神经元 TORC1通过 AMPK 调节寿命和线虫细胞线粒体动力学的非自主调节

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Neuronal TORC1 modulates longevity via AMPK and cell nonautonomous regulation of mitochondrial dynamics in C. elegans

Abstract

摘要

Target of rapamycin complex 1 (TORC1) and AMP-activated protein kinase (AMPK) antagonistically modulate metabolism and aging. However, how they coordinate to determine longevity and if they act via separable mechanisms is unclear. Here, we show that neuronal AMPK is essential for lifespan extension from TORC1 inhibition, and that TORC1 suppression increases lifespan cell non autonomously via distinct mechanisms from global AMPK activation. Lifespan extension by null mutations in genes encoding raga-1 (RagA) or rsks-1 (S6K) is fully suppressed by neuronal-specific rescues. Loss of RAGA-1 increases lifespan via maintaining mitochondrial fusion. Neuronal RAGA-1 abrogation of raga-1 mutant longevity requires UNC-64/syntaxin, and promotes mitochondrial fission cell nonautonomously. Finally, deleting the mitochondrial fission factor DRP-1 renders the animal refractory to the pro-aging effects of neuronal RAGA-1. Our results highlight a new role for neuronal TORC1 in cell nonautonomous regulation of longevity, and suggest TORC1 in the central nervous system might be targeted to promote healthy aging.

雷帕霉素复合物靶蛋白1(TORC1)和 AMP活化蛋白激酶(AMPK)拮抗代谢和衰老。然而,它们如何协调以确定寿命,以及它们是否通过可分离机制发挥作用,目前尚不清楚。在这里,我们表明,神经元 AMPK 是必不可少的寿命延长从 TORC1抑制,和 TORC1抑制增加寿命细胞非自主通过不同的机制从整体 AMPK 激活。由编码 RagA-1(RagA)或 rsks-1(S6K)的基因缺失突变延长寿命完全被神经元特异性拯救抑制。RAGA-1的缺失通过维持线粒体融合增加寿命。神经元 RAGA-1对 RAGA-1突变体寿命的抑制需要 UNC-64/syntaxin,并且非自主地促进线粒体分裂细胞。最后,删除线粒体分裂因子 DRP-1使动物对神经元 RAGA-1的促衰老作用无反应。我们的研究结果强调了神经元 TORC1在细胞非自主调节长寿方面的新作用,并提示中枢神经系统中的 TORC1可能是促进健康老龄化的靶点。https://doi.org/10.7554/eLife.49158.001“Open annotations. The current annotation count on this page is打开注释。此页面上的当前注释计数为0.

引言

Introduction

Aging is the single biggest risk factor for the majority of non-communicable complex diseases, including some of those with the greatest negative impact on human health outcomes worldwide (Escoubas et al., 2017). Work over the last two decades has uncovered molecular mechanisms that can be manipulated in model organisms to modulate the aging process and reduce overall disease risk in old age (Fontana et al., 2010). Many of these interventions have been linked to nutrient and energy sensing pathways, whose modulation genetically or pharmacologically mimics the effects of dietary restriction on healthy aging (Fontana and Partridge, 2015). One classic example of a nutrient sensor linked to longevity is TORC1, which promotes anabolic processes such as protein translation to provide macromolecules for growth and proliferation while inhibiting catabolic activities such as autophagy (Albert and Hall, 2015). TORC1 is activated by growth factors and amino acids, the latter of which act through sensors such as the sestrins to facilitate heterodimer formation of the Rag proteins (consisting of RagA, B, C and D in mammals), an essential step for TORC1 activation (Saxton and Sabatini, 2017). Suppression of TORC1 both genetically and pharmacologically, via rapamycin feeding, promotes longevity in multiple species from yeast to mice (Kennedy and Lamming, 2016). In contrast to TORC1, the conserved kinase AMPK is activated under low energy conditions. AMPK activation promotes catabolic processes that generate ATP, including the TCA cycle, fatty acid oxidation and autophagy (Burkewitz et al., 2014), and extends lifespan in C. elegans (Apfeld et al., 2004Greer et al., 2007Mair et al., 2011) and fruit flies (Stenesen et al., 2013). Given the antagonistic roles of TORC1 and AMPK in nutrient-sensing, metabolism and longevity, it is perhaps intuitive that they are mechanistically linked. Indeed, in mammals, AMPK is generally regarded as an upstream suppressor of TORC1 as it phosphorylates the TSC complex and raptor to inhibit TORC1 activity (Gwinn et al., 2008Inoki et al., 2003). Conversely, TORC1 signaling has been suggested to inhibit AMPK via p70 ribosomal protein S6 Kinase (S6K) (Dagon et al., 2012). However, much of the work elucidating how AMPK and TORC1 interact has been performed in vitro in cell culture studies and it remains unclear in multicellular organisms whether TORC1 and AMPK causally coordinate to modulate the aging process.

对于大多数非传染性复杂疾病,包括对全世界人类健康产生最大负面影响的一些疾病,老龄化是最大的单一风险因素(Escoubas 等人,2017年)。过去二十年的工作已经揭示了可以在模型生物中操纵的分子机制,以调节衰老过程并降低老年时的总体疾病风险(丰塔纳等人,2010年)。其中许多干预措施都与营养和能量传感途径有关,这些途径的基因或药理调控模拟了饮食限制对健康老龄化的影响(丰塔纳和帕特里奇,2015年)。一个与长寿有关的营养传感器的典型例子是 TORC1,它促进蛋白质翻译等合成代谢过程,为生长和增殖提供大分子,同时抑制自噬等分解代谢活动(Albert and Hall,2015)。TORC1被生长因子和氨基酸激活,后者通过传感器如 sestrins 起作用,促进 Rag 蛋白异二聚体的形成(包括哺乳动物中的 RagA、 b、 c 和 d) ,这是 TORC1激活的必要步骤(Saxton 和 Sabatini,2017年)。通过雷帕霉素的喂养,从遗传学和药理学上抑制 TORC1,促进从酵母到小鼠的多种物种的长寿(Kennedy 和 Lamming,2016)。与 TORC1相反,保守的激酶 AMPK 在低能量条件下被激活。AMPK 激活促进分解代谢过程,产生 ATP,包括 TCA 循环,脂肪酸氧化和自噬(伯克维茨等人,2014年) ,并延长线虫寿命(Apfeld 等人,2004年; 格里尔等人,2007年; Mair 等人,2011年)和果蝇(Stenesen 等人,2013年)。考虑到 TORC1和 AMPK 在营养感知、新陈代谢和寿命中的拮抗作用,也许直觉上它们是机械连接的。事实上,在哺乳动物中,AMPK 通常被认为是 TORC1的上游抑制因子,因为它磷酸化 TSC 复合体和 raptor 以抑制 TORC1的活性(Gwinn 等人,2008; Inoki 等人,2003)。相反,TORC1信号通过 p70核糖体蛋白质 s 6激酶抑制 AMPK (S6K)(Dagon 等,2012)。然而,许多阐明 AMPK 和 TORC1如何相互作用的工作已经在体外细胞培养研究中完成,在多细胞生物中,目前尚不清楚 TORC1和 AMPK 是否因果协同调节衰老过程。

Here, we elucidate the relationship between AMPK and TORC1 in the modulation of aging by discovering a critical role for neuronal AMPK in lifespan extension resulting from suppression of TORC1 components in C. elegans. We show that neuronal TORC1 pathway activity itself is critical for healthy aging. Restoring either raga-1 or rsks-1 (encoding the C. elegans orthologue of S6 Kinase) only in neurons fully suppresses lifespan extension in animals lacking the respective TORC1 component in all other cell types. However, the downstream effectors of AMPK longevity and TORC1 longevity appear separable; constitutive activation of CREB regulated transcriptional coactivator ‘CRTC’−1, which suppresses AMPK longevity, does not suppress lifespan extension by TORC1 suppression. We show that neuronal RAGA-1 regulates metabolic genes in distal tissues and critically requires UNC-64/syntaxin exocytosis to modulate systemic longevity. Using RNA-Seq and in vivo reporters, we identify mitochondrial network state as the downstream mechanism for lifespan regulation by neuronal RAGA-1. Neuronal RAGA-1 activity regulates mitochondrial networks in distal tissues cell nonautonomously and this regulation is required for the effect of neuronal RAGA-1 on the aging process. Together, our data provide key insights into the tissue-specific roles of TORC1 suppression in healthy aging and highlight the nervous system as a putative target site for future pharmacological interventions aimed at the TORC1 pathway.

在这里,我们阐明了 AMPK 和 TORC1之间的关系,通过发现神经元 AMPK 在延长寿命的关键作用,抑制 TORC1成分在线虫。我们发现神经元 TORC1通路的活动本身对于健康的衰老是至关重要的。只在神经元中恢复 raga-1或 rsks-1(编码 S6激酶的秀丽隐杆线虫直系亲属) ,完全抑制缺乏其他所有细胞类型中 TORC1成分的动物的寿命延长。然而,AMPK 寿命和 TORC1寿命的下游效应因子似乎是可分离的; CREB 调节的转录辅激活因子 CRTC’-1的组成性激活,抑制 AMPK 寿命,并不通过 TORC1抑制抑制寿命延长。我们发现神经元 RAGA-1调节远端组织的代谢基因,并且特别需要 UNC-64/syntaxin 的胞吐作用来调节系统的寿命。利用 RNA-Seq 和体内报告基因,我们确定线粒体网络状态是神经元 RAGA-1调控寿命的下游机制。神经元 RAGA-1活性对远端组织细胞线粒体网络的调控是神经元 RAGA-1在衰老过程中发挥作用的必要条件。总之,我们的数据为 TORC1抑制在健康老龄化中的组织特异性作用提供了关键的见解,并强调神经系统作为未来针对 TORC1通路的药理干预的假定靶点。

结果

Results

AMPK is required for longevity mediated by TORC1 suppression

AMPK 是 TORC1抑制介导的长寿所必需的

AMPK is canonically regarded as an upstream suppressor of TORC1. However, whether TORC1 suppression acts downstream of the pro-longevity effects of AMPK is unknown. Loss of the TORC1 target S6 Kinase increases lifespan in C. elegansand mouse (Selman et al., 2009) and results in transcriptional profiles in muscle that resemble those of active AMPK (Selman et al., 2009). Interestingly, AMPK activity is required for lifespan extension by loss of S6K in C. elegans(Selman et al., 2009), which does not support a model where AMPK acts linearly upstream of TORC1 to modulate aging.

AMPK 通常被认为是 TORC1的上游抑制基因。然而,是否 TORC1抑制行为的 AMPK 延长寿命的影响下游是未知的。TORC1目标 S6激酶的缺失增加了秀丽隐杆线虫和小鼠的寿命(Selman 等人,2009年) ,并导致肌肉类似于活跃 AMPK 的转录谱(Selman 等人,2009年)。有趣的是,AMPK 活性是必需的寿命延长的 S6K 损失秀丽隐杆线虫(Selman 等人,2009年) ,这不支持一个模型,其中 AMPK 行为线性上游的 TORC1调节。

S6K deletion has been shown to activate AMPK in both C. elegans and mice (Selman et al., 2009). To examine whether the activation of AMPK is specific to RSKS-1 (S6K) or pertains more broadly to the TORC1 pathway, we assayed the phosphorylation status of AMPK upon inhibition of multiple TORC1 components in C. elegans. We measured AMPK activity, using antibodies targeting phosphorylation of the α catalytic subunit at threonine 172, a site in the activation loop that is a hallmark of AMPK activation (Hardie et al., 2012). This phosphorylation site is conserved in C. elegans on the two AMPKα subunits, AAK-1 and AAK-2 (Apfeld et al., 2004) and its phosphorylation has been used to measure AMPK activity (Selman et al., 2009). To confirm conservation, we generated animals carrying a CRISPR induced threonine-to-alanine mutation at T243 on AAK-2, the residue homologous to mammalian T172. Lysates from both CRISPR generated AAK-2(T243A) mutation animals, and aak-2(ok524) mutants which harbor a 408 bp deletion spanning T243, completely lose recognition by antibody targeting phosphorylated AMPK T172 (Figure 1—figure supplement 1a), confirming conservation of this phosphorylation event (hereafter referred to as AMPK T172 phosphorylation) and specificity of the antibody to AAK-2. RNAi against let-363 (homolog of TOR) significantly increases AMPK T172 phosphorylation (Figure 1—figure supplement 1b,c) in whole worm lysates. In addition, we found significantly increased levels of AMPK T172 phosphorylation in animals carrying a null allele in raga-1 (Figure 1a,b), a gene encoding a homologous protein to RagA, which senses amino acids and activates TORC1 (Saxton and Sabatini, 2017). Taken together, these data suggest that in C. elegans, AMPK is activated when TORC1 signaling is decreased.

S6K 缺失已被证明能够激活线虫和小鼠中的 AMPK (Selman 等人,2009)。为了研究 AMPK 的激活是否特异于 RSKS-1(S6K)或更广泛地涉及 TORC1通路,我们分析了 AMPK 对多种 TORC1成分抑制作用的磷酸化状态。我们测量 AMPK 活性,使用抗体的目标磷酸化的 α 催化亚基在苏氨酸172,一个位点的激活环是 AMPK 活化的标志(哈迪等人,2012年)。这个磷酸化位点在秀丽隐杆线虫的两个 AMPKα 亚基上是保守的,AAK-1和 AAK-2(Apfeld 等人,2004年) ,它的磷酸化已经被用来测量 AMPK 活性(Selman 等人,2009年)。为了确认保护性,我们在 AAK-2基因的 T243处制备了 CRISPR 诱导的苏氨酸转丙氨酸突变动物,该突变与哺乳动物 T172基因同源。从 CRISPR 产生的 AAK-2(T243A)突变动物和 AAK-2(ok524)突变体中分离得到的裂解物,其中 T243缺失408bp,抗体靶向磷酸化 AMPK T172(图1ー图1a)完全失去识别能力,证实了这一磷酸化事件(以下简称 AMPK T172磷酸化)的保守性和抗体对 AAK-2的特异性。对 let-363(TOR 同源基因)的 rna 干扰显著增加了整个蠕虫裂解液中 AMPK T172的磷酸化(图1ー图补充1b,c)。此外,我们发现携带 RagA-1缺失等位基因(图1a,b)的动物中 AMPK T172磷酸化水平显著增加,该基因编码 RagA 的同源蛋白质,它能感知氨基酸并激活 TORC1(Saxton 和 Sabatini,2017)。综上所述,这些数据表明,在线虫中,当 TORC1信号减少时 AMPK 就被激活。Figure 1 图1 with 1 supplement 还有一种补充剂Download asset下载资产Open asset开放资产

Neuronal AMPK is required for TORC1-mediated longevity.神经元 AMPK 是 torc1介导的寿命所必需的(a, b) AMPK T172 phosphorylation is increased in raga-1(ok386) null mutants compared to wild type animals. Actin levels are used as loading controls (representative immunoblot and corresponding … see more(a,b)与野生型动物相比,raga-1(ok386)缺失突变体 AMPK T172磷酸化水平升高。肌动蛋白水平被用作负载控制(代表性免疫印迹和相应的… 更多https://doi.org/10.7554/eLife.49158.002
Figure 1—source data 1
图1ー源数据1

Figure 1b AMPK activity is increased in raga-1 mutants.

图1b 在 raga-1突变体中 AMPK 活性增加。https://doi.org/10.7554/eLife.49158.004Download elife-49158-fig1-data1-v2.xlsxFigure 1—figure supplement 1 图1ー图补充资料1Download asset下载资产Open asset开放资产

Conservation of critical residues in AAK-2 and their requirement for TORC1-mediated longevity.AAK-2中关键残基的保护及其对 torc1介导的寿命的要求(a) Western blot showing that a threonine-to-alanine mutation at T243 generated by CRISPR on aak-2completely loses recognition for antibodies against phosphorylation at the conserved residue T172 … see more(a)蛋白质印迹显示,在 aak-2上 CRISPR 产生的 T243上的苏氨酸转丙氨酸突变完全失去对保守残基 T172磷酸化抗体的识别… 更多https://doi.org/10.7554/eLife.49158.003
Figure 1—figure supplement 1—source data 1
图1ー图补充1ー源数据1

Figure 1—figure supplement 1cAMPK activity is increased by knockdown of TOR.

图1ー图补充1c AMPK 活性通过降低 TOR 而增加。https://doi.org/10.7554/eLife.49158.005Download elife-49158-fig1-figsupp1-data1-v2.xlsx

Figure 1—figure supplement 1—source data 2
图1ー图补充1ー源数据2

Figure 1—figure supplement 1fThe conserved S6K/Akt phosphorylation site serine 551 on AAK-2 modulates AMPK activity.

图1ー图补充1f AAK-2上保守的 S6K/Akt 磷酸化位点丝氨酸551调节 AMPK 活性。https://doi.org/10.7554/eLife.49158.006Download elife-49158-fig1-figsupp1-data2-v2.xlsx

We next tested whether AMPK activity is required for longevity resulting from TORC1 suppression. Adult-onset RNAi of let-363 (TOR) or raga-1 extended median lifespan by 20% in wild type animals, but this effect is suppressed in aak-2(ok524) mutants (Figure 1c,d). In addition, while null mutation of raga-1extends lifespan by 30% in an otherwise wild type background, it has no effect in aak-2(ok524) mutants (Figure 1e). Supporting the hypothesis that phosphorylation of AMPK at T172 is critical for extension of lifespan by TORC1 suppression, the extended lifespan of raga-1 mutants was also suppressed by AAK-2 T243A (Figure 1f). These results are unlikely due to the general sickness of the aak-2 mutants, since their lifespan can be extended by other interventions, such as dietary restriction in liquid culture or intermittent fasting (Greer and Brunet, 2009Honjoh et al., 2009Mair et al., 2009). LKB1 is the primary kinase that phosphorylates AMPK under energy stress (Tsou et al., 2011). We tested the requirement for PAR-4, the LKB1 homolog in C. elegans, for raga-1 mediated longevity using animals bearing a temperature sensitive allele par-4(it57) (Morton et al., 1992). par-4(it57) animals are refractory to the lifespan extension of raga-1 RNAi when housed at restrictive temperatures, suggesting AMPK T172 phosphorylation by LKB1 is required for TORC1-mediated longevity (Figure 1g).

我们接下来测试是否 AMPK 活性是需要的寿命产生的 TORC1抑制。Let-363(TOR)或 raga-1的成年起始 rna 干扰使野生型动物的中位寿命延长了20% ,但这种效应在 aak-2(ok524)突变体中被抑制(图1c,d)。此外,在其他野生型背景下,raga-1的零突变可使寿命延长30% ,但对 aak-2(ok524)突变体没有影响(图1e)。T172位 AMPK 的磷酸化是 TORC1抑制延长寿命的关键假设的支持,raga-1突变体的延长寿命也被 AAK-2 T243A 抑制(图1f)。由于 aak-2突变体普遍患病,这些结果不太可能出现,因为其寿命可以通过其他干预措施延长,例如限制饮食或间歇性禁食(Greer 和 Brunet,2009; Honjoh 等人,2009; Mair 等人,2009)。LKB1是能量胁迫下磷酸化 AMPK 的主要激酶(Tsou 等人,2011)。我们利用具有温度敏感等位基因 PAR-4(it57)的动物测试了 PAR-4(线虫 LKB1同源基因)对 raga-1介导的长寿的需求。Par-4(it57)动物在限制性温度下对 raga-1 rna 干扰的寿命延长是不敏感的,这表明,通过 LKB1磷酸化 AMPK T172是 torc1介导的寿命延长所必需的(图1g)。

S6K has been suggested to phosphorylate AMPK alpha 2 at serine 485, resulting in inhibition of AMPK activity (Dagon et al., 2012). We examined whether phosphorylation status at the C. elegans equivalent of AMPK alpha 2 serine 485 mediates lifespan extension by TORC1 pathway suppression. Although we found that this serine residue is conserved in C. elegans (serine 551 residue in AAK-2) (Figure 1—figure supplement 1d) and inhibits AMPK (Figure 1—figure supplement 1e,f), mutating S551 to alanine does not alter the longevity response to reduced TORC1 signaling (Figure 1—figure supplement 1g), suggesting that this phosphorylation event is not required for TORC1-mediated longevity. Taken together, these data suggest that in C. elegans, AMPK is activated and critically required for lifespan extension when TORC1 is inhibited.

S6K 被认为可以在丝氨酸485处磷酸化 AMPK α2,从而抑制 AMPK 活性(Dagon 等人,2012)。我们研究了与 AMPKα2丝氨酸485相当的秀丽隐杆线虫的磷酸化状态是否通过 TORC1途径抑制介导寿命延长。虽然我们发现这种丝氨酸残基在秀丽隐杆线虫(AAK-2中的丝氨酸551残基)中是保守的(图1ー图1d) ,并抑制 AMPK (图1ー图1e,f) ,但是突变 S551到丙氨酸并不改变 TORC1信号减少引起的寿命反应(图1ー图1g) ,这表明这种磷酸化事件并不是 TORC1寿命所必需的。综上所述,这些数据表明,在线虫中,当 TORC1受到抑制时,AMPK 被激活,并且是延长寿命的关键所需。

TORC1 and AMPK act via separable downstream mechanisms

TORC1和 AMPK 通过可分离的下游机制起作用

If AMPK acts as the key downstream effector of TORC1 mediated longevity, we reasoned that interventions that inhibit lifespan extension via AMPK activation might also block longevity resulting from suppression of TORC1. Previously, we have shown that phosphorylation of CREB-regulated transcriptional coactivator (CRTC)−1 in neurons is required for lifespan extension by constitutively activated AMPK (Burkewitz et al., 2015). To determine if lifespan extension via raga-1 deletion acts via a similar mechanism, we crossed a transgene that expresses a non-phosphorylatable variant of CRTC-1(S76A, S179A) only in neurons into raga-1(ok386) mutants. Although neuronal CRTC-1(S76A, S179A) fully suppresses AMPK mediated longevity (Burkewitz et al., 2015), it does not similarly suppress raga-1 mutant longevity (Figure 1h). Together these data suggest that, while AMPK is required for TORC1 mediated longevity, the mechanisms by which TORC1 suppression and AMPK activation promote healthy aging are likely separable.

如果 AMPK 作为 TORC1介导的长寿的关键下游效应器,我们推断通过 AMPK 激活抑制寿命延长的干预措施也可能阻止由 TORC1抑制导致的长寿。在此之前,我们已经证明,creb 调节的转录辅激活因子(CRTC)-1在神经元中的磷酸化是通过组成性激活 AMPK 延长寿命所必需的(Burkewitz 等人,2015)。为了确定是否通过 raga-1基因缺失来延长寿命,我们将一个表达 CRTC-1(S76A,S179A)非磷酸化变异体的转基因仅在神经元中转入 raga-1(ok386)突变体。虽然神经元 CRTC-1(S76A,S179A)完全抑制 AMPK 介导的长寿(Burkewitz 等人,2015年) ,但它并没有类似地抑制 raga-1突变体的长寿(图1h)。综合这些数据表明,尽管 AMPK 对于 TORC1介导的长寿是必需的,但是 TORC1抑制和 AMPK 激活促进健康老龄化的机制是可以分开的。

Neuronal AMPK is required for TORC1-mediated longevity

神经元 AMPK 是 torc1介导的寿命所必需的

To further explore why AMPK activity is required for lifespan extension by TORC1 suppression, we asked where AMPK acts to mediate this effect. We rescued aak-2(ok524) mutants in specific tissues with a truncated and constitutively active form of AAK-2aa1-321 (Mair et al., 2011). Ubiquitous expression of AAK-2aa1-321 fully restored the responsiveness of aak-2(ok524) mutant animals to the lifespan promoting effects of raga-1 RNAi (Figure 1i), whereas expression of AAK-2aa1-321 specifically in either the intestine or muscle did not (Figure 1j,k). Remarkably, however, expressing AAK-2aa1-321 solely in neurons fully restored lifespan extension by raga-1 RNAi (Figure 1l). We saw similar rescue using neuronal expression of full-length wild type aak-2 (Figure 1—figure supplement 1h). These data demonstrate that activity of AMPK specifically in neurons is a critical mediator of TORC1 longevity, and also suggest that neuronal TORC1 activity itself may impact healthy aging.

为了进一步探索为什么 AMPK 活性对 TORC1抑制延长寿命是必需的,我们询问 AMPK 在哪里起作用来介导这种效应。我们在特定组织中解救了 aak-2(ok524)突变体,该突变体具有 AAK-2aa1-321的截短和组成活性形式(Mair 等人,2011)。AAK-2aa1-321的无处不在的表达完全恢复了 aak-2(ok524)突变动物对 raga-1 rna 干扰(图1i)促进寿命的反应性,而 AAK-2aa1-321的表达特别是在肠道或肌肉中没有(图1j,k)。然而,只在神经元中表达 AAK-2aa1-321,通过 raga-1 rna 干扰完全恢复了寿命延长(图11)。我们看到类似的拯救使用全长野生型 aak-2的神经元表达(图1ー图补充1h)。这些数据表明 AMPK 特异性在神经元中的活性是 TORC1长寿的关键调节因子,并且提示神经元的 TORC1活性本身可能影响健康的衰老。

TORC1 acts in neurons to regulate aging

TORC1在神经元中起调节衰老的作用

Homozygous null mutations for many TORC1 components and regulators lead to developmental arrest (Albert and Riddle, 1988The C. elegans Deletion Mutant Consortium, 2012Long et al., 2002) and therefore do not facilitate longevity assays. However, null mutants for raga-1 or rsks-1 are viable and long-lived (Schreiber et al., 2010Selman et al., 2009). To directly examine the role of neuronal TORC1 activity on organismal aging, we rescued TORC1 pathway activity in raga-1 and rsks-1 mutants specifically in neurons, via ectopic expression regulated by the pan-neuronal rab-3 promoter. These animals therefore have reduced TORC1 signaling in all tissues except neurons. Strikingly, we found that expression of raga-1 in neurons fully suppressed the long lifespan of raga-1 mutant animals without affecting wild type lifespan (Figure 2a). Neuronal raga-1 fully suppressed the long lifespan both with and without the use of FUDR, a chemical inhibitor of DNA synthesis commonly used in C. eleganslifespan experiments to stop the production of progeny that can also lead to complex interactions with various genes and treatments in the regulation of lifespan (Anderson et al., 2016) (Figure 2—figure supplement 1). Similarly, neuronal rsks-1 expression rescued the lifespan extension of rsks-1 deletion mutants (Figure 2b), suggesting that neuronal regulation of longevity is not specific to RAGA-1 and extends to other TORC1 pathway components.

许多 TORC1组分和调节因子的纯合性缺失突变导致发育停滞(Albert and Riddle,1988; The c. elegans Deletion Mutant Consortium,2012; Long et al. ,2002) ,因此不能促进长寿试验。然而,缺失突变体 raga-1或 rsks-1是可行的和长寿的(Schreiber 等人,2010; Selman 等人,2009)。为了直接研究神经元 TORC1活性在生物体衰老中的作用,我们通过泛神经元 rab-3启动子调控的异位表达,挽救了 raga-1和 rsks-1突变体的 TORC1通路活性。因此,这些动物在除神经元以外的所有组织中减少了 TORC1信号。引人注目的是,我们发现 raga-1在神经元中的表达完全抑制了 raga-1突变动物的长寿命,而不影响野生型动物的寿命(图2a)。Raga-1完全抑制了长寿命,无论是否使用 FUDR,一种 DNA 合成的化学抑制剂,通常用于线虫寿命试验,以停止后代的产生,也可能导致复杂的相互作用,与各种基因和治疗调节寿命(Anderson 等人,2016年)(图2ー图补充1)。类似地,神经元 rsks-1的表达拯救了 rsks-1缺失突变体的寿命延长(图2b) ,表明神经元对长寿的调节不是 RAGA-1特有的,而是延伸到其他 TORC1途径的组成部分。Figure 2 图2 with 3 supplements 有三种补充剂Download asset下载资产Open asset开放资产

TORC1 signaling is required in neurons to regulate lifespan.神经元需要 TORC1信号来调节寿命(a) The raga-1(ok386) deletion increases lifespan (p<0.0001). However, when raga-1 is expressed in the nervous system via extrachromosomal array using the rab-3 promoter, raga-1(ok386) does not … see more(a) raga-1(ok386)缺失可以增加寿命(p < 0.0001)。然而,当 raga-1通过 rab-3启动子的染色体外阵列在神经系统中表达时,raga-1(ok386)就不会… 见更多https://doi.org/10.7554/eLife.49158.007
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Figure 2e qPCR of raga-1 expression in SCIs and extrachromosomal lines.

图2e qPCR 的 raga-1表达在 SCIs 和染色体外线。https://doi.org/10.7554/eLife.49158.011Download elife-49158-fig2-data1-v2.xlsxFigure 2—figure supplement 3 图2ー图补充3Download asset下载资产Open asset开放资产

Neuronal raga-1does not rescue development delay of raga-1(ok386)mutants.神经元 raga-1不能解救 raga-1(ok386)突变体的发育延迟Stacked bar graph showing percent of animals at the noted developmental stage after 72 hr at 20°C. Shown are averaged values from two independent experiments. Error bars denote SEM. ‘neuronal raga-1’… see more堆叠的条形图显示了在20 ° c 下72小时之后动物发育阶段的百分比。显示的是两个独立实验的平均值。误差条表示扫描电镜。神经元 raga-1… 看到更多https://doi.org/10.7554/eLife.49158.010
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Figure 2—figure supplement 3Developmental stages of raga-1 rescue lines.

图2ー图补充3 raga-1营救系的发育阶段。https://doi.org/10.7554/eLife.49158.012Download elife-49158-fig2-figsupp3-data1-v2.xlsxFigure 2—figure supplement 2 图2ー图补充资料2Download asset下载资产Open asset开放资产

Elimination of raga-1 in the intestine by RNAi does not impair rescue by the extrachromosomal neuronal raga-1 array.Rna 干扰消除肠道中的 raga-1并不损害外染色体神经元 raga-1阵列的拯救作用(araga-1RNAi eliminates non-specific expression of the rab-3p::raga-1::SL2::mCherryextrachromosomal transgene in the intestine but preserves expression in neurons. Images were taken from adult … see more(a) raga-1 rna 干扰消除了 rab-3p: : raga-1: : SL2: : mCherry 外染色体转染基因在肠道的非特异性表达,但保留了神经元的表达。图片来自成人… 更多https://doi.org/10.7554/eLife.49158.009

Figure 2—figure supplement 1 图2ー图补充1Download asset下载资产Open asset开放资产

Neuronal raga-1expressed by extrachromosomal transgene suppresses raga-1mutant longevity when animals are not treated with FUDR to prevent progeny development.当不用 FUDR 处理动物时,染色体外基因表达的神经元 raga-1抑制 raga-1突变体的长寿,从而阻止后代的发育Details on strains and lifespan replicates can be found in Supplementary file 6.关于菌株和寿命复制的详细信息可以在补充文件6中找到。https://doi.org/10.7554/eLife.49158.008

Since many neuronal-specific promoters show leakiness in the intestine, especially in adult C. elegans, we used an RNAi protocol to eliminate transgene expression in the intestine while leaving neuronal expression intact (Figure 2—figure supplement 2a). Critically, in animals in which intestinal leakiness of the promoter was removed via raga-1 RNAi, rab-3p::raga-1 still fully suppressed raga-1 mutant lifespan (Figure 2—figure supplement 2b). Additionally, we generated single copy knock-ins of raga-1 cDNA using the SKI LODGE system (Silva-García et al., 2019). We used CRISPR to insert raga-1 cDNA sequences into an intergenic cassette which uses both the rab-3 promoter and rab-3 3’ UTR sequences to drive gene expression more specifically in the nervous system (Silva-García et al., 2019). We also knocked raga-1 cDNA into a cassette that uses the eft-3 promoter to express raga-1 across all somatic tissues. Remarkably, raga-1 expressed in the nervous system by single copy transgene effectively rescued raga-1 lifespan, despite its more restricted and lower level of expression (Figure 2c,d). In contrast, expressing raga-1directly in the intestine by multicopy array using the intestine-specific ges-1promoter did not suppress raga-1 mutant longevity (Figure 2e). Expression of raga-1 in the nervous system by single copy transgene did not rescue other phenotypes resulting from loss of RAGA-1 function, including reduced body size (Figure 2f) and developmental delay (Figure 2—figure supplement 3). Together, these results show that TORC1 activity specifically in neurons modulates systemic aging, and this activity can be uncoupled from its regulation of growth and development.

由于许多神经元特异性启动子在肠道中表现出泄漏性,特别是在成年线虫中,我们使用 rna 干扰技术来消除肠道中的转基因表达,同时保持神经元表达的完整(图2ー图补充2a)。至关重要的是,在通过 raga-1 rna 干扰去除启动子肠道泄漏的动物中,rab-3p: : raga-1仍然完全抑制 raga-1突变体的寿命(图2ー图2b)。此外,我们使用 SKI LODGE 系统(Silva-García et al. ,2019)生成了 raga-1基因的单拷贝敲入。我们利用 CRISPR 技术将 raga-1基因序列插入到基因间盒中,该盒使用 rab-3启动子和 rab-33’ UTR 序列来驱动神经系统中更具体的基因表达(Silva-García et al. 2019)。我们还将 raga-1基因敲入一个盒中,利用 eft-3启动子在所有体细胞组织中表达 raga-1。值得注意的是,raga-1通过单拷贝转基因在神经系统中表达,有效地挽救了 raga-1的寿命,尽管它的表达更加受限和更低水平(图2c,d)。相比之下,利用肠道特异性启动子 ges-1多拷贝阵列直接在肠道中表达 raga-1并不会抑制 raga-1突变体的长寿(图2e)。RAGA-1通过单拷贝转基因在神经系统中的表达并不能挽救其他因 RAGA-1功能丧失而导致的表型,包括体型缩小(图2f)和发育迟缓(图2ー图3)。总之,这些结果表明,TORC1活动特别是在神经元调节系统老化,这种活动可以从其生长和发育的调节解偶联。

Neuronal RAGA-1 modulates aging via neuropeptide signaling

神经元 RAGA-1通过神经肽信号调节衰老

To identify mechanisms specifically coupled to neuronal RAGA-1 regulation of lifespan, we examined the transcriptomes of wild type (‘WT’), raga-1 mutant (‘mutant’) and raga-1 mutant with extrachromosomal neuronal raga-1 expression (‘rescue’) C. elegans by RNA-Seq (Supplementary files 13). We performed a cluster analysis that takes into account trends in gene expression across all three conditions to identify genes that change in the raga-1 mutant and are reversed by neuronal rescue. As expected given the specific nature of neuronal raga-1 rescue of lifespan but not other raga-1 phenotypes, the majority of differentially expressed genes changed similarly in the raga-1 mutant and in the rescue line (Figure 3—figure supplement 1a). However, a small number of genes that showed increased or decreased expression in the raga-1 mutant were rescued by the neuronal raga-1 array (Figure 3aFigure 3—figure supplement 1a). Interestingly, functional analysis of the genes within these clusters reveals an enrichment for Gene Ontology (GO) terms related to organelle organization, organelle fission, and unfolded protein/ER stress pathways that are upregulated in the raga-1 mutant but not in rescue, whereas GO terms related to neuronal function, including synaptic structure and signaling as well as regulation of dauer entry are enriched in the cluster of genes that show reduced expression in raga-1 but not in rescue (Figure 3b). These GO terms are also revealed in pairwise comparisons designed to identify biological processes that differ between wild type and raga-1, but not between wild type and neuronal rescued animals (Figure 3—figure supplement 1b,c).

为了确定神经元 RAGA-1调控寿命的特异性机制,我们利用 RNA-Seq (补充文件1-3)检测了野生型(WT)、 RAGA-1突变型(突变型)和 RAGA-1突变型的非染色体神经元 RAGA-1表达(拯救型)线虫的转录组。我们进行了一项基因数据聚类研究,该研究考虑了所有3种情况下的基因表达趋势,以确定 raga-1突变体中发生变化的基因,并通过神经元拯救来逆转这些基因。鉴于神经元 raga-1拯救寿命的特殊性,而非其他 raga-1表型,raga-1突变体和拯救系的大多数差异表达基因都发生了相似的变化(图3ー图1a)。然而,少数表现出 raga-1突变体表达增加或减少的基因被神经元 raga-1阵列拯救(图3a,图3ー图1a 补充)。有趣的是,对这些簇内基因的功能分析表明,与细胞器组成、细胞器分裂和未折叠蛋白/er 应激通路有关的基因本体(GO)项在 raga-1突变体中上调,而与神经元功能有关的 GO 项,包括突触结构和信号传导以及调节信号通路,在基因簇中富集,raga-1的表达减少而在抢救中没有表达(图3b)。这些 GO 术语也在成对的比较中被揭示出来,这些比较旨在确定野生型和 raga-1之间的生物学过程不同,但是野生型和神经拯救动物之间不同(图3ー图1b,c)。Figure 3 图3 with 3 supplements 有三种补充剂Download asset下载资产Open asset开放资产

Neuronal TORC1 modulates aging via changes to organelle organization and neuropeptide signaling.神经元 TORC1通过改变细胞器结构和神经肽信号通路来调节衰老(a) Cluster analysis identified 59 genes that show increased expression in raga-1(ok386) that is reversed by neuronal rescue array (Cluster 3, top) and 107 genes that show decreased expression in rag… see more(a)数据聚类发现59个基因在 raga-1(ok386)中表达增加,而神经元拯救阵列(Cluster 3,top)和107个基因在 rag 中表达减少… 见更多https://doi.org/10.7554/eLife.49158.013
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Figure 3c and d qPCR of daf-28 and ins-6.

图3c 和 daf-28和 ins-6的 d qPCR。https://doi.org/10.7554/eLife.49158.017Download elife-49158-fig3-data1-v2.xlsxFigure 3—figure supplement 3 图3ー图补充资料3Download asset下载资产Open asset开放资产

Neuronal raga-1regulates ins-6expression in adults.神经元 raga-1调控成人胰岛素6的表达Relative expression of ins-6in animals at day 1 of adulthood as determined by qPCR. Points plotted are from independent biological samples. Error bars denote mean + /- SEM. Pvalues determined by … see more定量 pcr 检测成年第1天动物胰岛素 -6的相对表达。绘制的点是来自独立的生物样本。误差条表示均值 +/-SEM。由… 决定的 p 值见更多https://doi.org/10.7554/eLife.49158.016
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Figure 3—figure supplement 3qPCR of ins-6 in day 1 adults.

图3ー图补充3日龄成人胰岛素 -6的 qPCR。https://doi.org/10.7554/eLife.49158.019Download elife-49158-fig3-figsupp3-data1-v2.xlsxFigure 3—figure supplement 2 图3ー图补充资料2Download asset下载资产Open asset开放资产

Validation of changes identified by RNA-seq in independent biological samples.RNA-seq 在独立生物样本中识别变化的验证Independent biological samples were collected and tested for gene expression changes identified by RNA-Seq by qPCR. Three new samples for each strain used in the RNA-Seq experiment (wild type (N2), r…see more采集独立的生物标本,用 qPCR 方法检测 RNA-Seq 基因表达变化。在 RNA-Seq 实验中,每个菌株分别采用3个新样品(野生型(N2) ,r。.更多https://doi.org/10.7554/eLife.49158.015
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Figure 3—figure supplement 2qPCR validation of RNA seq results.

图3ー图补充2 qPCR 验证 RNA 序列分析结果。https://doi.org/10.7554/eLife.49158.018Download elife-49158-fig3-figsupp2-data1-v2.xlsxFigure 3—figure supplement 1 图3ー图补充1Download asset下载资产Open asset开放资产

Gene clusters and differentially represented GO terms identified by analysis of RNA-seq.利用 RNA-seq 分析鉴定基因簇和差异表达 GO 术语(a) Plot depicting the profiles of gene expression defining each cluster of differentially expressed genes. Most differentially expressed genes in raga-1(ok386)are not rescued by the neuronal raga-1… see more(a)绘制基因表达谱图,定义每一组差异表达基因。大多数 raga-1(ok386)的差异表达基因并不是由神经元 raga-1拯救的… 更多https://doi.org/10.7554/eLife.49158.014

Among the genes represented in Cluster 4 (decreased in raga-1, increased in rescue) are multiple insulin-like peptide genes (daf-28ins-6ins-26ins-30) including two, daf-28 and ins-6, that when overexpressed are sufficient to suppress the lifespan extension resulting from loss of chemosensory function (Artan et al., 2016) (Supplementary file 2). We verified increased expression of daf-28 and ins-6, as well as other identified changes in gene expression, in L4 animals rescued either by extrachromosomal array or by single copy transgene knock-in by qPCR (Figure 3c,dFigure 3—figure supplement 2). In adults, the levels of ins-6 expression induced by neuronal or somatic expression of raga-1are indistinguishable, suggesting its regulation is entirely neuronal (Figure 3—figure supplement 3). These data suggest that neuronal TORC1 might mediate systemic longevity cell nonautonomously via secretion of insulin-like peptides or other raga-1 dependent neuronal signals.

在集群4中表达的基因(raga-1减少,挽救中增加)是多个胰岛素样肽基因(daf-28,ins-6,ins-26,ins-30) ,包括两个 daf-28和 ins-6,当过度表达时足以抑制化学感受功能丧失导致的寿命延长(Artan 等人,2016)(补充文件2)。我们证实,在 L4动物中 daf-28和 ins-6的表达增加,以及其他已确定的基因表达变化,无论是通过染色体外基因芯片还是通过 qPCR 单拷贝转基因敲入(图3c,d,图3ー图2)获救。在成年人中,由神经元或体细胞表达的 raga-1诱导的 ins-6表达水平是无法区分的,这表明它的调节完全是神经元的(图3ー图补充3)。这些数据表明,神经元 TORC1可能通过分泌胰岛素样肽或其他 raga-1依赖性神经元信号介导系统性长寿细胞的非自主性活动。

Neuropeptides are released from dense core vesicles (DCVs) (Li, 2008). If neuronal RAGA-1 suppresses raga-1 longevity via expression of insulin-like peptides or other neuropeptide signals, we reasoned the lifespan of raga-1neuronal rescue worms might be de-repressed by blocking neuropeptide release. We utilized mutants for unc-64, a homolog of mammalian syntaxin, an essential plasma membrane receptor for DCV exocytosis to test whether impairing neuronal function in this way would block the ability of the neuronal extrachromosomal raga-1 array to rescue. Hypomorphic unc-64(e246) mutant animals are defective for dense core vesicle docking (Zhou et al., 2007) and remarkably completely remove suppression of raga-1 lifespan by neuronal RAGA-1. raga-1; unc-64 double mutant animals with the neuronal rescue array live more than 40% longer than raga-1 neuronal rescue animals (Figure 3e). The longevity effects by unc-64mutation on raga-1 neuronal rescue animals, combined with the RNA seq results, strongly suggest that neuronal TORC1 actively causes the release of neuropeptide signals to limit longevity cell nonautonomously.

神经肽从致密核心囊泡(DCVs)中释放(Li,2008)。如果神经元 RAGA-1通过表达胰岛素样肽或其他神经肽信号抑制 RAGA-1的寿命,我们推测 RAGA-1神经营救蠕虫的寿命可能通过阻断神经肽释放而解除。我们利用同源哺乳动物同细胞表面受体素 unc-64的突变体来检测神经元功能受损是否会阻碍染色体外基因 raga-1芯片的拯救能力。亚致密基因 unc-64(e246)突变体在致密核心囊泡对接中存在缺陷(Zhou 等人,2007) ,并且显著地消除了神经元 RAGA-1对 RAGA-1寿命的抑制。具有神经营救阵列的双突变动物比 raga-1神经营救动物的寿命长40% 以上(图3e)。Unc-64突变对 raga-1神经营救动物的长寿效应,结合 RNA seq 结果,强烈提示神经元 TORC1主动引起神经肽信号的释放,非自主地限制了细胞的长寿。

Neuronal RAGA-1 drives peripheral mitochondrial fragmentation in aging animals

神经元 RAGA-1对衰老动物线粒体外周细胞断裂的影响

Since genes linked to organelle organization were upregulated in the raga-1mutant but not in rescue, we explored whether this might be causal to longevity of the raga-1 mutants.

由于与细胞器结构相关的基因在 raga-1突变体中被上调而不是被拯救,我们探讨了这是否可能是导致 raga-1突变体长寿的原因。

Mitochondria can dynamically move between fused and fragmented networks in response to changes in the cellular environment, mediated in C. elegans by the GTPases: FZO-1 (fusion) and DRP-1 (fission) (Wai and Langer, 2016). We examined mitochondrial networks in young and old C. elegans in multiple tissues using reporters expressing GFP fused with a fragment from the mitochondrial outer membrane protein TOMM-20, which includes a transmembrane domain that anchors the fusion protein to mitochondria (Weir et al., 2017). For neurons and muscle, we developed an ImageJ/FIJI macro, ‘MitoMAPR’, to characterize and quantify changes in mitochondrial architecture and morphology in C. elegans cells. MitoMAPR uses several pre-existing image enhancement filters to augment the signal clarity of images, converting the mitochondrial signals into skeleton-like binary backbones (Figure 4—figure supplement 1), and quantifying various attributes of the mitochondrial network as described in (Figure 4—figure supplement 2). Coupled with a high-throughput batch processing mode, this macro allows us to characterize changes in mitochondrial object length, distribution, network coverage and complexity in a large number of images acquired from the samples in question (Figure 4Figure 4—figure supplement 3Figure 4—figure supplement 5). Age induces fragmentation of mitochondria in neurons, intestine, and muscle, in wild type animals but degeneration is not seen in long lived raga-1 mutants (Figure 4Figure 4—figure supplement 3Figure 4—figure supplement 4Figure 4—figure supplement 5).

线粒体可以动态地在融合和破碎的网络之间移动,以响应细胞环境的变化,在秀丽隐杆线虫中通过 GTPases 介导: FZO-1(融合)和 DRP-1(裂变)(Wai 和 Langer,2016)。我们使用报告表达 GFP 融合于线粒体外层膜蛋白 TOMM-20片段的方法,检测了年轻和年老的秀丽隐杆线虫在多个组织中的线粒体网络,其中包括一个将融合蛋白锚定到线粒体的跨膜结构域。对于神经元和肌肉,我们开发了一个 ImageJ/FIJI 宏,“ MitoMAPR” ,用于描述和量化线虫细胞线粒体结构和形态的变化。MitoMAPR 使用几种已有的图像增强滤波器来增强图像的信号清晰度,将线粒体信号转换成骨架状的二进制骨架(图4ー图补充1) ,并对线粒体网络的各种属性进行量化(图4ー图补充2)。与高通量批处理模式相结合,这个宏允许我们描述线粒体对象长度、分布、网络覆盖范围和复杂性的变化,这些变化是从有关样本中获得的(图4,图4ー图3,图4ー图补充,图5)。年龄引起野生型动物神经元、肠道和肌肉中线粒体的断裂,但在长寿命 raga-1突变体中未见变性(图4,图4ー图补充3,图4ー图补充4,图4ー图补充5)。Figure 4 图4 with 6 supplements 还有6种补充剂Download asset下载资产Open asset开放资产

Neuronal RAGA-1 drives mitochondrial fragmentation in muscle cells.神经元 RAGA-1促进肌细胞线粒体断裂(a) Representative pictures showing that loss of raga-1 preserves muscle mitochondrial content during aging, while neuronal RAGA-1 reverses these effects as seen in the corresponding skeletonized … see more(a)具有代表性的图片显示,RAGA-1的缺失在衰老过程中保留了肌肉线粒体的含量,而神经元 RAGA-1则逆转了这些影响,如相应的骨骼化图片所示… 见更多https://doi.org/10.7554/eLife.49158.020
Figure 4—source data 1
图4ー源数据1

Figure 4c-h and Figure 4—figure supplement 5 Effects of neuronal raga-1 rescue on parameters of muscle mitochondrial morphology.

图4c-h 和图4ー图补充5神经元 raga-1拯救对肌肉线粒体形态参数的影响。https://doi.org/10.7554/eLife.49158.027Download elife-49158-fig4-data1-v2.xlsxFigure 4—figure supplement 6 图4ー图补充6Download asset下载资产Open asset开放资产

Effects of the unc-64hypomorphic allele on muscle mitochondria morphology.Unc-64等位基因对肌肉线粒体形态学的影响(a) Representative pictures showing that unc-64(e246)mutants preserve mitochondrial morphology during aging. The mitochondrial backbone (red) is overlaid on binary images. TOMM-20aa1-49::GFP … see more(a)有代表性的图片显示 unc-64(e246)突变体在衰老过程中保持线粒体形态。线粒体骨干(红色)覆盖在二进制图像上。TOMM-20aa1-49: : GFP… 见更多https://doi.org/10.7554/eLife.49158.026
Figure 4—figure supplement 6—source data 1
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Figure 4—figure supplement 6c-fMitochondria network characteristics of muscle mitochondria in unc-64mutants.

图4ー图补充6c-f 突变体肌肉线粒体网络特征。https://doi.org/10.7554/eLife.49158.030Download elife-49158-fig4-figsupp6-data1-v2.xlsxFigure 4—figure supplement 5 图4ー图补充5Download asset下载资产Open asset开放资产

Neuronal raga-1expression alters muscle mitochondrial architecture.神经元 raga-1表达改变了肌肉线粒体结构Quantification showing that neuronal raga-1rescue animals also have decreased network count (a) and number of junction points (b) indicating that the mitochondrial architecture in these animals are … see more量化显示神经元 raga-1救援动物也减少了网络计数(a)和连接点的数量(b) ,表明这些动物的线粒体结构是… 见更多https://doi.org/10.7554/eLife.49158.025

Figure 4—figure supplement 4 图4ー图补充4Download asset下载资产Open asset开放资产

raga-1deletion prevents mitochondria fragmentation in intestine.Raga-1基因缺失可防止肠道线粒体破裂(a) Mitochondrial architecture in the intestine can be categorized manually into fragmented, intermediate and fused network states. (b) Representative pictures showing that loss of raga-1preserves … see more(a)肠道中的线粒体结构可以人工分为片段、中间和融合的网络状态。(b)有代表性的图片显示拉加 -1号保护区的损失… 见更多https://doi.org/10.7554/eLife.49158.024
Figure 4—figure supplement 4—source data 1
图4ー图补充4ー源数据1

Figure 4—figure supplement 4cNetwork states of intestinal mitochondria in raga-1mutants.

图4ー图补充4c raga-1突变体肠线粒体的网络状态。https://doi.org/10.7554/eLife.49158.029Download elife-49158-fig4-figsupp4-data1-v2.xlsxFigure 4—figure supplement 3 图4ー图补充3Download asset下载资产Open asset开放资产

raga-1deletion affects mitochondrial network states in neurons.Raga-1缺失影响神经元的线粒体网络状态(a–b) Representative pictures (a) showing that loss of raga-1preserves mitochondrial content during aging, in neurons as seen in the overlay images (Left) post MitoMAPR processing. The … see more(a-b)具有代表性的图片(a)显示 raga-1的缺失在衰老过程中保留了线粒体的含量,在神经元中可以看到覆盖图像(左) MitoMAPR 处理后。更多信息https://doi.org/10.7554/eLife.49158.023
Figure 4—figure supplement 3—source data 1
图4ー图补充3ー源数据1

Figure 4—figure supplement 3c-fEffects of neuronal raga-1rescue on parameters of neuronal mitochondrial morphology.

图4ー图补充3c-f 神经元 raga-1营救对神经元线粒体形态参数的影响。https://doi.org/10.7554/eLife.49158.028Download elife-49158-fig4-figsupp3-data1-v2.xlsxFigure 4—figure supplement 2 图4ー图补充资料2Download asset下载资产Open asset开放资产

Examples of networks analyzed by MitoMAPR.MitoMAPR 分析的网络实例Using Networks (N) and Junction Point (JP) values, it is possible to estimate the degree of complexity of the mitochondrial network. As illustrated the four cells (A–D) have their mitochondria … see more利用网络(n)和结点(JP)值,可以估计线粒体网络的复杂程度。如图所示,这四个细胞(a-d)有自己的线粒体https://doi.org/10.7554/eLife.49158.022

Figure 4—figure supplement 1 图4ー图补充1Download asset下载资产Open asset开放资产

Workflow for MitoMAPR analysis.MitoMAPR 分析工作流程A Region of Interest (15 × 15 um) was processed by the MitoMAPR macro. The ROI is enhanced for signal intensity by Enhance Local Contrast (CLAHE) followed by conversion to Binary. The binary image … see more利用 MitoMAPR 宏处理感兴趣区域(15 × 15 um) 。通过增强局部对比度(CLAHE) ,然后转换为二进制,提高了感兴趣区域的信号强度。二进制图像… 请看更多https://doi.org/10.7554/eLife.49158.021

To test whether mitochondria in peripheral tissues might be affected by signals generated by neuronal TORC1 activity cell nonautonomously, we examined mitochondrial morphology in body wall muscle in wild type, raga-1 mutant and raga-1 neuronal rescue animals (Figure 4a,b). Mitochondrial networks in wild type muscle cells showed decreased total coverage (measured by percentage of cell area covered by mitochondria) (Figure 4c) and size (measured by length and area of each mitochondria particle) with age (Figure 4d,e). Further suggesting an increase in fragmentation of mitochondria in WT muscle cells with age as seen previously (Weir et al., 2017), we saw an increased object number normalized to area (Figure 4f). Strikingly, loss of raga-1 attenuates both the age-related decline in mitochondrial coverage and the increase in fragmentation (Figure 4c,d,e,f). Interestingly, the mitochondrial architecture in raga-1mutants contains fewer networks and junction points compared to WT at all ages tested, suggesting that mitochondria are longer but less interconnected (Figure 4—figure supplement 5). Next, we examined the impact of neuronal raga-1 on mitochondria in muscle cells. raga-1 neuronal rescue suppressed the effect of raga-1 deletion on muscle mitochondria in both young and old animals, causing reduced coverage, decreased size and a higher degree of fragmentation (Figure 4c,d,e,f). Further, network counts in raga-1 neuronal rescue animals are not significantly different to wild type in young or old animals (Figure 4—figure supplement 5). Together, these data suggest that loss of raga-1 specifically in neurons can maintain youthful mitochondrial network states with age in non-neuronal tissues.

为了检测神经元 TORC1活性细胞产生的信号是否会非自主地影响外周组织中的线粒体,我们检测了野生型、 raga-1突变型和 raga-1神经元拯救动物体壁肌肉中的线粒体形态(图4a,b)。野生型肌肉细胞中的线粒体网络显示,随着年龄的增长,线粒体覆盖的总面积(以被线粒体覆盖的细胞面积百分比衡量)和大小(以每个线粒体粒子的长度和面积衡量)减少(图4d,e)。进一步表明,随着年龄的增长,WT 肌肉细胞中线粒体的碎片化程度增加,正如前面所见(Weir 等人,2017年) ,我们看到物体数目增加,归一化为面积(图4f)。引人注目的是,raga-1的缺失既减少了与年龄相关的线粒体覆盖率的下降,也减少了线粒体碎片化的增加(图4c,d,e,f)。有趣的是,在所有测试年龄段中,raga-1突变体的线粒体结构包含较少的网络和连接点,这表明线粒体较长但相互联系较少(图4ー图5)。接下来,我们研究了神经元 raga-1对肌肉细胞线粒体的影响。Raga-1神经元拯救抑制了 raga-1缺失对年轻和老年动物肌肉线粒体的影响,导致覆盖率降低、体积减小和更高程度的破碎(图4c,d,e,f)。此外,raga-1神经营救动物的网络计数与野生型年轻或老年动物的网络计数没有显著差异(图4ー图补充5)。总之,这些数据表明,raga-1特异性在神经元中的缺失可以随着年龄的增长在非神经元组织中维持年轻的线粒体网络状态。

Next, we examined the effect of loss of UNC-64/syntaxin function, which de-represses the effects of neuronal TORC1 on longevity, on muscle mitochondria (Figure 4—figure supplement 6a,b). In young unc-64 mutants, mitochondria coverage and area were decreased in muscle cells and the networks show a higher degree of fragmentation compared to wild type. However, in unc-64 mutants, unlike for WT, neither mitochondrial coverage nor area show significant alterations with age (Figure 4—figure supplement 6c,d,e,f). Taken together, our data suggest that neuronal signaling driven by TORC1 can cell nonautonomously drive mitochondrial fragmentation in peripheral tissues.

接下来,我们研究了 UNC-64/syntaxin 功能的丧失对肌肉线粒体的影响(图4ー图补充6a,b) ,该功能可以去抑制神经元 TORC1对长寿的影响。在年轻的 unc-64突变体中,肌肉细胞线粒体的覆盖率和面积减少,网络的破碎程度高于野生型。不同于 WT 的是,unc-64突变体的线粒体覆盖率和面积均不随年龄的增长而显著变化(图4ー图6c,d,e,f)。综上所述,我们的数据表明 TORC1所驱动的神经元信号可以不自主地驱动周围组织中的线粒体碎片。

raga-1 deletion specifically requires a fused mitochondrial network to promote longevity

Raga-1缺失特别需要融合的线粒体网络来延长寿命

We sought to determine whether the changes we observed in mitochondrial network state were causally associated with raga-1 longevity. First, we asked whether raga-1 mutant animals require a fused mitochondrial network to extend lifespan. We crossed the raga-1(ok386) mutants with animals carrying a null allele of fzo-1, which encodes a protein orthologous to mammalian mitofusins. fzo-1(tm1133) mutant animals are therefore defective in mitochondrial fusion and consequently have fragmented mitochondria (Breckenridge et al., 2008Ichishita et al., 2008). fzo-1(tm1133) significantly suppresses the lifespan extension seen in raga-1 mutants, which suggests that mitochondrial fusion is required for raga-1 mediated longevity (Figure 5a). Mutations in fzo-1 suppress the long lifespan both with and without the use of FUDR (Figure 5—figure supplement 1). Deleting the C. elegans dynamin-related protein 1 (DRP-1), which is required for mitochondrial fission, does not block raga-1(ok386) longevity (Figure 5b), indicating that raga-1 longevity specifically requires mitochondria fusion. In addition to the previous data on the effects of neuronal CRTC-1, these data emphasize how AMPK and TORC1 modulate aging in C. elegans by distinct mechanisms: AMPK longevity requires both fusion and fission (Weir et al., 2017), while lifespan extension by in raga-1 mutants specifically requires mitochondrial fusion.

我们试图确定我们观察到的线粒体网络状态的变化是否与 raga-1的长寿有因果关系。首先,我们询问 raga-1突变的动物是否需要一个融合的线粒体网络来延长寿命。我们将 raga-1(ok386)突变体与携带 fzo-1缺失等位基因的动物杂交,fzo-1能编码与哺乳动物细胞分裂素直系同源的蛋白。Fzo-1(tm1133)突变的动物因此在线粒体融合方面存在缺陷,从而使线粒体碎片化(布雷肯里奇等人,2008; Ichishita 等人,2008)。Fzo-1(tm1133)显著抑制了 raga-1突变体的寿命延长,这表明线粒体融合是 raga-1介导的长寿所必需的(图5a)。Fzo-1的突变抑制使用 FUDR 和不使用 FUDR 的长寿命(图5ー图1)。删除秀丽隐杆线虫动力相关蛋白1(DRP-1) ,这是需要的线粒体分裂,并不阻止 raga-1(ok386)寿命(图5 b) ,表明 raga-1的寿命特别需要线粒体融合。除了以前关于神经元 CRTC-1影响的数据,这些数据强调 AMPK 和 TORC1如何通过不同的机制调节线虫的衰老: AMPK 的寿命需要融合和裂变(Weir 等人,2017年) ,而 raga-1突变体的寿命延长特别需要线粒体融合。Figure 5 图5 with 1 supplement 还有一种补充剂Download asset下载资产Open asset开放资产

raga-1 deletion requires a fused mitochondrial network to promote longevity.Raga-1缺失需要一个融合的线粒体网络来延长寿命(afzo-1(tm1133) raga-1(ok386) double mutants, which are deficient in mitochondrial fusion, have significantly shortened lifespan compared to raga-1(ok386) single mutants (p<0.0001). However, raga-1… see more(a)线粒体融合缺陷的 fzo-1(tm1133) raga-1(ok386)双突变体与单突变体相比,寿命明显缩短(p < 0.0001)。然而,raga-1… 看到更多https://doi.org/10.7554/eLife.49158.031

Figure 5—figure supplement 1 图5ー图补充1Download asset下载资产Open asset开放资产

fzo-1(tm1133)suppresses extended lifespan of raga-1(ok386)without the use of FUDR.Fzo-1(tm1133)在不使用 FUDR 的情况下抑制 raga-1(ok386)的延长寿命The lifespan of fzo-1(tm1133) raga-1(ok386)double mutant is not longer than control animals (p=0.8252). n = 2 independent biological replicates; sample sizes range between 109–153 deaths per … see moreFzo-1(tm1133) raga-1(ok386)双突变体的寿命不长于对照动物(p = 0.8252)。N = 2个独立的生物复制品; 样本大小在每109-153人死亡之间… 更多https://doi.org/10.7554/eLife.49158.032

We examined which tissues require fused mitochondrial networks to facilitate lifespan extension via raga-1 deletion. We restored mitochondrial fusion specifically in neurons in fzo-1(tm1133) mutants via rescue of fzo-1 cDNA with a pan-neuronal rab-3 promoter. This neuronal rescue failed to restore lifespan extension by loss of raga-1 (Figure 5c). Similarly, expression of fzo-1 in muscles failed to restore lifespan extension (Figure 5d), suggesting that mitochondrial fusion in neither neurons nor muscle underpin lifespan extension by TORC1 suppression. In contrast however, restoring mitochondrial fusion in intestine by expressing fzo-1 in the fzo-1(tm1133) mutants enabled raga-1deletion to significantly extend lifespan (Figure 5e). These data support the hypothesis that neuronal RAGA-1 activity modulates lifespan via cell nonautonomous regulation of mitochondrial dynamics in distal tissues.

我们研究了哪些组织需要融合的线粒体网络,以促进寿命延长通过 raga-1缺失。我们通过拯救含有泛神经元 rab-3启动子的 fzo-1 cDNA,在 fzo-1(tm1133)突变体中特异性地恢复了线粒体融合。这种神经元拯救没有通过失去 raga-1来恢复寿命延长(图5c)。类似地,fzo-1在肌肉中的表达未能恢复寿命延长(图5d) ,这表明线粒体融合在神经元和肌肉中并不通过 TORC1的抑制来促进寿命延长。然而,相反,通过在 fzo-1(tm1133)突变体中表达 fzo-1,恢复肠道线粒体融合,使 raga-1缺失显著延长寿命(图5e)。这些数据支持的假设,神经元 RAGA-1活动调节寿命通过细胞非自主调节线粒体动态远端组织。

Neuronal RAGA-1 suppresses lifespan cell nonautonomously via mitochondrial fission

神经元 RAGA-1通过线粒体分裂非自主抑制寿命细胞

Finally, having shown that mitochondrial fusion is required for lifespan extension in raga-1(ok386) mutants, we tested whether neuronal rescue of raga-1suppresses lifespan via promoting peripheral mitochondrial fragmentation. We generated animals carrying three perturbations: null mutation in raga-1, rescue of raga-1 in neurons, and null mutation of drp-1, which we reasoned would block the ability of neuronal TORC1 to induce mitochondrial fragmentation in peripheral tissues. Notably, driving mitochondrial fusion by drp-1 fully restores longevity in neuronal raga-1 rescue animals to that of single raga-1mutants (Figure 5f). These data suggest that indeed, neuronal TORC1 modulates lifespan via cell nonautonomous effects on mitochondrial dynamics. In summary, our results highlight a critical role of neuronal TORC1 activity on healthy aging, and suggest that lifespan extension by reduced TORC1 signaling requires a fused mitochondrial network which itself can be modulated via TORC1 activity in neurons.

最后,在证明了 raga-1(ok386)突变体延长寿命需要线粒体融合之后,我们测试了 raga-1的神经元拯救是否通过促进外周线粒体断裂来抑制寿命。我们生成的动物进行了三个方面的干扰: raga-1基因的缺失突变、神经元的 raga-1基因的缺失突变和 drp-1基因的缺失突变,我们推测这可能阻断神经元 TORC1诱导外周组织线粒体断裂的能力。值得注意的是,通过 drp-1促进线粒体融合,神经元 raga-1拯救动物的寿命完全恢复到单个 raga-1突变体的寿命(图5f)。这些数据表明,神经元 TORC1确实通过细胞对线粒体动力学的非自主影响来调节寿命。总而言之,我们的研究结果强调了神经元 TORC1活性在健康老化中的关键作用,并提出通过减少 TORC1信号延长寿命需要一个融合的线粒体网络,这个网络本身可以通过神经元中的 TORC1活性来调节。Discussion讨论

Taken together, our data suggest that AMPK and TORC1 can coordinate in neurons to modulate systemic organismal aging cell nonautonomously. Although primarily AMPK is regarded as an upstream suppressor of TORC1, our data suggest that for aging, lifespan extension via suppression of TORC1 activates and requires functional AMPK in neurons. However, lifespan extension by TORC1 suppression and AMPK activation in C elegans differ in two key aspects. First, expression of CRTC-1S76A, S179A in neurons completely suppresses AMPK longevity but has little effect on raga-1 mutant lifespan. Second, whereas AMPK longevity requires both mitochondrial fusion and fission mechanisms to be functional (Weir et al., 2017), lifespan extension by raga-1 deletion only requires mitochondrial fusion. Therefore, although both AMPK and TORC1 can modulate longevity and mitochondria dynamics cell nonautonomously in C. elegans, both the origin of those neuronal signals and the functional role mitochondria play in responding to them appear to differ.

综上所述,我们的研究结果表明 AMPK 和 TORC1在神经元中可以协同作用,非自主地调节系统性器官衰老细胞。尽管 AMPK 主要被认为是 TORC1的上游抑制因子,但我们的数据表明,对于衰老而言,通过 TORC1的抑制延长寿命,需要神经元中有功能的 AMPK 激活。然而,通过 TORC1抑制和 AMPK 激活延长秀丽隐杆线虫寿命在两个关键方面存在差异。首先,CRTC-1S76A、 S179A 在神经元中的表达完全抑制 AMPK 的寿命,但对 raga-1突变体的寿命影响不大。其次,虽然 AMPK 的长寿需要线粒体融合和分裂机制的功能(Weir 等人,2017年) ,寿命延长 raga-1缺失只需要线粒体融合。因此,虽然 AMPK 和 TORC1都可以非自主地调节线虫的寿命和线粒体动力学细胞,但这些神经元信号的来源和线粒体对它们的反应功能似乎有所不同。

Many of the described experiments used FUDR, a chemical inhibitor of thymidine synthesis, to block the production of progeny. While the actions of FUDR in postmitotic adult C. elegans are thought to act primarily in the germline to inhibit DNA replication in the dividing germ cells, there is also evidence that FUDR can act in somatic tissue and can lead to complex interactions with various genes and treatments in the regulation of lifespan (Anderson et al., 2016). As such, we chose a relatively small dose (40 μM) to use for our experiments, and importantly, verified key findings in lifespan experiments that did not use FUDR.

许多上述实验使用 FUDR,一种胸腺嘧啶合成的化学抑制剂,来阻止后代的产生。虽然 FUDR 在有丝分裂后成年线虫中的作用被认为主要是在种系中抑制分裂生殖细胞中的 DNA 复制,但也有证据表明 FUDR 可以在体细胞组织中起作用,并且可以导致与各种基因和寿命调控治疗的复杂互动(Anderson 等人,2016)。因此,我们选择了一个相对较小的剂量(40微米)用于我们的实验,而且重要的是,验证了在没有使用 FUDR 的寿命实验中的关键发现。

Beyond requiring LKB1/PAR-4, how TORC1 suppression activates AMPK in C. elegansremains unclear. S6K inhibits AMPK in mouse hypothalamus via S485 (or 491 depending on the isoform) (Dagon et al., 2012). Although our data suggest the conserved serine 551 residue is an inhibitory phosphorylation site in C. elegans, phosphorylation of AAK-2 S551 is not required for TORC1 mediated longevity. Several additional mechanisms have been recently identified that modulate AMPK activity, including an increasing number of post-translational modifications of the α subunit, expression levels of γ subunits and recruitment of LKB1 to lysosomal surface by the scaffold protein AXIN (Hardie, 2014Tullet et al., 2014Zhang et al., 2016). It remains to be tested whether these mechanisms are utilized by TORC1 to modulate AMPK in neurons, and whether they are relevant in the context of aging. Interestingly, rsks-1 mutants require the creatine kinase, ARGK-1, which is primarily expressed in glial cells, to activate AMPK (McQuary et al., 2016). S6K1 has also been shown to inhibit AMPK in the hypothalamus in mice (Dagon et al., 2012). Together these data support a hypothesis that AMPK mediates TORC1 longevity in neurons.

除了需要 LKB1/PAR-4之外,TORC1抑制如何激活线虫中的 AMPK 还不清楚。S6K 抑制 AMPK 在小鼠下丘脑通过 S485(或491取决于异构体)(大衮等人,2012)。虽然我们的数据表明,保守的丝氨酸551残基是一个抑制秀丽隐杆线虫磷酸化位点,磷酸化的 AAK-2 S551是不需要的 TORC1介导的长寿。最近又发现了一些其他的调控 AMPK 活性的机制,包括越来越多的 α 亚基翻译后修饰、 γ 亚单位表达水平和 LKB1在溶酶体表面的补充,由骨架蛋白 AXIN 研究(Hardie,2014; Tullet et al. ,2014; Zhang et al. ,2016)。这些机制是否被 TORC1利用来调节神经元中的 AMPK,以及它们是否与衰老有关还有待检验。有趣的是,rsks-1突变体需要肌酸激酶 ARGK-1激活 AMPK (McQuary 等人,2016) ,该蛋白主要在神经胶质细胞中表达。S6K1也被证明可以抑制小鼠下丘脑中的 AMPK (大衮等人,2012)。这些数据共同支持了 AMPK 介导神经元 TORC1长寿的假设。

While AMPK has been shown to act in neurons to promote longevity (Burkewitz et al., 2015Ulgherait et al., 2014), one highlight of our study is that TORC1 itself has critical functions in neurons to modulate lifespan. It is especially striking that when TORC1 signaling is suppressed in all the major metabolically active tissues in C. elegans but active in neurons, animals are not long lived. raga-1 or rsks-1 neuronal rescued animals share non-aging related phenotypes with raga-1 or rsks-1 mutants, such as smaller body size, delayed development and reduced brood size (data not shown). These results suggest that neuronal TORC1 might modulate lifespan through a specific mechanism that is uncoupled from the broad effects of TORC1 on growth and anabolism, for example via its regulation of insulin-like or other neuropeptides that act systemically to regulate longevity. Understanding where and how RAGA-1 acts in the nervous system and whether suppressing TORC1 signaling only in neurons either genetically or pharmacologically is sufficient to promote healthy aging is now a key future goal.

虽然 AMPK 已被证明在神经元中起到延年益寿的作用(Burkewitz 等人,2015; Ulgherait 等人,2014) ,我们研究的一个亮点是 TORC1本身在神经元中有调节寿命的关键功能。尤其引人注目的是,当 TORC1信号在秀丽隐杆线虫所有主要代谢活跃组织中被抑制,而在神经元中被激活时,动物的寿命并不长。Raga-1或 rsks-1神经元获救动物与 raga-1或 rsks-1突变体具有非衰老相关的表型,如体型较小、发育延迟和育雏体积减小(数据未显示)。这些结果表明,神经元 TORC1可能通过一个特定的机制来调节寿命,这个机制与 TORC1对生长和合成代谢的广泛影响是分离的,例如通过它对胰岛素样或其他神经肽的调节作用来全面调节寿命。了解 RAGA-1在神经系统中的作用位置和作用方式,以及无论从遗传学还是药理学上抑制神经元中的 TORC1信号是否足以促进健康衰老,现在是一个关键的未来目标。

Our data suggest that neuropeptide signals may act downstream of neuronal TORC1 to communicate with peripheral tissues. Indeed, we identified two insulin-like peptides, INS-6 and DAF-28, that are regulated by neuronal expression of raga-1. Both peptides have been shown to be regulated by food cues, and interestingly, when overexpressed are sufficient to suppress the long lifespan caused by loss of sensory signaling through the TAX-2/TAX-4 cyclic nucleotide gated channel (Artan et al., 2016). Whether these or other neuropeptides mediate the effects of neuronal TORC1 signaling on lifespan remains to be explored. With recent findings that neuropeptides influence aging in mammals (Riera et al., 2014), our study provides a critical starting point to investigate the identity and regulation of the neuropeptides and/or neurotransmitters that directly modulate TORC1 longevity.

我们的数据表明,神经肽信号可能作用于神经元 TORC1的下游,与外周组织进行通信。事实上,我们确定了两个胰岛素样肽,INS-6和 DAF-28,它们受到神经元表达 raga-1的调节。这两种肽已经被证明是由食物线索调节的,有趣的是,当过度表达足以抑制通过 TAX-2/TAX-4环核苷酸门控通道的感觉信号丢失导致的长寿命(Artan 等人,2016)。这些神经肽或其他神经肽是否介导神经元 TORC1信号通路对寿命的影响还有待于进一步研究。最近的研究发现神经肽影响哺乳动物的衰老(Riera et al. ,2014) ,我们的研究提供了一个关键的起点,调查身份和调节神经肽和/或神经递质,直接调节 TORC1的寿命。

Rapamycin suppresses TORC1 and alleviates a plethora of age-related pathologies and functional decline in mice, slowing age-related degenerative or neoplastic changes in liver, endometrium, heart and bone marrow (Chen et al., 2009Flynn et al., 2013Wilkinson et al., 2012). Moreover, short term rapamycin administration has significant anti-aging effects (Bitto et al., 2016). In addition, metformin, an anti-diabetic drug which both activates AMPK and inhibits TORC1 via AMPK-dependent and -independent mechanisms (Howell et al., 2017Kalender et al., 2010), extends lifespan in nematodes and mice (Martin-Montalvo et al., 2013Onken and Driscoll, 2010). The critical site of action for the anti-aging effects of rapamycin and metformin remain unknown. Our findings raise the exciting possibility that their effects on life- and healthspan could be mediated through their regulation of TORC1 and AMPK in the nervous system.

雷帕霉素抑制 TORC1并减轻老年相关疾病和老鼠功能下降,减缓与年龄相关的肝脏、子宫内膜、心脏和骨髓的退行性或肿瘤性改变(Chen 等人,2009; Flynn 等人,2013; Wilkinson 等人,2012)。此外,短期雷帕霉素给药具有显著的抗衰老作用(Bitto et al. ,2016)。此外,二甲双胍,一种抗糖尿病药物,通过 AMPK 依赖和非依赖机制激活 AMPK 并抑制 TORC1(Howell 等人,2017; Kalender 等人,2010) ,延长线虫和小鼠的寿命(Martin-Montalvo 等人,2013; Onken 和 Driscoll,2010)。雷帕霉素和二甲双胍抗衰老作用的关键部位仍然未知。我们的发现提出了一个令人兴奋的可能性,即它们对生命和健康的影响可以通过调节神经系统中的 TORC1和 AMPK 来调节。

Finally, we identified mitochondria as the downstream ‘receivers’ of TORC1 mediated neuronal signals important to directly influence lifespan. Mitochondria are major organelles for energy and intermediate metabolite production, with critical roles in the aging process (López-Lluch, 2017). Our finding that raga-1 mutant animals have hyperfused mitochondria is consistent with the in vitro mitochondrial hyperfusion induced by starvation or mTORC1 inhibition, which potentially allows more efficient ATP production and prevents healthy mitochondria from being degraded by mitophagy (Gomes et al., 2011). We further show that driving mitochondrial fission blocks the longevity of raga-1mutants, and driving fusion de-represses longevity in the presence of neuronal RAGA-1. Our results therefore unravel two critical aspects regarding the role of mitochondrial fusion in TORC1-mediated longevity: first, mitochondrial fission in peripheral tissues can be driven in a cell nonautonomous manner by neuronal RAGA-1; second, mitochondrial fusion is causally linked to raga-1longevity.

最后,我们确定线粒体是 TORC1介导的神经信号的下游接收者,这些信号直接影响寿命。线粒体是能量和中间代谢产物的主要细胞器,在衰老过程中起着关键作用(López-Lluch,2017)。我们发现 raga-1突变动物具有高灌注的线粒体,这与体外饥饿或 mTORC1抑制所诱导的线粒体低灌注相一致,这可能允许更有效的 ATP 产生,并防止健康的线粒体被线粒体降解(Gomes 等人,2011)。我们进一步证明,驱动线粒体分裂阻断了 RAGA-1突变体的寿命,并且在神经元 RAGA-1存在的情况下驱动融合去抑制寿命。因此,我们的结果阐明了关于线粒体融合在 torc1介导的长寿中的作用的两个关键方面: 第一,神经元 RAGA-1可以以非自治的方式驱动外周组织中的线粒体分裂; 第二,线粒体融合与 RAGA-1长寿有因果关系。

These and other recent work highlight how mitochondrial fission and fusion influence aging in a context-dependent manner: several longevity interventions require fusion (Chaudhari and Kipreos, 2017), while promoting fission can also extend lifespan in Drosophila (Rana et al., 2017), and dietary restriction and AMPK mediated longevity in C. elegans require both fusion and fission (Weir et al., 2017). Comparative analysis of the functional roles different mitochondrial network states play in AMPK and TORC1 longevity provide a unique opportunity to dissect out how mitochondrial networks might be modulated to promote healthy aging. Together, our data emphasize the role of TORC1 in the nervous system in modulating whole body metabolism and longevity. Further studies will help to elucidate the molecular identities of the neuronal signals and periphery receptors that underlie neuronal TORC1 activity to influence aging, and whether neuronal TORC1 might modulate aging in organisms beyond C. elegans.

这些和其他最近的工作强调了线粒体分裂和融合是如何以一种依赖环境的方式影响老化的: 一些长寿干预需要融合(Chaudhari 和 Kipreos,2017年) ,同时促进裂变也可以延长寿命果蝇(Rana 等人,2017年) ,饮食限制和 AMPK 介导的长寿线虫需要融合和裂变(Weir 等人,2017年)。比较分析不同的线粒体网络状态在 AMPK 和 TORC1长寿中的功能作用,可以提供一个独特的机会来剖析线粒体网络如何被调节以促进健康老龄化。总之,我们的数据强调了 TORC1在神经系统中调节全身新陈代谢和长寿的作用。进一步的研究将有助于阐明神经元 TORC1活动影响衰老的神经元信号和外围受体的分子特性,以及神经元 TORC1是否可能调节秀丽隐杆线虫之外的生物体的衰老。Materials and methods材料和方法Key resources table 关键资源表

Reagent type试剂类型
(species) or resource (物种)或资源
Designation名称Source or reference 来源或参考资料Identifiers 标识符Additional 附加
information 信息
Strain 紧张
(Caenorhabditis elegans 秀丽隐桿线虫)
N2Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# N2_(ancestral), RRID: WB Cat # N2 _ (祖先) ,RRID:WB-STRAIN:N2_(ancestral)WB-STRAIN: N2 _ (祖先)Laboratory reference strain 实验室参考菌株
Strain ( 应变(C. elegans 线虫属)VC222Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# VC222, RRID: 222,RRID:WB-STRAIN:VC222 WB-STRAIN: VC222Genotype: 基因型:raga-1(ok386) II.
Strain 紧张
(C. elegans 线虫属)
RB754Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# RB754, RRID: WB Cat # RB754,RRID:WB-STRAIN:RB754 菌株: RB754Genotype: 基因型:aak-2(ok524) X.
Strain ( 应变(C. elegans 线虫属)WBM997This study 这项研究Genotype: 基因型:aak-2(wbm20) X.
Strain 紧张
(C. elegans 线虫属)
RB1206Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# RB1206, RRID: WB Cat # RB1206,RRID:WB-STRAIN:RB1206 菌株: RB1206Genotype: 基因型:rsks-1(ok1255) III.
Strain ( 应变(C. elegans 线虫属)WBM536536This study 这项研究Genotype: 基因型:wbmEx238[rab-3p::raga-1 cDNA::SL2::mCherry::unc-54 3’UTR] 238[ rab-3p: : raga-1 cDNA: : SL2: : mCherry: : : unc-543‘ UTR ]
Strain ( 应变(C. elegans 线虫属)WBM772772This study 这项研究Genotype: 基因型:wbmEx333 [rab-3p::rsks-1 cDNA::SL2::mCherry::unc-54 3’UTR] wbmEx333[ rab-3p: : rsks-1 cDNA: : SL2: : mCherry: : : unc-543‘ UTR ]
Strain ( 应变(C. elegans 线虫属)WBM11671167This study 这项研究Genotype: 基因型:wbmIs79[eft-3p::3XFLAG::raga-1::SL2::wrmScarlet::unc-54 3’UTR, *wbmIs67] 2: : wrmScarlet: : unc-543‘ UTR,* wbmIs67]
Strain ( 应变(C. elegans 线虫属)WBM11681168This study 这项研究Genotype: 基因型:wbmIs80[rab-3p::3XFLAG::raga-1::SL2::wrmScarlet::rab-3 3’UTR, *wbmIs68] 80[ rab-3p: : 3XFLAG: : raga-1: : SL2: : wrmScarlet: : rab-3‘ UTR,* wbmIs68]
Strain 紧张
(C. elegans 线虫属)
WBM650This study 这项研究Genotype: 基因型:wbmEx271 [ges-1p::raga-1 cDNA::SL2::mCherry::unc-54 3’UTR; rol-6 (su1006)] wbmEx271[ ges-1p: : raga-1 cDNA: : SL2: : mCherry: : unc-543‘ UTR; rol-6(su1006)]
Strain ( 应变(C. elegans 线虫属)WBM671PMID:29107506Genotype: 基因型:wbmEx289 [myo-3p::tomm20 aa1-49::GFP::unc54 3’UTR] wbmEx289[ myo-3p: : tomm20 aa1-49: : GFP: : unc543‘ UTR ]
Strain ( 应变(C. elegans 线虫属)WBM955955This study 这项研究Genotype: 基因型:
wbmEx373 [rab-3p::tomm-20 aa1-49::GFP::unc-54 3’UTR, rol-6] wbmEx373[ rab-3p: : tomm-20 aa1-49: : GFP: : unc-543‘ UTR,rol-6]
Strain ( 应变(C. elegans 线虫属)WBM926926PMID:29107506Genotype: 基因型:wbmEx367[ges-1p::tomm20 aa1-49::GFP::unc-54 3’UTR] wbmEx367[ ges-1p: : tomm20 aa1-49: : GFP: : unc-543‘ UTR ]
Strain ( 应变(C. elegans 线虫属)CU5991Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# CU5991, RRID: WB Cat # CU5991,RRID:WB-STRAIN:CU5991 菌株: CU5991Genotype: 基因型:fzo-1 (tm1133) II. Fzo-1(tm1133) II
Strain ( 应变(C. elegans 线虫属)CU63726372Caenorhabditis Genetics Center 昆虫遗传学研究中心WB Cat# CU6372, RRID: WB Cat # CU6372,RRID:WB-STRAIN:CU6372 菌株: CU6372Genotype: 基因型:drp-1(tm1108) IV.
Strain ( 应变(C. elegans 线虫属)WBM861861This study 这项研究Genotype: 基因型:fzo-1(tm1133) II; wbmEx335 [rab-3p:3xFLAG fzo-1 cDNA: unc54 3’UTR, myo-3p:mCherry] Fzo-1(tm1133) II; wbmEx335[ rab-3p: 3xFLAG fzo-1 cDNA: unc543‘ UTR,myo-3p: mCherry ]
Strain ( 应变(C. elegans 线虫属)WBM612612PMID:29107506Genotype: 基因型:fzo-1 (tm1133) II; wbmEx258 [pHW11 (myo-3p::3xFLAG::fzo-1 cDNA::unc-54 3’UTR) + pRF4 (rol-6(SU1006))] Fzo-1(tm1133) II; wbmEx258[ pHW11(myo-3p: : 3xFLAG: : fzo-1 cDNA: : : unc-543‘ UTR) + pRF4(rol-6(SU1006)]
Strain 紧张
(C. elegans 线虫属)
WBM639639PMID:29107506Genotype: 基因型:fzo-1(tm1133) II; wbmEx276 [pHW18 (ges-1p::3xFLAG::fzo-1 Fzo-1(tm1133) II; wbmEx276[ pHW18(ges-1p: : 3xFLAG: : fzo-1 
cDNA::unc54 3’UTR) + cDNA: unc543’ UTR) + 
pRF4(rol-6(SU1006))]
Antibody 抗体Phospho-AMPKα (Thr172) antibody 磷酸化 ampkα (Thr172)抗体Cell Signaling Technology 细胞信号技术Cat# 2535, RRID: 2535,RRID:AB_331250331250
Antibody 抗体Beta actin antibody β 肌动蛋白抗体Abcam 女名女子名Cat# ab8226, RRID: 8226,RRID:AB_306371306371
Software 软件MitoMAPR 米特/欧洲/美国This study 这项研究Source code provided as源代码提供作为Source code 1 源代码1
Commercial 商界
assay or kit 化验或试剂盒
TruSeq Stranded mRNA LT – Set A kit TruSeq 受困 mRNA LT-Set a kitIllumina Illumina 公司RS-122–2101 RS-122-2101

Worm strains

蠕虫菌株

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Worms were grown at 20°C on nematode growth media (NGM) plates seeded with E. coli strain OP50-1(CGC) with standard techniques (Brenner, 1974). Information for all strains used is in Supplementary file 4.

在线虫生长介质(NGM)板上,用标准技术接种大肠杆菌 OP50-1(CGC) ,在20 ° c 温度下培养蠕虫(Brenner,1974)。所有菌株的信息都在补充文件4中。

RNAi feeding

Rna 干扰饲养

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Feeding RNAi clones were obtained from the Ahringer or Vidal RNAi libraries and sequence-verified before using. To use, bacteria were grown overnight in LB broth with 100 μg/mL carbenicillin and 12.5 μg/mL tetracycline, seeded on NGM plates with 100 μg/mL carbenicillin (NG Carb) and allowed 48 hr to grow at room temperature. At least 4 hr before use, 0.1M IPTG solution with 100 μg/mL carbenicillin and 12.5 μg/mL tetracycline was added to the bacterial lawn to induce dsRNA expression.

利用 Ahringer 或 Vidal RNAi 文库获得饲用 rna 干扰克隆,并在使用前进行序列验证。用100μg/mL 的卡本西林和12.5 μg/ml 的四环素在 LB 培养液中培养一夜,接种于100μg/mL 卡本西林(NG Carb)的 NGM 培养板上,室温下培养48h。在使用前至少4小时,将0.1 m IPTG 溶液加入100 μg/ml 的卡本西林和12.5 μg/ml 的四环素,诱导 dsRNA 的表达。

Lifespan experiments

寿命实验

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All worms were kept fed for at least two generations on OP50-1 bacteria. Before the start of each lifespan experiment, gravid adult worms were bleached and eggs were fed HT115 bacteria until adulthood to either start the lifespan experiment or a timed egg lay to obtain synchronized populations. Day 1 of lifespan marks the onset of egg laying. In the cases where FUDR is used, plates were seeded with bacteria, allowed 24 hr to grow and 100 μl of 1 mg/mL FUDR solution was seeded on top of the bacteria lawn for each plate containing 10 mL NGM (for a final concentration of 40 μM). FUDR was allowed 24 hr to diffuse to the whole plate before plates were used. When combining FUDR with RNAi treatments, to overcome potential inhibition of FUDR on dsRNA expression in bacteria, plates were induced with IPTG solution 18 hr after seeding; FUDR was applied 24 hr after seeding; IPTG was applied again 4 hr before use.

用 OP50-1细菌饲喂所有蠕虫至少两代。在每个寿命实验开始之前,对妊娠成虫进行漂白,并在成年前喂食 HT115细菌卵,以便开始寿命实验或定时产卵以获得同步种群。寿命的第一天标志着产卵的开始。在使用 FUDR 的情况下,在细菌培养板上接种细菌,生长24小时,在细菌草坪上接种100μl 1mg/mL FUDR 溶液,每个培养板含10ml NGM (最终浓度为40μM)。在使用钢板前,FUDR 可以扩散到整个钢板24小时。结合 FUDR 和 RNAi 处理,克服 FUDR 对 dsRNA 表达的潜在抑制作用,接种后18小时用 IPTG 溶液诱导平板,接种后24小时用 FUDR,接种前4小时再用 IPTG。

Western blots

西部污染指数

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More than 500 day one adults were used for each sample. Worms were collected in M9 buffer with 0.01% Tween-20 and washed three times in M9. Liquids were removed after centrifugation and samples were frozen in liquid nitrogen. For worm lysis, RIPA buffer containing protease inhibitors (Sigma, MI, USA #8340) and phosphatase inhibitors (Roche, Basel, Switzerland #4906845001) was added to each sample at the same volume as the worm pellet. Worms were lysed via sonication (Qsonica, CT, USA Q700). Protein concentration was measured using Pierce BCA protein assay kit (Thermo Fisher Scientific, MA, USA PI23227) following manufacturer’s instructions. To denature the proteins, 5X RSB was added and samples were heated to 95°C for 5 min. Samples containing 20–30 μg protein were loaded to 10% Tris-Glycine gels (Thermo Fisher Scientific #XP00100) for SDS-PAGE. Proteins were transferred to PVDF membranes (Thermo Fisher Scientific, #LC2005) and blocked with 5% BSA in TBST. Primary antibodies and dilutions are: phosphor-AMPK (Cell signaling, MA, USA #2535) 1:1000; beta actin (Abcam, Cambridge, UK #8226) 1:1000. Antibody signals were developed using ECL Western Blotting Detection Reagent (GE Healthcare, IL, USA Catalog number: 95038–560) and bands were quantified with Gel Doc system (Bio Rad) and Image Lab software (Version 4.1).

每个样本使用500多天一个成年人。在0.01% 吐温 -20的 M9缓冲液中采集蠕虫,在 M9中洗涤3次。离心后去除液体,样品在液氮中冷冻。对于蠕虫裂解,在每个样品中加入含有蛋白酶抑制剂(Sigma,MI,USA # 8340)和磷酸酶抑制剂(Roche,Basel,Switzerland # 4906845001)的 RIPA 缓冲液,体积与蠕虫裂解液相同。蠕虫经超声裂解(Qsonica,CT,美国 Q700)。蛋白质浓度测量使用 Pierce BCA 蛋白质检测试剂盒(Thermo Fisher Scientific,MA,USA PI23227)按照制造商的说明书。为了使蛋白质变性,加入5X RSB,并将样品加热到95 ° c 5min。含20-30 μg 蛋白质的样品用10% Tris-Glycine 凝胶(Thermo Fisher Scientific # xp00100)进行 SDS-PAGE 分析。蛋白质转移到聚偏氟乙烯膜(Thermo Fisher Scientific,# lc2005) ,并在 TBST 中被5% BSA 封闭。主要的抗体和稀释剂是: phosphor-AMPK (Cell signal,MA,USA # 2535)1:1000; beta actin (Abcam,Cambridge,UK # 8226)1:1000。抗体信号用 ECL 西方墨点法检测试剂(GE Healthcare,IL,USA Catalog number: 95038-560)开发,条带用 Gel Doc 系统(Bio Rad)和 Image Lab 软件(Version 4.1)量化。

Genotyping of deletion alleles

缺失等位基因的基因分型

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Worms were individually lysed in single worm lysis buffer and lysates were used as templates for PCR reactions with a combination of 2–3 primers that will produce bands of different sizes for wild type and mutant alleles. Primers and PCR conditions for each deletion allele are listed in Supplementary file 5.

在单一的蠕虫裂解液中对蠕虫进行单独裂解,并以裂解物为模板进行 PCR 反应,通过2-3引物的组合,产生不同大小的野生型和突变型等位基因带。引物和 PCR 条件为每个删除等位基因列在补充文件5。

Generation of transgenes

转基因的产生

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To generate transgenic animals expressing raga-1 in neurons, raga-1 cDNA was PCR amplified and cloned using standard techniques into a plasmid where a 3X FLAG tag was added to the N terminus and a gpd-2 SL2 sequence with mCherry ORF was added between raga-1 stop codon and unc-54 3’UTR. rab-3 promoter was subsequently PCR amplified and inserted. To express wild type and mutated forms of aak-2, both 3 kb promoter region before the aak-2 gene and the 6.7 kb coding region were amplified from N2 genomic DNA and cloned into a plasmid, where a 3X FLAG tag and unc-54 3’UTR were added to the C terminus. Serine-to-alanine mutation was generated using QuikChange II XL Site-directed Mutagenesis Kit (Agilent Technologies, CA, USA 200522) following manufacturer’s instructions. Transgenic strains were generated via microinjection. Detailed information on strains used is in Supplementary file 4.

为了获得表达 raga-1的转基因动物,利用标准技术将 raga-1 cDNA 扩增并克隆到质粒中,在质粒的 n 端加入3X FLAG 标签,在 raga-1终止密码子和 unc-543’ UTR 之间加入含 mCherry ORF 的 gpd-2 SL2序列。利用 PCR 扩增和插入 rab-3启动子。为了表达 aak-2基因的野生型和突变型,从 N2基因组 DNA 中扩增 aak-2基因前3kb 启动子区和6.7 kb 编码区,并将其克隆到一个质粒中,在 c 端加入3X FLAG 标签和 unc-543’ UTR。按照制造商的说明,使用 QuikChange II XL 定点突变试剂盒(安捷伦科技有限公司,加利福尼亚州,美国200522)产生丝氨酸到丙氨酸的突变。通过显微注射获得转基因菌株。关于所用菌株的详细信息见补充文件4。

Generation of single copy raga-1 transgene knock-in strains

单拷贝 raga-1转基因敲入菌株的建立

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Strains WBM1167 N2, wbmIs79[eft-3p::3XFLAG::raga-1::SL2::wrmScarlet::unc-54 3’UTR, *wbmIs67] and WBM1168 N2, wbmIs80[rab-3p::3XFLAG::raga-1::SL2::wrmScarlet::rab-3 3’UTR, *wbmIs68] were generated by CRISPR according to Silva-García et al. (2019). Specifically, a homology repair template (HR) containing raga-1::SL2 sequences was amplified from plasmid pYZ30 using primers

据 crispra silva 等人(2019年)介绍,菌株 WBM1167 N2,wbmIs79[ eft-3p: : 3XFLAG: : : raga-1: : SL2: : wrmScarlet: : : unc-543‘ UTR,* wbmIs67]和 WBM1168 N2,wbmIs80[ rab-3p: : 3XFLAG: : raga-1: : SL2: : : wrmScarlet: rab-3‘ UTR,* wbmIs68]均由 crispra 生成。利用引物从质粒 pYZ30中扩增出一个含有 raga-1: : SL2序列的同源修复模板(HR)

‘5’ TATAAAGATCATGACATCGATTACAAGGATGACGAcGAtAAGTCTT CAAAACGAAAAGTT’

5’ tatagatatcatcatacatcacatcacatcacaggatacacgatgatgatacgatagtctt caaaacgaagtt

and ‘

和’

5’ AACGCATGAACTCCTTGATAACTGCCTCTCCCTTGCTGACC ATGATGCGTTGAAGCAGTT’,

5’ aacgcatactcttgatactctctctctctctctctctctctctctctctctctctctctcgacc atgatcgttgaagcagtt’ ,

followed by a second round of amplification using ‘5’ CCGGGATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGG and ‘5’ CGTGTCCGTTCATGGATCCCTCCATGTGGACCTTGAAACGCATGAACTCCTTGATA’ to extend the HR arms. A CRISPR mix containing the raga-1::SL2 HR template, and a 3x flag 3’ crRNA [5’ TTACAAGGATGACGATGACA 3’] was prepared according to Paix et al. (2015)Silva-García et al. (2019) and injected into strains WBM1143 and WBM1144 (Silva-García et al., 2019). Resulting CRISPR edited alleles, wbmIs79 and wbmIs80, were outcrossed 6 and 5 times to N2 respectively to generate strains WBM1167 and WBM1168. To introduce wbmIs79 and wbmIs80 single copy transgenes into a raga-1 mutant background the strains were crossed into WBM499 (an outcrossed raga-1(ok386) allele) and genotyped for the presence of the transgene by the visible expression of wrmScarlet, and for presence of the ok386 deletion by PCR, using primers 5’ TTCAAGTCCGAAACAGTCAATTCTC and 5’ GGAACTGAAGCGATCACACCGAC. raga-1 rescue strains are WBM1169 raga-1 (ok386) II; wbmIs79[eft-3p::3XFLAG::raga-1::SL2::wrmScarlet::unc-54 3’UTR, *wbmIs67] and WBM1170 raga-1 (ok386) II; wbmIs80[rab-3p::3XFLAG::raga-1::SL2::wrmScarlet::rab-3 3’UTR, *wbmIs68].

然后进行第二轮扩增,使用‘5’ cgggggatactacaaagacacacattgatgatcggatcacacacatcacagg 和‘5’ cggtctctctctctctctctctctcatcatcatcatgatgatgatgatgatcatcatcatcatgatgatgatgatgatgatgatgatgatgatgatcatcatcatcatactccccccctctctctctctctctgatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgattctctcatgatgatgatgatgatgatgatgatgatgatgatgatgatgatgat。根据 Paix 等人(2015年) ; Silva-García 等人(2019年)制备了含有 raga-1: : SL2 HR 模板和3倍标记3’ crRNA [5’ ttacaggatgacgatgaca 3’]的 CRISPR 混合物,并注射到菌株 WBM1143和 WBM1144中(Silva-García 等人,2019年)。分别将编码 CRISPR 的等位基因 wbmIs79和 wbmIs80与 N2杂交6次和5次,得到菌株 WBM1167和 WBM1168。为了将 wbmIs79和 wbmIs80单拷贝转基因导入 raga-1突变体背景中,利用5’ ttcaccgaactattcctc 和5’ ggaacgatcaccgac 两个引物,将菌株与 WBM499(一个异交 raga-1(ok386)等位基因杂交,通过 wrmScarlet 可见表达检测转基因的存在,并利用 PCR 检测 ok386的缺失。Raga-1营救菌株分别是 wb1169 raga-1(ok386) II; wbmIs79[ rab-3p: : 3XFLAG: : raga-1: : SL2: wrmScarlet: : unc-543‘ UTR,* wbmIs67]和 WBM1170 raga-1(ok386) II; wbmIs80[ rab-3p: : 3XFLAG: raga-1: SL2: : wrmScarlet: rab-3‘‘ UTR,* wbmIs68]。

Determination of developmental rate

发育速度的测定

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Strains were fed and passaged for multiple generations on NG Carb plates seeded with HT1115 bacteria. At the start of the experiment, adult hermaphrodites were allowed to lay eggs for ~2 hr, after which, 50 eggs from each strain were picked to each of 2 new plates (100 eggs total) and left to develop at 20°C for 72–73 hr. The developmental stage was scored by eye under a light microscope on the basis of larval and adult stage specific hallmarks.

菌株在接种 HT1115菌的 NG 碳水化合物培养板上饲养传代数代。实验开始时,雌雄同体成虫产卵约2小时,然后每株50枚卵分别取到2个新的培养板上(共100枚卵) ,在20 °c 温度下发育72ー73小时。在光学显微镜下,根据幼虫和成虫阶段特有的标志,用眼睛记录发育阶段。

Comparison of body size

体型比较

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Strains were fed and passaged for multiple generations on NG Carb plates seeded with HT1115 bacteria. To synchronize animals, L4-staged larvae were picked to a new plate and aged for two days at 20°C. Day two adult animals were picked to a new plate and anaesthetized in a drop of 0.4 mg/mL tetramisole for ~15 mins. Once the animals stopped moving, they were aligned into groups according to genotype, and imaged with an Axiocam camera on a Zeiss Discovery V8 dissection microscope.

菌株在接种 HT1115菌的 NG 碳水化合物培养板上饲养传代数代。为了使动物同步化,l4阶段的幼虫被挑选到一个新的平板上,在20 ° c 的温度下放置2天。第二天将两只成年动物取出,置于新盘中,滴入0.4毫克/毫升四咪唑,麻醉15分钟。一旦动物停止移动,它们就根据基因型排列成组,并在蔡司发现 V8解剖显微镜上用 Axiocam 摄像机成像。

Microscopy

显微镜

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Worms of desired stage/age were anesthetized in 0.5 mg/mL tetramisole (Sigma, T1512) diluted in M9 and mounted to 2% agarose pads. For imaging of the mitochondrial TOMM-20 reporter in the muscle, images were taken using a Zeiss Imager.M2 microscope. Apotome optical sectioning was used to acquire fluorescence and one picture with best focus was chosen for each worm for quantification (as described in Weir et al., 2017). For imaging of mitochondria in neurons and in intestine, images were taken in the Sabri Ulker imaging lab using a Yokogawa CSU-X1 spinning disk confocal system (Andor Technology, South Windsor, CT, USA) combined with a Nikon Ti-E inverted microscope (Nikon Instruments, Melville, NY, USA). Images were taken using a 100x/1.45 oil Plan Apo objective lens, Zyla cMOS (Zyla 4.2 Plus USB3) camera and 488 nm Laser for GFP. Optical slice thickness was 0.2 µm. NIS elements software was used for acquisition parameters, shutters, filter positions and focus control. For images shown of the intestine, images were taken as a z stack and each plane was then threaded together by concatenation of the stack. These concatenated stacks were then rendered into 3d by the 3d viewer function of FIJI.

所需阶段/年龄的蠕虫用0.5 mg/mL 四咪唑(Sigma,T1512)稀释于 M9中麻醉,并安装在2% 琼脂糖垫上。为了对肌肉中的线粒体 TOMM-20报告者进行成像,图像是用蔡司成像仪 m2显微镜拍摄的。光学切片被用来获得荧光,并且为每个蠕虫选择了一张最佳焦点的图片进行量化(见 Weir 等人2017年的文章)。在 Sabri Ulker 成像实验室,我们使用横河 CSU-X1旋转圆盘共聚焦系统(Andor Technology,South Windsor,CT,USA)和尼康 Ti-E 倒置显微镜(Nikon Instruments,Melville,NY,USA)对神经元和肠道中的线粒体进行成像。采用100x/1.45 oil Plan Apo 物镜、 Zyla cMOS (Zyla 4.2 Plus USB3)相机和 GFP 的488nm 激光拍摄。光学薄片厚度为0.2 μm。NIS 元素软件用于获取参数,百叶窗,过滤器位置和焦点控制。对于所显示的肠道图像,将图像作为 z 堆栈进行拍摄,然后通过堆栈的连接将每个平面螺纹连接在一起。然后,FIJI 的3 d 查看器函数将这些连接起来的栈渲染为3 d。

C. elegans mitochondrial analysis using MitoMAPR

线虫线粒体的 MitoMAPR 分析

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The images were analysed using a novel macro called MitoMAPR (Source code 1) in ImageJ or FIJI (Schindelin et al., 2012). Briefly, worm muscle cells and neurons were selected as ROIs, processed and filtered using the CLAHE plugin (Zuiderveld, 1994) with median filter and unsharp mask to increase the local contrast and particle distinctiveness. The ROI is then converted to a binary image to generate a 2D skeleton using the Skeletonize3D plugin (Lee et al., 1994). This skeleton image is then dissected using the AnalyzeSkeleton function (Arganda-Carreras et al., 2010) in FIJI to generate Tagged and Labelled skeletons. Labelled skeletons allow the program to count distinct mitochondrial objects while the tagged skeleton provides information about junction points, branch counts and other pre-configured attributes. MitoMAPR uses the values obtained from the AnalyzeSkeleton function to quantify previously defined aspects of the mitochondrial network (Koopman et al., 2006). The workflow of the macro is illustrated in Figure 4—figure supplement 1. Analysis was performed in batch mode using the MitoMAPR_Batch (Source code 1, part B) while keeping all the parameters constant. While the user is required to select a region of interest (ROI) in case of images processed singly, MitoMAPR_Batch imports the saved ROIs generated while cropping the images for cells. A separate macro called CropR (Source code file 1, part C) was written to select and crop large data sets in batch mode. The cropped dataset is then used as a batch input for MitoMAPR_Batch. The codes for the macros can be found as supplementary notes.

图像分析使用一个新的宏称为 MitoMAPR (源代码1)在 ImageJ 或 FIJI (Schindelin 等人,2012年)。简要地说,蠕虫肌肉细胞和神经元被选为 ROIs,处理和过滤使用 CLAHE 插件(Zuiderveld,1994)与中值滤波器和非锐化掩膜,以增加局部对比度和粒子的明显性。然后将 ROI 转换为二进制图像,使用 Skeletonize3D 插件生成2D 骨架(Lee 等人,1994)。这个骨骼图像然后被解剖使用分析 keleton 功能(Arganda-Carreras 等人,2010年)在斐济生成标记和标记的骨骼。标记的骨架允许程序计算不同的线粒体对象,而标记的骨架提供关于连接点、分支计数和其他预先配置的属性的信息。MitoMAPR 使用从 AnalyzeSkeleton 函数获得的值来量化线粒体网络之前定义的方面(Koopman 等人,2006)。宏的工作流如图4ー图补充1所示。在保持所有参数不变的情况下,使用 MitoMAPR _ batch (源代码1,b 部分)以批处理模式进行分析。在单独处理图像时,用户需要选择感兴趣的区域(ROI) ,而 MitoMAPR _ batch 导入在为单元裁剪图像时生成的保存的 ROI。编写了一个单独的宏 CropR (源代码文件1,c 部分) ,以批处理模式选择和裁剪大型数据集。裁剪后的数据集用作 MitoMAPR _ batch 的批量输入。宏的代码可以作为补充说明找到。

The attributes used here to describe alterations in the mitochondrial architecture are listed in Supplementary file 7. Additionally, to determine the complexity of the mitochondrial network, we focused on the Network and Junction Point attribute. As illustrated in Figure 4—figure supplement 2, greater number of junction points in individual mitochondrial networks point towards higher complexity. The output data is kept as an array in a. CSV file that lists the values of all the above-mentioned attributes.

这里用来描述线粒体结构变化的属性列在补充文件7中。另外,为了确定线粒体网络的复杂性,我们重点研究了网络和连接点属性。如图4ー图补充2所示,单个线粒体网络中连接点的数量越多,其复杂性越高。输出数据以数组的形式保存在.CSV 文件中,该文件列出了上述所有属性的值。

RNA seq sample collection, RNA extraction and library preparation

RNA 标本采集、 RNA 提取及文库制备

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Worms were bleached to HT115 bacteria carrying L4440 vector. Synchronized populations were obtained via timed egg lay. four biological replicate samples were collected for each genotype. For each sample, an independent egg lay was performed and 1000 mid-L4 stage progeny were harvested. For the raga-1, rab-3p::raga-1::SL2::mCherry strain, only worms carrying the transgene were picked out to a new NGM plate using a fluorescent dissecting microscope for subsequent collection. For N2 and raga-1 mutant animals, worms were directly washed off the plates. M9 buffer with 0.01% Tween-20 were used to wash worms off the plates. Samples were centrifuged at 2,500 rpm to pellet the worms and washed three times with M9 buffer to remove bacteria. QIAzol lysis reagent (Qiagen, Venlo, Netherlands, #79306) was added to each sample before snap-freezing in liquid nitrogen. All samples were stored in −80°C freezer until RNA extraction.

将蠕虫漂白成携带 L4440载体的 HT115细菌。通过定时产卵获得同步种群。每个基因型收集4个生物复制样本。每个样本进行一个独立的产卵,并收获1000个 l4期中期后代。对于 raga-1,rab-3p: : raga-1: : SL2: : mCherry 菌株,只有携带转基因的蠕虫被挑选出来放在一个新的 NGM 平板上,用荧光解剖显微镜进行后续采集。对于 N2和 raga-1基因突变的动物,虫子被直接从培养皿中冲洗掉。用含0.01% 吐温 -20的 M9缓冲液冲洗虫体。样品在每分钟2500转的速度下离心,使蠕虫颗粒状,并用 M9缓冲液洗涤三次以去除细菌。在液氮中速冻前,每个样品加入恰佐裂解试剂(芬洛,# 79306)。所有样品均在 -80 °c 冰箱中冷冻至 RNA 提取。

To break the worm cuticle and improve RNA yield, all samples underwent five freeze-thaw cycles. In each cycle, samples were thawed at 37 ºC and then snap-frozen in liquid nitrogen. RNA extraction was performed immediately using QIAGEN RNeasy Mini Kit (QIAGEN, #74104) following manufacturer’s instructions. RNA quality was confirmed using Agilent Bioanalyzer 2100 (Agilent Technologies). All samples passed the quality control standard of RIN > 8.0. mRNA libraries were prepared using TruSeq Stranded mRNA LT – Set A kit (Illumina, CA, USA RS-122–2101) following manufacturer’s instructions and linked to different adaptors to enable pooling. Library quality was checked using 2200 High Sensitivity D1000 Tape Station (Agilent Technologies). Libraries were pooled and sequenced with Illumina HiSeq 2500 using 50-cycle, pair-end settings.

为了打破蠕虫表皮,提高 RNA 产量,所有样品都经历了五次冻融循环。在每个循环中,样品在37 °c 下解冻,然后在液氮中快速冻结。RNA 提取立即使用 QIAGEN RNeasy Mini Kit (QIAGEN,# 74104)按照制造商的说明。使用安捷伦生物分析仪2100(安捷伦科技有限公司)确认了 RNA 的质量。所有样品均通过 RIN > 8.0的质量控制标准。根据生产商的说明,使用 TruSeq 的受困 mRNA LT-Set a 试剂盒(Illumina,CA,USA RS-122-2101)制备了 mRNA 库,并与不同的适配器连接,以便集中使用。使用2200高灵敏度 D1000磁带站(安捷伦科技有限公司)检查了库的质量。库是汇集和测序与 Illumina HiSeq 2500使用50周期,对端设置。

RNA seq gene expression and functional analysis

RNA seq 基因表达与功能分析

Read processing and quantification

阅读处理和量化

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All samples were processed using an RNA-seq pipeline implemented in the bcbio-nextgen project (https://bcbio-nextgen.readthedocs.org/en/latest/). Raw reads were examined for quality issues using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) to ensure library generation and sequencing data were suitable for further analysis. Reads were aligned to the Ensembl94 build of the C. elegans genome using STAR (Dobin et al., 2013). Quality of alignments was assessed by checking for evenness of coverage, rRNA content, genomic context of alignments, complexity and other quality checks. Expression quantification was performed with Salmon (Patro et al., 2017) to identify transcript-level abundance estimates and then collapsed down to the gene-level using the R Bioconductor package tximport (Soneson et al., 2015).

所有样本都使用 bcbio-nextgen 项目( https://bcbio-nextgen.readthedocs.org/en/latest/)实施的 RNA-seq 管道进行处理。原始阅读是检查质量问题使用 FastQC ( http://www.bioinformatics.babraham.ac.uk/projects/FastQC/) ,以确保图书馆生成和测序数据适合进一步分析。该片段使用 STAR (Dobin 等人,2013)与秀丽线虫(C.elegans)基因组的 ensemblebl94构建对齐。通过检查覆盖均匀度、 rRNA 含量、比对的基因组背景、复杂性和其他质量检查来评估比对的质量。表达量化进行了与 Salmon (Patro 等人,2017年) ,以确定转录水平丰度估计,然后塌陷到基因水平使用 r 生物导体包装 tximport (Soneson 等人,2015年)。

Principal components analysis (PCA) and hierarchical clustering methods validated clustering of samples from the same sample group. Differential expression was performed at the gene level using the R Bioconductor package DESeq2 (Love et al., 2014). Differentially expressed genes were identified using the Likelihood Ratio Test (LRT) and significant genes were obtained using an FDR threshold of 0.01. Significant genes were separated into clusters based on similar expression profiles across the defined sample groups. Gene lists for each cluster was used as input to the R Bioconductor package clusterProfiler (Yu et al., 2012) to perform an over-representation analysis of Gene Ontology (GO) biological process terms. A secondary pairwise analysis was also performed for all pairs of sample groups using the Wald test.

主成分分析(PCA)和层次聚类方法验证了来自同一样本组的样本的聚类。用 r Bioconductor 软件包 DESeq2(Love et al. ,2014)在基因水平上进行差异表达。用似然比检验(LRT)鉴定差异表达基因,用0.01的 FDR 阈值获得重要基因。重要的基因被分离成簇的基础上相似的表达谱在所定义的样本群体。每个集群的基因列表被用作 r Bioconductor 包 clusterProfiler (Yu 等人,2012)的输入,以执行基因本体(GO)生物过程术语的过度表示分析。次要的配对分析也进行了所有对样本组使用瓦尔德试验。

Quantitative real-time PCR

实时荧光定量 PCR

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RNA was isolated from ~200 L4 staged (for validation of RNASeq in independent samples) or Day one adult (determination of raga-1 and ins-6 expression in adults) C. elegans samples using method described above. cDNA was synthesized from 30 μg of RNA with SuperScript VILO Master Mix (ThermoFisher Scientific, 11755050) following manufacturer’s instructions. 5 ng cDNA was used as template for each RT-PCR reaction. 2 or three independent biological replicates were used for each genotype/condition and always run in parallel with Taqman control probe to invariant control gene Y45F10D.4 (Ce02467253_g1) (Heintz et al., 2017), for normalization on a 96 well plate. RT-PCR was performed on the StepOne Plus qPCR Machine (Life Technologies, MA, USA) using Taqman Universal Master Mix II (Life Technologies, 4440040). Taqman probes used to target each gene of interest are as follows: Ce02445578_g1 (C28A5.2), Ce02484227_g1 (F35E12.5 (irg-5)), Ce02421566_m1 (Y39G10AR.6 (ugt-31)), Ce02488119_g1 (K10G4.5), Ce02433249_g1 (ZK84.6 (ins-6)), Ce02489787_g1 (Y116F11B.1 (daf-28)) and Ce02439068_g1 (raga-1). Relative expression levels were calculated using ΔΔCt method.

用上述方法分别从 ~ 200l4(用于独立样品 RNASeq 的验证)和第1天(用于成虫 raga-1和 ins-6表达的测定)样品中分离到 RNA。根据制造商的指示,用上标 VILO Master Mix (ThermoFisher Scientific,11755050)从30μg RNA 合成 cDNA。5ng 的 cDNA 为模板进行 RT-PCR 反应。每个基因型/条件采用2或3个独立的生物复制,始终与 Taqman 控制探针平行运行于不变控制基因 Y45F10D. 4(Ce02467253 _ g1)(Heintz 等,2017) ,在96孔板上进行归一化。RT-PCR 是在 StepOne Plus qPCR Machine (Life Technologies,MA,USA)上使用 Taqman Universal Master Mix II (Life Technologies,4440040)进行的。用于靶向每个相关基因的 Taqman 探针如下: Ce02445578 _ g1(C28A5.2) ,Ce02484227 _ g1(F35E12.5(irg-5)) ,Ce02421566 _ m1(Y39G10AR. 6(ugt-31)) ,Ce02488119 _ g1(K10G4.5) ,Ce02433249 _ g1(ZK84.6(ins-6)),ce02489787 _ g1(Y116F11B. 1(daf-28))和 Ce02439068 _ g1(raga-1)。用 δδct 方法计算相对表达水平。

Statistics and reproducibility

统计学和可重复性

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Statistical methods are indicated in the figure legends. Statistical tests were performed using Graphpad Prism versions 7.0b and 8.0 for Mac. No animals or samples were excluded from the analyses. For lifespan analyses, p values were calculated using Log-rank (Mantel-Cox) test. Data from all lifespan experiments are included in Supplementary file 6 without excluding any lifespan replicates. For qPCR, statistical significance was determined by two-tailed t-test unless otherwise indicated. Data are presented as mean ± s.e.m. P value: NS no significance, *<0.05, **<0.01, ***<0.001, ****<0.0001 relative to controls.

统计方法在图形图例中表示。统计测试使用 Graphpad Prism 版本7.0 b 和 Mac 版本8.0进行。分析中没有排除任何动物或样品。对于寿命分析,p 值是使用 Log-rank (Mantel-Cox)检验计算的。所有寿命试验的数据都包含在补充文件6中,不排除任何寿命复制。对于 qPCR,除非另有说明,否则统计学意义通过双尾 t 检验确定。数据以平均 ± s.e.m. p 值表示: NS 无显著性,* < 0.05,* * < 0.01,* * < 0.001,* * < 0.0001。

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