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  1. Reviewer #3 (Public Review):

    Jenny I. Aguilar et. al. present a manuscript that methodically investigates the behavioral, structural, functional, and physiological consequences of a Cys substitution at R445 in the human dopamine transporter. Parkinson's disease is a common progressive neurodegenerative disorder that affects millions of people worldwide. In most patients, the underlying cause their disease is unknown, but some genetic forms of Parkinsonism have been identified. In this manuscript, the authors investigate the effect of a mutation in the gene that encodes the dopamine transporter that was identified in a patient with infantile Parkinsonism-Dystonia. Using a Drosophila model and an abundance of tools, the data show that the mutation produces: 1) a reduction in spontaneous motor activity, movement vigor, compromised flight initiation, and impaired coordinated movements; 2) a decrease in dopamine content and the number of tyrosine hydroxylase containing neurons in fly brain; 3) a decrease in amphetamine-induced dopamine efflux and dopamine uptake; 4) altered dopamine transporter structure leading to increased probability of open conformations on both sides of the transporter; 5) a reduction in dopamine transporter surface expression and transport capacity. Chloroquine, used as means to limit dopamine transporter lysosomal degradation, increased the ratio of mature to immature dopamine transporter and improved flight initiation. So why does a decrease in dopamine reuptake promote a dopamine-deficient Parkinson phenotype? The Authors conclude that an overall reduction in dopamine transporter would deplete dopamine stores by promoting excessive extracellular dopamine. The decrease in vesicular release would be further exacerbated by DA stimulation of presynaptic dopamine-D2 receptors on dopamine axons. This rather novel counterintuitive hypothesis appears to be supported by the outcome of this investigation. Overall, the study may highlight the mechanism underlying a rare type of Parkinsonism that can affect children as well as adults.

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  2. Reviewer #2 (Public Review):

    I have reviewed Psychomotor Impairments and Therapeutic Implications Revealed by a Mutation Associated with Infantile Parkinsonism-Dystonia by Aguilar et al. The authors first express hDAT in the dDAT loss of function background to explore in vivo effects. The comparison of hDAT rescue flies to wild type flies and the DAT mutant provide a nice control for the functionality of the hDAT transgene. A better control might have been rescue using dDAT with the same driver but this is a very minor concern since the wild type flies and the hDAT rescue look so similar. They then show that the R445C mutant decreases "movement vigor" and flight initiation. They use HPLC and immunolabeling to convincingly show deficits in both total tissue DA and a decrease in the number of detectable DA cells and use amperometry in the fly brain to quantify defects in efflux. Amperometry in the fly brain is technically impressive since few other labs have accomplished this without fouling the carbon electrode. In the second section of the paper, the authors perform a structural analysis, using LeuT to model DAT. The combination of Rosetta modeling, X-ray crystallography and EPR spectroscopy further adds to the technical strength of the paper. They show that substitution at the position in LeuT R375 analogous to DAT R445 disrupts a previously identified salt bridge and the IC vestibule. They then generate X-ray crystal structures of LeuT WT, LeuT R375A and LeuT R375D at resolutions of 2.1-2.6 Å. Their analysis confirms that substitution at LeuT-R375 disrupt salt bridge formation consistent with Rosetta modeling. They further conform the disruption of the interaction between R375 and its partner using a variant of EPR and show that substitutions at this site bias toward open conformations. In the final figure of the paper they heterologously express the DAT mutants in cell culture and show that cell surface expression, transport and efflux are compromised, similar to previously published findings from another lab. Finally, they show that chloroquine can rescue some of the behavioral deficits in the fly.

    The authors present a remarkably comprehensive and technically sophisticated analysis of the structure, function and behavioral sequelae of a mutation in the DAT (hDAT R445C). The analysis is translationally relevant since the mutation was identified in a patient suffering from a rare movement disorder relevant to Parkinson's disease. The combination of behavioral and biochemical analysis in a transgenic animal with X-ray crystallography and modeling is extremely unusual and from a technical standpoint the paper is unusually strong. The insight gained from comparing the structural and functional halves of the paper is also useful. The partial pharmacologic rescue of the behavioral deficits further elevates this work.

    Concerns It might be argued that the insights obtained from comparing the various data on modeling, structural analysis, biochemical assays and the behavior of the R445 mutant may not always be consistent with one another, making it difficult to determine the physiological relevance of each effect. This concern is balanced by the idea that we cannot know which aspects of any given mutant will or will not conform to expectations without the comprehensive analysis used here. As such, the paper provides an important example of examining a risk allele in a variety of different ways to determine which molecular deficits may be relevant to the observed phenotype and to the function of the transporter. That said, the authors should add text to acknowledge that some of the molecular defects they observe may be overshadowed by others and/or may not be as relevant to the in vivo defects in activity. For example, the idea that efflux may play a role in the R445 phenotype similar to other mutants and neuropsychiatric illness in general is provocative, but seems difficult to reconcile with the observation that relatively low levels of protein are present at the cell surface.

    The behavioral analysis is elegant and takes advantage of high-speed video recording to determine subtle defects in movement. The specificity of the defect is also interesting since grooming is not affected. However, it is difficult to determine whether the data represent a true deficit in movement versus wakefulness or overall activity of the animal. Dopamine is well known to be required for sleep in the fly and it is unclear whether the "deficits in movement vigor" are caused by the flies being "sleepy". Alternatively, higher order decision making processes rather than movement per se might be compromised. These explanations for the observed deficits would not take away from the importance of the findings. Indeed, as the authors acknowledge, the non-motor symptoms of PD are just as important as the motor symptoms. However, it seemed at times that authors felt compelled to fit their data into a motor paradigm rather than taking a more general view on the relationship of the observed defects to other problems that accompany PD. The authors should address these issues with additional text. Additional experiments to address this issue are likely beyond the scope of the current manuscript which is already quite lengthy.

    Minor points:

    The authors discuss a model in which loss or DAT reuptake and an increase in extracellular DA could down regulate TH. Since they use TH labeling to count DA cells they should acknowledge the possibility the cells are not absent in the mutant (even if they are functionally compromised) but are simply not detectable.

    It is unclear why (Brand and 147 Perrimon, 1993); are cited on line 146.

    Typo in "Initiate" on Y axis of Fig 3B.

    State somewhere in the text or in the Fig 3 legend that HPLC was used to measure tissue concentrations of DA to make it more obvious that amperometry was not used

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  3. Reviewer #1 (Public Review):

    The authors generated new transgenic fly lines with the human dopamine transporter (hDAT-WT) and the hDAT with the R445C mutation (hDAT-R445C). Studies in the hDAT-R445C flies show a decrease of tissue DA content and a loss of TH+ PPL1 neurons indicating an effect of the DAT mutation on dopamine neuron phenotype or cell survival rather than general DA levels per se. The motor phenotypes observed in the fly include a decrease in the time to initiate flight and in the velocity of locomotion (vigor) but not in the velocity of locomotion initiation or grooming behavior. These behaviors are consistent with the bradykinesia observed in patients. This model system could potentially be used to assay for specific modulators of the mutant to restore surface expression, TH expression and motor behavior.

    In the recombinant cell culture system (HEK Cells), the major consequence of the mutation is a decrease in cell surface expression (there is a decrease in conversion to the mature form). A change in the Km is difficult to ascertain with such a dramatic change in the cell surface expression level but looks to be dramatically decreased (higher affinity). These data differ somewhat from those reported in the study by Ng et al, 2014 where the Bmax for CFT was slightly reduced and the affinity was significantly decreased (Km was ~8 fold higher) as was the Ki for DA inhibition of CFT. It should be noted that the decrease in cell surface expression of R445C reported by Ng et al was also not as dramatic as what the same group demonstrated for the other mutation, R87L, that was compound heterozygous in this family. Differences in the transport properties between the two studies should be discussed.

    X-ray crystallography and molecular modeling provide novel insights into how the mutation (and other substitutions at this site) affects structure-function relationships of the transporter with respect to gating, uptake and efflux. This information could be used to design modulators of the transporter mutants to rescue cell surface expression or function.

    The behavioral effect of CQ on the mutant flies was on the time to flight initiation, which decreased. Locomotion was not tested.

    The value of the study is the creation of the flies for screening and the crystallography and molecular modeling studies which examined the impact of this residue on function in detail. The weakness of the study was the limited characterization of the transport properties and cell surface expression in the flies. Being able to tie together the different studies into a cohesive understanding of what happens in patients and thus what needs to be corrected in patients is an important goal of the study. Some of the key questions needed to achieve this understanding were not fully addressed.

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  4. Evaluation Summary:

    Infantile parkinsonism-dystonia is a rare but devastating condition that leads to early mortality. Mutations in the dopamine transporter that decrease its transport activity or cell surface expression have been identified as potential causes of this disease. Here, Aguilar et al perform a series of experiments to examine the effect of one of the mutations, R445C, on properties of the transporter in cell culture and on motor function in newly generated transgenic flies. They also explore structure function relationships of the mutation using X-ray crystallography of LeuT, a bacterial homolog, and molecular modeling. Lastly, they show blocking lysosomal degradation rescues a motor deficit in the flies. Insights from the work could lead to new approaches to specifically modulate the transporter structure to restore surface expression and function of the mutant dopamine transporter in this disorder. This elegant and technically sophisticated analysis is of interest to readers in the fields of neurobiology, behavior, and movement disorders, as the work provides an excellent example of using a variety of different approaches to determine the relationship between transporter structure and activity and potentially underlying pathology in human disease.

    (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 #1 agreed to share their name with the authors.)

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