the exact effects of AMPKα on autophagic flux in neurons accumulating α-syn.

AMPKα1和 α2过表达的神经元对 α-syn 过表达所引起的变化反应更加敏感。所观察到的响应不能代表 α 同位素引起的改变的挽救效应。Ampkα 过表达的神经元可能迅速发生适应性变化,以应对 α-syn 诱导的应激反应。Ampkα 表达的神经元通过增加自噬体的总数来响应 α-syn 的过度表达,同时限制自溶体的积累(见图6e) ,这可能表明了一个改进的自噬过程。这些作用与 AMPK 调节溶酶体活性的作用一致[60,61]。然而,ampkα 对累积 α-syn 的神经元自噬通量的确切影响还有待进一步研究。

In vivo overexpression of α-syn has dramatic effects on mitochondrial morphology in nigral neurons, as seen by electron microscopy (Fig. 3). This effect of α-syn overexpression can be rescued by AMPKα2. Furthermore, neurons exposed to α-syn-induced stress show increased mitochondrial mass when overexpressing AMPKα2, both in vitro and in vivo. These changes are likely to represent adaptations to the effects of human α-syn on mitochondria. Indeed, AMPK has been shown to promote mitochondrial biogenesis, and in the same time may promote mitochondrial fission and the autophagic turnover of defective organelles [62,63,64]. The overall increase in mitochondrial mass may therefore contribute to preserving mitochondrial function in neurons overexpressing α-syn. This effect could contribute to neuron survival, in particular in the dopaminergic neurons of the SNpc, which have been reported to have a low mitochondrial content [65]. It is however surprising that AMPKα is unable to rescue the decreased number of mtDNA copies observed in neurons exposed to α-syn, which may contribute to the low reserve respiratory capacity observed in these conditions. This suggests that α-syn may affect the amount of mtDNA via mechanisms that are downstream of AMPK. Nuclear α-syn has been reported to block the transcriptional activity of PGC-1α [55], which drives expression of mitochondrial transcription factor A (TFAM), a key factor in mtDNA replication [58]. Methylation of the PGC-1α promoter is also associated with sporadic PD cases [56]. Furthermore, other pathogenic factors can affect mtDNA, such as mitochondrial unfolded protein response (UPRMT) which leads to TFAM clearance [66], or α-syn-induced changes in the dynamics of mitochondria, such as enhanced fragmentation [4547]. Remarkably, an increase in mitochondrial mass has been observed in mice with a conditional knockout of Tfam [6768], suggesting that similar compensatory mechanisms may take place in neurons co-expressing α-syn and AMPKα. In addition, it is worth noting that neurons expressing the T172Dα1 variant kept constant mitochondrial mass and low mtDNA content, both in the presence and absence of α-syn. Therefore, α-syn overexpression does not seem to affect these mitochondrial parameters in neurons with constitutive low AMPK activity.

电镜观察表明,在黑质神经元中 α-syn 的过表达对线粒体形态有显著影响(图3)。AMPKα2可以挽救 α-syn 过表达的这种效应。此外,过表达 AMPKα2的神经元在体内外都表现出线粒体质量的增加。这些变化可能代表了对人 α- 同位素对线粒体影响的适应。事实上,AMPK 已经被证明可以促进线粒体的生物合成,同时可以促进线粒体分裂和有缺陷细胞器的自噬转换。因此,线粒体质量的总体增加可能有助于保护过度表达 α-syn 的神经元的线粒体功能。这种效应可能有助于神经元存活,特别是在多巴胺能神经元的 SNpc,已报告有一个低线粒体含量[65]。然而,令人惊讶的是,ampkα 无法挽救在暴露于 α- 同位素的神经元中观察到的线粒体 dna 拷贝的减少,这可能有助于在这些条件下观察到的低储备呼吸能力。这表明 α-syn 可能通过 AMPK 的下游机制影响线粒体 dna 的数量。核的 α-syn 已经被报道能够阻断 pgc-1α 的转录活性,而 pgc-1α 能够促进线粒体 dna 复制的关键因子线粒体转录因子 a (TFAM)的表达[58]。Pgc-1α 启动子的甲基化也与散发性 PD 病例相关[56]。此外,其他致病因素也会影响线粒体 dna,如导致 TFAM 清除的线粒体未折叠蛋白反应(UPRMT) ,或者 α-syn 诱导的线粒体动态变化,如加强碎裂[45,47]。值得注意的是,在条件性敲除 Tfam [67,68]的小鼠中观察到线粒体质量的增加,这表明在共表达 α-syn 和 ampkα 的神经元中可能存在类似的补偿机制。此外,值得注意的是,表达 T172Dα1变异体的神经元在有无 α-syn 存在的情况下,线粒体质量保持不变,线粒体 dna 含量较低。因此,在 AMPK 活性较低的神经元中,α-syn 过表达似乎不影响线粒体参数。

Modulating AMPK activity: A neuroprotective approach in PD?

调节腺苷酸活化蛋白激酶活性: 帕金森病的神经保护途径?

A growing body of evidence suggests that AMPK signaling is crucial in the process of neurodegeneration associated with many diseases [1669]. However, data on the potentially neuroprotective effects of AMPK signaling appear ambiguous, sometimes even contradictory [70,71,72,73,74]. In the context of PD, inhibition of AMPK with compound C has been shown to accelerate the process of neurodegeneration following MPTP intoxication, both in vivo [75] and in vitro [76]. On the other hand, activation of AMPK promotes neuronal degeneration in toxin models of PD, both in vivo [77] and in vitro [78], as well as in PIKE-null mice overexpressing α-syn [28]. Additionally, AMPK activation was shown to accelerate the formation of α-syn oligomers in primary neurons [27]. Overall, the beneficial effects of AMPK signaling are mainly obtained when the insult is chronic and mild, as well as slowly progressing. Conversely, in acute and severe models, such as stroke or exposure to neuronal toxins, over-activation of AMPK signaling might even exacerbate the process of neurodegeneration [5370747778].

越来越多的证据表明,AMPK 信号在与许多疾病相关的神经退行性疾病过程中起着至关重要的作用。然而,有关 AMPK 信号传导的潜在神经保护作用的数据似乎并不明确,有时甚至是矛盾的[70,71,72,73,74]。在帕金森病的背景下,复合 c 对 AMPK 的抑制已经被证明可以加速 MPTP 中毒后神经退行性疾病的过程,无论是在体内[75]和体外[76]。另一方面,AMPK 的激活促进了 PD 毒素模型中的神经元退化,无论是在体内[77]和体外[78] ,还是在过度表达 α-syn [28]的 PIKE-null 小鼠中。此外,AMPK 活化可以加速初级神经元 α- 合成寡聚体的形成[27]。总的来说,AMPK 信号转导的有益作用主要是在慢性轻度损伤和缓慢进展时获得的。相反,在急性和严重的模型中,如中风或暴露在神经毒素中,AMPK 信号的过度激活甚至可能加剧神经退行性疾病过程[53,70,74,77,78]。

Conclusions

结论

It has been suggested that AMPK signaling progressively decreases with age [17]. Therefore, chronic activation of AMPK might serve as an effective preventive therapy when applied at the pre-symptomatic stage of neurodegenerative diseases associated with aging. Here, we provide in vivo evidence that overexpression of the AMPKα subunit can protect neurons at early stages of the α-syn pathology. In particular, the T172Dα1 variant with chronic low AMPK activity has the highest neuroprotective effects in a genetic rat model of progressive α-syn toxicity.

有人认为 AMPK 信号随着年龄的增长而逐渐减少。因此,慢性激活腺苷酸活化蛋白激酶(AMPK)可能作为一种有效的预防性治疗方法应用于与衰老相关的神经退行性疾病的前症状阶段。在这里,我们提供体内证据表明 ampkα 亚基的过度表达可以保护神经元在早期的 α- 突变。特别是慢性低 AMPK 活性的 T172Dα1变异体在进行性 α- 同位素毒性遗传大鼠模型中具有最高的神经保护作用。

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