Dopamine neuron morphology and output are differentially controlled by mTORC1 and mTORC2

  1. Polina Kosillo
  2. Kamran M Ahmed
  3. Erin E Aisenberg
  4. Vasiliki Karalis
  5. Bradley M Roberts
  6. Stephanie J Cragg
  7. Helen S Bateup

  • Curated by eLife

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    Evaluation Summary:

    This manuscript by Kosillo and colleagues presents a series of carefully carried out experiments evaluating the impact of perturbing the mTORC1 and mTORC2 protein complexes selectively in mouse dopamine neurons. By utilizing dopamine neuron-specific Raptor and Rictor cKO mice, this paper elucidated which of these mTOR complexes are responsible for the regulation of dopamine neuronal functions, revealing the importance of mTORC1/2 signaling for the structure and function of dopamine neurons. This paper provided comprehensive data including structural, physiological, and biochemical alterations by genetic deletion of Raptor/Rictor in dopamine neurons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor , which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

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  1. Version published to 10.7554/elife.75398 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Kosillo et al. used dopamine neuron-specific cKO mice to examine the contributions of mTORC1 (Raptor cKO) and mTORC2 (Rictor cKO) to dopamine neuron dendrite and axon morphology, neuronal electrophysiological properties and dopamine release. Overall, Raptor cKO mice have stronger deficits as compared to Rictor cKO, while double cKO had additional deficits. These results suggest that mTORC1 is more critical to dopamine neuron function, and that there is some functional redundancy between mTORC1 and mTORC2.

    The data presented is generally of high quality, and the conclusions drawn are consistent with the data presented. I have the following concerns:

    1. Conclusion point 3: "mTORC2 inhibition leads to distinct cellular changes not observed following mTORC1 suppression, suggesting some independent actions of the two mTOR complexes in DA neurons." Currently, data supporting this conclusion is weak.

    We appreciate the reviewer’s comment. Although we do see some distinct cellular changes, we agree that these are minor and we have removed this sentence from the main conclusions paragraph in the discussion.

    Specifically, WT SNc DA neurons do not have typical morphology (very different from all other neurons, including WT neurons in Fig 1o), making the observed increase in proximal dendrite morphology hard to interpret.

    We have replaced the example images in Fig. 2m with better representative examples of WT SNc DA neurons

    Data presented in Figure 1o suggest no significant increase in total dendrite length. Are there changes in primary dendrite number?

    We do find that deletion of Rptor from SNc and VTA neurons causes a reduction in total dendrite length (new Fig. 1u,v), which is observable in the example images (Fig. 1o,p) showing shorter dendrites in DA-Raptor KO cells. We have now quantified primary dendrite number in both DA-Raptor and DA-Rictor KO neurons and find that there are no significant differences compared to control neurons. These new data have been added to Figures 1 and 2

    The electrophysiological results presented in Fig 5 are inconsistent with increased dendrite arborization. The authors need to either provide more evidence showing significant increase in dendrite morphology in Rictor cKO mice, or reinterpret their results.

    While we do find that DA-Rictor KO neurons have increased dendritic complexity at 50-100µm from the soma by Sholl analysis, total dendrite length is not significantly changed (new Fig. 2mt). Since the cell body size of Rictor cKO DA neurons is significantly reduced, we believe that this is responsible for the reduced membrane capacitance and slightly increased resistance observed in these cells (Fig. 5a,f,g)

    1. The manuscript is repetitive in some places, and the discussion largely reiterates the results. Could the authors please discuss why mTORC1 signaling contributes more to dopamine neuron function, as compared to mTORC2, based on existing knowledge of gene function and expression. Another point of interest is how the different parameters they measure are related, i.e. which parameters may be more causal than others in terms of changes in dopamine neuron function.

    We thank the reviewer for this suggestion. We have significantly revised the discussion section and added further discussion of mTORC1 and mTORC2 functions as they relate to DA neuron properties.

    Reviewer #3 (Public Review):

    In this paper, Kosillo et al. investigated the structural and functional alterations of dopamine neurons in dopamine neuron-specific Raptor and Rictor KO mice. Physiological functions and cellular structures were broadly and markedly affected in Raptor cKO mice, while Rictor cKO mice exhibited marginal changes, indicating that each adaptor protein of mTOR in either mTORC1 or mTORC2 may play both similar or distinct roles in the maintenance of dopaminergic structures and functions. Non-specific activation or inhibition of mTOR pathways in the previous literatures have hampered the understanding of molecular mechanisms behind the functions of mTOR pathways in dopamine neurons and related brain diseases. By utilizing dopamine neuron-specific Raptor and Rictor cKO mice, this paper elucidated which of these mTOR complexes are responsible for the regulation of dopamine neuronal functions, revealing the importance of mTORC1/2 signaling for the structure and function of dopamine neurons. Providing comprehensive data including structural, physiological, and biochemical alterations by genetic deletion of Raptor/Rictor in dopamine neurons is another strong point of this paper. However, lack of mechanistic evidence directly (or indirectly) linking the deletion of Raptor (or Rictor) to the alterations in TH/DAT/p-DAT/neuronal structures is a weak point of this manuscript. Overall, the conclusion of this paper is unbiased, just reflecting the data presented.

    We appreciate the reviewer’s positive comments on our manuscript. The goal of this study was to provide a thorough characterization of the effects of Raptor or Rictor loss on dopamine neuron properties. We acknowledge that while we have identified significant changes in dopamine neuron structure and function driven by mTORC1 deficiency, we have not yet probed the downstream mechanisms that may be responsible. We believe this is beyond the scope of the current manuscript but would be of interest for future studies.

  3. eLife

    Evaluation Summary:

    This manuscript by Kosillo and colleagues presents a series of carefully carried out experiments evaluating the impact of perturbing the mTORC1 and mTORC2 protein complexes selectively in mouse dopamine neurons. By utilizing dopamine neuron-specific Raptor and Rictor cKO mice, this paper elucidated which of these mTOR complexes are responsible for the regulation of dopamine neuronal functions, revealing the importance of mTORC1/2 signaling for the structure and function of dopamine neurons. This paper provided comprehensive data including structural, physiological, and biochemical alterations by genetic deletion of Raptor/Rictor in dopamine neurons.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Kosillo et al. used dopamine neuron-specific cKO mice to examine the contributions of mTORC1 (Raptor cKO) and mTORC2 (Rictor cKO) to dopamine neuron dendrite and axon morphology, neuronal electrophysiological properties and dopamine release. Overall, Raptor cKO mice have stronger deficits as compared to Rictor cKO, while double cKO had additional deficits. These results suggest that mTORC1 is more critical to dopamine neuron function, and that there is some functional redundancy between mTORC1 and mTORC2.

    The data presented is generally of high quality, and the conclusions drawn are consistent with the data presented. I have the following concerns:

    1. Conclusion point 3: "mTORC2 inhibition leads to distinct cellular changes not observed following mTORC1 suppression, suggesting some independent actions of the two mTOR complexes in DA neurons." Currently, data supporting this conclusion is weak. Specifically, WT SNc DA neurons do not have typical morphology (very different from all other neurons, including WT neurons in Fig 1o), making the observed increase in proximal dendrite morphology hard to interpret. Data presented in Figure 1o suggest no significant increase in total dendrite length. Are there changes in primary dendrite number? The electrophysiological results presented in Fig 5 are inconsistent with increased dendrite arborization. The authors need to either provide more evidence showing significant increase in dendrite morphology in Rictor cKO mice, or reinterpret their results.
    2. The manuscript is repetitive in some places, and the discussion largely reiterates the results. Could the authors please discuss why mTORC1 signaling contributes more to dopamine neuron function, as compared to mTORC2, based on existing knowledge of gene function and expression. Another point of interest is how the different parameters they measure are related, i.e. which parameters may be more causal than others in terms of changes in dopamine neuron function.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript by Kosillo and colleagues presents a series of carefully carried out experiments evaluating the impact of perturbing the mTORC1 and mTORC2 protein complexes selectively in mouse dopamine neurons. The work presents a substantial set of data and represents a significant advance in our understanding of the respective roles of these two protein complexes in dopamine neuron structure and function.

    Conceptually, it is not particularly surprising to see that in dopamine neurons, like in most other neuronal types, inhibition of these pathways and in particular of mTORC1 induces reduced morphological development and reduced neurotransmitter release.

    The value of the work is more in detailing the origin of this effect on dopamine release by showing that it is likely to be due to reduced dopamine synthesis and reduced axonal density. The work also adds to the literature by clarifying the respective contributions of the mTORC1 and mTORC2 pathways to the integrity and functions of SNc and VTA dopamine neurons.

    The authors conclude that there is no change in the number of dopamine neurons in the two lines of conditional KO mice. However, this is based on quantification of cell number in a very small subset of mice and using a technique this is not state of the art.

  6. eLife

    Reviewer #3 (Public Review):

    In this paper, Kosillo et al. investigated the structural and functional alterations of dopamine neurons in dopamine neuron-specific Raptor and Rictor KO mice. Physiological functions and cellular structures were broadly and markedly affected in Raptor cKO mice, while Rictor cKO mice exhibited marginal changes, indicating that each adaptor protein of mTOR in either mTORC1 or mTORC2 may play both similar or distinct roles in the maintenance of dopaminergic structures and functions. Non-specific activation or inhibition of mTOR pathways in the previous literatures have hampered the understanding of molecular mechanisms behind the functions of mTOR pathways in dopamine neurons and related brain diseases. By utilizing dopamine neuron-specific Raptor and Rictor cKO mice, this paper elucidated which of these mTOR complexes are responsible for the regulation of dopamine neuronal functions, revealing the importance of mTORC1/2 signaling for the structure and function of dopamine neurons. Providing comprehensive data including structural, physiological, and biochemical alterations by genetic deletion of Raptor/Rictor in dopamine neurons is another strong point of this paper. However, lack of mechanistic evidence directly (or indirectly) linking the deletion of Raptor (or Rictor) to the alterations in TH/DAT/p-DAT/neuronal structures is a weak point of this manuscript. Overall, the conclusion of this paper is unbiased, just reflecting the data presented.

  7. Version published to 10.1101/2021.11.07.467637v1 on bioRxiv