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Nanotechnology in Neuroscience Reveals Membrane Mobility Matters Sandra J. Rosenthal
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Department of Chemistry, Department of Physics and Astronomy, Department of Pharmacology, and the Department of Chemical and Biomolecular Engineering, and Vanderbilt Institute of Nanoscale Science and Engineering Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235, United States ABSTRACT: Quantum dots are nanometer-sized semiconductors that have size-tunable, narrow emission bands, high quantum yields, and are resistant to photobleaching. Ligand-conjugated quantum dots enable the real time visualization of membrane proteins and have revealed that membrane diffusion dynamics are intrinsic to protein regulation, are susceptible to the level of membrane cholesterol, and are altered in genetic variants linked to disease, suggesting a mise en place approach to neuropsychopharmacology. KEYWORDS: Quantum dots, serotonin, dopamine, neuropsychopharmacology, mis en place
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gated this system by depleting cholesterol in the membrane, triggered SERT phosphorylation through activation of protein kinase G by 8-Br-cGMP, and stimulated SERT activation by the inflammatory cytokine IL-1B. For both pathways of SERT activation, we found that the SERT molecules had higher velocities and larger diffusion coefficients while remaining bound within the membrane microdomain. We demonstrated that SERT activation increased the mobility of SERT in the membrane microdomain. SERT activation leads to elevated serotonin reuptake; by extension, higher mobility while confined to the membrane microdomain is pro-depressive. Membrane cholesterol profoundly influenced SERT membrane mobility.2,4 In the SERT experiments described above, acute depletion of membrane cholesterol with methyl-βcyclodextrin disrupted membrane microdomains and rendered the entire population of SERTs freely diffusing. Statins are cholesterol-lowering drugs that can cross the blood-brain barrier. There are conflicting studies regarding the prevalence of depression in patient populations taking statins. We have examined the effect of chronic cholesterol depletion on SERT membrane dynamics with mevastatin in primary midbrain neurons in culture.4 SERTs treated with mevastatin display a shift toward a more freely diffusing population, and had larger diffusion coefficients than control or 8-Br-cGMP treated neurons, though less than the diffusion coefficients for acute depletion of membrane cholesterol. We have observed that mobility affects SERT activity,2 and that membrane cholesterol affects SERT mobility,2,4 and cholesterol can also affect SERT conformation,4 so cholesterol plays multiple roles in SERT regulation. Genetic variants of DAT and SERT that we have studied include (1) DAT Arg615Cys, (R615C);3 (2) DAT Ala559Val (A559V);6 and (3) SERT Gly56Ala (G56A).5 The DAT R615C mutation is associated with ADHD, DAT A559V, is
uantum dots are so named because their size-tunable absorption and emission properties originate from quantum mechanics.1 Quantum dots are chemically synthesized, and the production method yields quantum dots that have bound organic surface molecules available for surface derivatization. The dots are highly bright and photostable, making possible single quantum dot tracking experiments. My research group has largely used home grown surface ligands; we find a drug that is selective to the desired target, and we modify it with a linker arm terminated in a biotin tail to attach to a streptavidin coated quantum dot. (Figure 1). Labeling is generally a two-step process: (1) the ligand is incubated with the cells, (2) the cells are washed, (3) the cells are incubated with the streptavidin quantum dots for a short time and then (4) washed again. For every new experiment (new target and new ligand) we first demonstrate that ligand-conjugated quantum dot labeling is specific (no labeling in absence of target, no labeling in absence of ligand, and no labeling in the presence of inhibitor) Membrane transporter proteins for serotonin (SERT) and dopamine (DAT) terminate signaling by clearing the synapse of the neurotransmitter through reuptake. SERT is the target of the selective serotonin reuptake inhibitor antidepressants, such as Prozac or Paxil. By blocking reuptake by SERT, synaptic levels of serotonin increase. This is currently the most effective way to treat depression, but by no means an optimal one. The opposite of blocking reuptake, enhancing serotonin reuptake, I term “pro-depressive”. DAT is the site of action for both cocaine and methamphetamine, and genetic variants of DAT have been implicated in bipolar disorder, schizophrenia, and attention deficit hyperactivity disorder (ADHD). We have studied the membrane diffusion dynamics of both SERT and DAT with ligand-conjugated quantum dots.2−6 SERT membrane diffusion dynamics were determined using the custom ligand IDT318, predominantly selective for SERT, and an immortalized serotonergic neural cell line, RN46A.2 We found two populations of SERT, a small fraction that is freely diffusing and a larger fraction localized to cholesterol and GM1 ganglioside-enriched microdomains (Figure 2). We interro© XXXX American Chemical Society
Received: September 17, 2018 Accepted: September 20, 2018
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DOI: 10.1021/acschemneuro.8b00495 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience
Figure 2. Depiction of SERT regulation that emerged from single quantum dot tracking studies of SERT.2 Cholesterol depletion disrupts the lipid rafts (membrane microdomains) and yields freely diffusing SERT with greatly reduced uptake activity (left). P38 MAPK activation releases SERT from cytoskeleton interactions leading to increased diffusion confined to the membrane microdomain (right). This enhanced mobility coincides with greater SERT uptake.
Figure 1. Quantum dot labeling strategy. The custom ligand contains a SERT selective drug, a hydrophobic spacer enabling the drug to reach the binding site, a PEG spacer for biocompatibility and reduced nonspecific binding, and a biotin tail for use in conjunction with streptavidin functionalized quantum dots.1
associated with ADHD and bipolar disorder, and SERT G56A is a gain of function variant that has been associated with autism spectrum disorder. Figure 3 displays the cumulative probability plots depicting diffusion rate distributions for each of these coding variants versus wild type controls. Immediately apparent is that each of the variants has a frequency distribution located at higher diffusion coefficients. The genetic variants were more mobile than their wild type counterpart. The reasons behind the variants’ increased mobility may be different for each mutant. For example, DAT R615C has its variation near the cytoskeleton and decreased proteincytoskeleton interactions may render the variant more mobile. Alternatively, DAT A559V is near the extracellular portion of transmembrane domain 12. DAT possibly exists as a dimer or oligomer and this variation may weaken the interactions within the complex, increasing mobility.6 Finally in SERT G56A the alanine replacement may also disrupt SERT membrane microdomain localization. These variant studies and additional reports in the literature suggest that spatiotemporal membrane
Figure 3. Cumulative probability plots depicting diffusion rate distributions for wild type and the genetic variants DAT Arg615Cys (R615C, top), DAT Ala559Val (A559V, middle), and SERT Gly56Ala (G56A, bottom). For these experiments, model cell systems were used: DAT, HEK cells and SERT, CHO cells. The inset on the lower right identifies the point of each mutation. The increase in diffusion coefficients is remarkably similar for the three genetic variations.
disorganization of neuronal proteins is emerging as a central molecular pathological theme in brain disorder research.5 Mise en place is a term from French cooking that means “everything in its place”. It refers to having all of the ingredients prepared and in their proper place prior to starting to cook. But mise en place neuropsychopharmacology? We have seen that membrane mobility matters. In neuropsycohpharmacology, we tend to agonize or antagonize; we do not often think about keeping the targets in their place. The results of single quantum dot tracking studies suggest we should. B
DOI: 10.1021/acschemneuro.8b00495 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
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ACS Chemical Neuroscience Further, as the underlying mechanisms for altered mobility may be different for different genetic variants, single quantum dot tracking studies also underscore the need for precision medicine in psychiatry.
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AUTHOR INFORMATION
ORCID
Sandra J. Rosenthal: 0000-0002-4576-5854 Funding
NIH Grant EB003728. Notes
The author declares no competing financial interest.
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ACKNOWLEDGMENTS S.J.R. acknowledges a long-standing collaboration with Randy Blakely and inspiration from Alton Brown. REFERENCES
(1) Rosenthal, S. J., Chang, C. J., Kovtun, O., McBride, J. R., and Tomlinson, I. D. (2011) Biocompatible Quantum Dots for Biological Applications. Chem. Biol. 18, 10. (2) Chang, J. C., Tomlinson, I. D., Warnement, M. R., Carneiro, A. M. D., Ustione, A., Piston, D. W., Blakely, R. D., and Rosenthal, S. J. (2012) Single Molecule Analysis of Serotonin Transporter Regulation Using Antagonist-Conjugated Quantum Dots Reveals Restricted, p38 MAPK-Dependent Mobilization Underlying Uptake Activation. J. Neurosci. 32, 8919. (3) Kovtun, O., Sakrikar, D., Tomlinson, I. D., Chang, J., ArzetaFerrer, X., Blakely, R. D., and Rosenthal, S. J. (2015) Single-Quantum Dot Tracking Reveals Altered Membrane Dynamics of an AttentionDeficit/Hyperactivity-Disorder-Derived Dopamine Transporter Coding Variant. ACS Chem. Neurosci. 6, 526. (4) Bailey, D. M., Catron, M. A., Kovtun, O., Macdonald, R. M., Zhang, Q., and Rosenthal, S. J. (2018) Single Quantum Dot Tracking Reveals Serotonin Transporter Diffusion Dynamics are Linked to Cholesterol-Sensitive Threonine 276 Phosphorylation Status in Primary Midbrain Neurons. ACS Chem. Neurosci., DOI: 10.1021/ acschemneuro.8b00214. (5) Kovtun, O., Tomlinson, I. D., Bailey, D. M., Thal, L. B., Harris, L., Frankland, M. P., Ferguson, R., Greer, J., IV, Glaser, Z., and Rosenthal, S. J. (2018) Single Quantum Dot Tracking Illuminates Neuroscience at the Nanoscale. Chem. Phys. Lett. 706, 741. (6) Thal, L. B., Tomlinson, I. D., Quinlan, M., Kovtun, O., Blakely, R. D., and Rosenthal, S. J. (2018) Single Quantum Dot Imaging Reveals PKCβ-Dependent Alterations in Membrane Diffusion and Clustering of an Attention-Deficit Hyperactivity Disorder/Autism/ Bipolar Disorder-Associated Dopamine Transporter Variant. ACS Chem. Neurosci., DOI: 10.1021/acschemneuro.8b00350.
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DOI: 10.1021/acschemneuro.8b00495 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX
