Subcellular proteomics of dopamine neurons in the mouse brain
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Evaluation Summary:
In this work, the authors provide a useful compendium of proteins labeled within SNpc dopaminergic cells using a novel approach. AAV virus was used to conditional express the APEX2 enzyme in dopaminergic neurons (based on DAT-1 Cre genetic technology) to rapidly biotinylate nearby proteins in oriented sections of brain whereby the striatonigral circuit can be spatially parsed for proteomic dissection. In addition to providing a useful new database of proteins for investigators interested in this circuit, the results also provide a more general approach to examining a compartment proteome in neurons and what might be expected in that analysis.
(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. The reviewers remained anonymous to the authors.)
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Abstract
Dopaminergic neurons modulate neural circuits and behaviors via dopamine (DA) release from expansive, long range axonal projections. The elaborate cytoarchitecture of these neurons is embedded within complex brain tissue, making it difficult to access the neuronal proteome using conventional methods. Here, we demonstrate APEX2 proximity labeling within genetically targeted neurons in the mouse brain, enabling subcellular proteomics with cell-type specificity. By combining APEX2 biotinylation with mass spectrometry, we mapped the somatodendritic and axonal proteomes of midbrain dopaminergic neurons. Our dataset reveals the proteomic architecture underlying proteostasis, axonal metabolism, and neurotransmission in these neurons. We find that most proteins encoded by DA neuron-enriched genes are localized within striatal dopaminergic axons, including ion channels with previously undescribed axonal localization. These proteomic datasets provide a resource for neuronal cell biology, and this approach can be readily adapted for study of other neural cell types.
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Evaluation Summary:
In this work, the authors provide a useful compendium of proteins labeled within SNpc dopaminergic cells using a novel approach. AAV virus was used to conditional express the APEX2 enzyme in dopaminergic neurons (based on DAT-1 Cre genetic technology) to rapidly biotinylate nearby proteins in oriented sections of brain whereby the striatonigral circuit can be spatially parsed for proteomic dissection. In addition to providing a useful new database of proteins for investigators interested in this circuit, the results also provide a more general approach to examining a compartment proteome in neurons and what might be expected in that analysis.
(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 …
Evaluation Summary:
In this work, the authors provide a useful compendium of proteins labeled within SNpc dopaminergic cells using a novel approach. AAV virus was used to conditional express the APEX2 enzyme in dopaminergic neurons (based on DAT-1 Cre genetic technology) to rapidly biotinylate nearby proteins in oriented sections of brain whereby the striatonigral circuit can be spatially parsed for proteomic dissection. In addition to providing a useful new database of proteins for investigators interested in this circuit, the results also provide a more general approach to examining a compartment proteome in neurons and what might be expected in that analysis.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The results provide a useful compendium of proteins IDed in SNpc dopaminergic cells using a very rigorous and powerful approach. In short, an engineered peroxidase that rapidly biotinylates nearby proteins is coupled to restricted expression in dopaminergic cells, particularly those dopaminergic cells previously transduced in the midbrain with AAV vectors. The spatial aspect to proximity IDs are the main strength, which is a well-known feature harnessed in many studies to understand proteomic compositions of particular circuits.
It is unclear how the different concentrations of the peroxidase factor into the relative abundance calculations of IDed proteins. Further, it would be useful to assess unexpected protein IDs at the synapse with an orthogonal measure instead of full reliance of peroxidase labeling, …
Reviewer #1 (Public Review):
The results provide a useful compendium of proteins IDed in SNpc dopaminergic cells using a very rigorous and powerful approach. In short, an engineered peroxidase that rapidly biotinylates nearby proteins is coupled to restricted expression in dopaminergic cells, particularly those dopaminergic cells previously transduced in the midbrain with AAV vectors. The spatial aspect to proximity IDs are the main strength, which is a well-known feature harnessed in many studies to understand proteomic compositions of particular circuits.
It is unclear how the different concentrations of the peroxidase factor into the relative abundance calculations of IDed proteins. Further, it would be useful to assess unexpected protein IDs at the synapse with an orthogonal measure instead of full reliance of peroxidase labeling, for example, with in situ analysis or immunohistochemistry. Finally, the associations with GWAS lists from Parkinson's disease with lists of compartment-restricted proteins are somewhat unconvincing and might benefit from a more rigorous analysis.
The strengths of the study include the in vivo catalogue of the cytosolic proteome of an important cell type using a suitable and current methodology. Most of the data is consistent with prior expectations, although there are some apparently postsynaptic proteins that are enriched in the synaptosomes that may represent more novel observations.
Set against these positives, there are three principal weaknesses. First, only dopamine neurons are evaluated. Hence, we do not know which observations are specific to this cell type vs being generic to other projection neurons. While we see the expected accumulation of dopamine synthesis and cytosolic handling, much of the rest of the catalogue would be expected in all neurons, including the accumulation of postsynaptic machinery in the axonal projection compartment. A particularly intriguing observation was the potential enrichment of autophagy machinery in the axonal proteome but we don't know if this is specific to DA neurons or more generally true, which impact both interpretability and level of novel insight.
Second, there is a lack of orthogonal approaches to validate or invalidate some of the major observations. This is particularly crucial in evaluating unexpected accumulation of postsynaptic proteins, for which the authors proffer up to four possible explanations including some that would appear to represent artifacts of the labeling procedure. Thus showing that some of these enrichments can be recapitulated in situ would strongly improve interpretation.
Third, the enrichment of PD genes and GWAS hits suffers from a lack of precision as to what is considered a true PD gene or not and how one considers those genes that are not enriched in the dataset. Here, again, the lack of comparator datasets is an omission as what we don't know is whether there would be a similar level of enrichment in any neuronal context - or what the non-neuronal proteome might be. On a more technical level, the inference here is that this set of genes shows enrichment in axonal vs somatic compartments whereas a better question is whether this gene set (29) is enriched over total detected (>1700) proteins more than chance alone. As presented, the conclusion that this dataset provides insights into PD genetics is not supported.
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Reviewer #2 (Public Review):
The manuscript by Hobson et al. uses APEX2, a proximity biotinylation approach, to resolve the subcellular proteomes of midbrain dopaminergic neurons. They used both viral and genetic tools to restrict expression of APEX2 to specific sets of dopaminergic neurons, then exploited the anatomy of the cells to separate the soma and dendrites from axons and axon terminals in the striatum. It is a clever approach and their experimental results are quite impressive. Since APEX2 cannot be used in vivo, they developed a brain-slice method that is very robust. They next purified biotinylated proteins from multiple controls and replicates - the studies were quite rigorous. As an independent approach, they also prepared striatal synaptosomes and performed proximity biotinylation using the synaptosomes. The results were …
Reviewer #2 (Public Review):
The manuscript by Hobson et al. uses APEX2, a proximity biotinylation approach, to resolve the subcellular proteomes of midbrain dopaminergic neurons. They used both viral and genetic tools to restrict expression of APEX2 to specific sets of dopaminergic neurons, then exploited the anatomy of the cells to separate the soma and dendrites from axons and axon terminals in the striatum. It is a clever approach and their experimental results are quite impressive. Since APEX2 cannot be used in vivo, they developed a brain-slice method that is very robust. They next purified biotinylated proteins from multiple controls and replicates - the studies were quite rigorous. As an independent approach, they also prepared striatal synaptosomes and performed proximity biotinylation using the synaptosomes. The results were remarkably and impressively similar to the results obtained with the biotinylation in slices. The similarity gives great confidence in the robustness of their approaches and data sets. The final aspect of the paper focused on combining several different resources including single cell RNAseq, GWAS studies of PD patients, and then comparing these to the subcellular dopaminergic neuron proteomes. They identified a set of proteins linked to PD that are preferentially enriched in axons.
I found this paper to be extremely interesting and important not only for the insight into differential protein localization and enrichment, but also because it points to candidate proteins that may play especially important and previously unrecognized roles in dopaminergic axons. This will no doubt serve as a resource for neurobiologists and PD researchers. Finally, the study design and execution is excellent and provides a model for how to carry out these kinds of experiments in the rodent brain using APEX2 for other questions.
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