A motor neuron disease-associated mutation produces non-glycosylated Seipin that induces ER stress and apoptosis by inactivating SERCA2b

  1. Shunsuke Saito
  2. Tokiro Ishikawa
  3. Satoshi Ninagawa
  4. Tetsuya Okada
  5. Kazutoshi Mori

  • Curated by eLife

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    Seipin is a multifunctional Endoplasmic Reticulum localised protein associated with seemingly unrelated human diseases. Here the authors establish a correlation between the expression of a particular mutant form of Seipin associated in humans with motor neuron disease and altered intracellular calcium dynamics and allied proteotoxic stress. The paper is noted for the clues it provides into how these cellular defects arise and for offering a plausible, but yet unproven hypothesis for the cellular pathology that may account for the human disease phenotype.

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Abstract

A causal relationship between endoplasmic reticulum (ER) stress and the development of neurodegenerative diseases remains controversial. Here, we focused on Seipinopathy, a dominant motor neuron disease, based on the finding that its causal gene product, Seipin, is a protein that spans the ER membrane twice. Gain-of-function mutations of Seipin produce non-glycosylated Seipin (ngSeipin), which was previously shown to induce ER stress and apoptosis at both cell and mouse levels albeit with no clarified mechanism. We found that aggregation-prone ngSeipin dominantly inactivated SERCA2b, the major calcium pump in the ER, and decreased the calcium concentration in the ER, leading to ER stress and apoptosis in human colorectal carcinoma-derived cells (HCT116). This inactivation required oligomerization of ngSeipin and direct interaction of the C-terminus of ngSeipin with SERCA2b, and was observed in Seipin-deficient neuroblastoma (SH-SY5Y) cells expressing ngSeipin at an endogenous protein level. Our results thus provide a new direction to the controversy noted above.

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  1. Version published to 10.7554/elife.74805 on eLife
  2. eLife

    eLife assessment

    Seipin is a multifunctional Endoplasmic Reticulum localised protein associated with seemingly unrelated human diseases. Here the authors establish a correlation between the expression of a particular mutant form of Seipin associated in humans with motor neuron disease and altered intracellular calcium dynamics and allied proteotoxic stress. The paper is noted for the clues it provides into how these cellular defects arise and for offering a plausible, but yet unproven hypothesis for the cellular pathology that may account for the human disease phenotype.

  3. eLife

    Reviewer #1 (Public Review):

    This work characterizes at the mechanistic level the relationship between ER stress and the lack of glycosylation of two seipin mutants observed in seipinopathy, N152S and S154L (nomenclature of the long form of seipin). In short, the authors find that lack of glycosylation (ngSeipin) decreases ER calcium levels, and that it does so in an aggregation-dependent way, with no effect at low protein expression (or when oligomerization is impeded) and a significant effect (also leading to apoptosis) when high amounts of non-glycosylated seipin are expressed in the cell. The authors show that this causes ER stress, using BiP, XBP1 and CHOP as markers, and that this is attenuated when SERCA2b is overexpressed. They also identify the C-terminus of seipin as the region directly interacting with SERCA2b.

    The work is carefully described, with extensive controls, and the conclusions are supported by the data presented. In addition, the results have important consequences in several fields. First, they clarify the relationship between ER stress and nerurodegenerative diseases in general, and seipinopathy in particular. Second, by identifying the mechanism through which seipin and SERCA2b interact, they raise interesting mechanistic considerations about the relationship between lipid accumulation and calcium homeostasis; third, they hint at potential therapeutic approaches for ER-stress associated neurodegenerative diseases.

    Weaknesses

    The major weakness of this work is that it lacks an assessment of the relevance of the findings in vivo. This originates from two issues. First, the phenotype observed depends on the amount of non-glycosylated protein, and the investigation of the amount of protein in different cell types (especially neurons) is beyond the scope of this work. Also, the use of a double mutant (N152S, S154L) rather than of two single mutants (that are clinically relevant) makes a direct comparison with the pathological scenario quite difficult.

    In addition, the authors describe that in glycosylated seipin, deletion of the N-terminus and modification of the TM helices has a very large effect on ER calcium levels (Figure 6C), but no mechanistic explanation for this observation is provided.

  4. eLife

    Reviewer #2 (Public Review):

    The authors used a cell based system to investigate how expression of disease-associated Seipin glycosylation mutants (ngSeipin) impact on endoplasmic reticulum (ER) homeostasis. In particular, they focus their attention on SERCA, previously shown to interact genetically and biochemically with Seipin. They show that endogenous SERCA interacts with both overexpressed WT and ngSeipin. Using reporters monitoring calcium levels in the cytosol and ER lumen, it is shown that overexpression of ngSeipin (but not WT seipin) results in lower ER calcium levels, increase ER stress and eventually apoptosis. Based on the analysis of several Seipin mutants, the authors conclude that the toxicity of ngSeipin requires oligomerization (via the luminal domain) and the presence of its C-terminal domain. It is proposed that low ER calcium resulting from inhibition of SERCA by ngSeipin is a key event in Seipinopathies.

    Despite the large amount of data presented, these not always lay support to the main conclusions of the study. Critical flaws are:

    1- All conclusions are based on experiments where Seipin is overexpressed to levels are are unlikely to be physiological, even in the disease context. Importantly, as shown at several points (for example Figure 3), the effects of ngSeipin are drastically different depending of the expression levels.

    2- The conclusions about ngSeipin aggregation are unjustified. The PLA assay is not suitable to assess protein aggregation or to distinguish between aggregation and oligomerization.

    3- The effects of ngSeipin on UPR activation or calcium levels are modest, in particular considering that the levels to which it is overexpressed in relation to endogenous Seipin (see for example Figure 1Ec or 3Ac).

  5. eLife

    Reviewer #3 (Public Review):

    Here, Saito et al. studied the mechanism underlying Seipinopathy, a dominant motor neuron neurodegenerative disease, showing that non-glycosylated Seipin dominantly inactivates ER calcium pump SERCA2b and subsequently causes ER stress and apoptosis. Seipin is a key regulator of lipid metabolism and involves in the biogenesis of lipid droplets. This manuscript provides valuable insights into the role of non-glycosylated Seipin in ER calcium homeostasis and ER stress-induced apoptosis, which is important for a better understanding of the pathogenesis of Seipinopathy and the role of ER calcium in neurodegenerative diseases.

    1. The biochemical and genetic evidence from HCT116 cells showed in this manuscript strongly supports the function of non-glycosylated Seipin in ER stress and cell apoptosis by disrupting ER calcium homeostasis. However, a concern about this study is the colorectal carcinoma cell line HCT116 used. Neuron cell expresses a much higher level of Seipin than HCT116 cells. Although a higher level of non-glycosylated Seipin was expressed in HCT116 Seipin knockout cells to mimic the physiological level in neuron cells, whether non-glycosylated Seipin exhibits the same mechanism in neuron cells is still unclear. Further studies in neurons or cell lines with comparable Seipin level will help to understand its actual role in neurodegenerative disease.
    2. A higher level of non-glycosylated Seipin expression causes aggregates/clusters of Seipin on ER, as shown in Figure 1C. Since non-glycosylated Seipin physically interacts with SERCA2, it is important to know whether non-glycosylated Seipin expression changes the localization of calcium pump SERCA2b on ER.

  6. Version published to 10.1101/2021.10.22.465502v1 on bioRxiv