1. Insel, Thomas R., Faulty Circuits, Sci. Am. 44-52, April 2009.
2. Hyman, S.E., Obsessed with Grooming, Nature 448: 871-872 (2007).
3. Devlin, B. et al., Autism Serotonin Transporter: The Long and Short of It, Mol. Psych. 10: 1110-1016 (2005).
4. Shandana, S.R. et al., Significance of Abnormalities in Developmental Trajectory and Asymmetry of Corticol Serotonin Synthesis in Autism, Int, J, Devl. Neuroscience 23:171-182 (2005).
5. Singh, S.K. et al., Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters Nature 448: 952-956 (2007).
6. Zhou et al., Antidepressant Specificity of Serotonin Transporter Suggested bt Three Leu-T-SSRI Structures, Nat. Struct, Mol. Biol 16:652-658 (2009).
7. Ressler, K.S. & Maybery, H., Targeting Abnormal Neural Circuits in Mood and Anxiety Disorders: from The Laboratory to the Clinic. Nat. Neuroscience 10: 1116-1124 (2007).
www.rcsb.org Rutgers Center for Structural Biology. Database of biological structures.
Concise answers to the Study Guide questions and molecular images resulting from the modeling exercises are available on the Proteopedia website. Simply type in ‘autism’ in the search bar.
1. Mental Illness & Global Brain Circuits. In Study Guide No. 7 we encountered the locus coeruleus-noradrenergc system (LC-NR) that arises in the brain stem and projects axons to nearly every region of the brain as part of the stress response system of the body. The synaptic terminals release norepinephrin at the target sites. This is an example of a global circuit that synchronizes and coordinates activity across regions widely separated across the brain.
Tom Insel, the Director of the National Institutes of Mental Health (NIMH), discusses three other types of global circuits that link psychological behavior to specific patterns of brain activation (‘circuits’). Note that the LC-NR system responds to physiological stress, whereas Insel is discussing behavioral manifestations of faulty circuits that regulate the fear response, life pressures, and action planning. Briefly describe the brain structures imvolved in each of the three examples that Insel gives (major depressive disorder, obsessive compulsive behavior, and post traumatic stress disorder). Do the three disorders share any common brain regions? How are fhese brain regions affected by life experiences (as revealed by brain imaging) or laboratory fear paradigms. How did Helen Mayberry (as portrayed by Insel) show that Region 25 mediates the ‘depression circuit’? Does her work offer any promises of relief from chronic depression? Given what we have talked about in this course, can you provide a plausible explanation for her observations (HINT: synaptic plasticity). Is there a genetic link to the serotonin system in major depression disorder? (HINT: long and short arms).
Steven Hyman, Provost of Harvard University and former Director of the NIMH, discusses a mouse model for OCD. What is the function of the protein involved in this genetic manipulation? The mice exhibit extreme self-grooming to the extent that they injure themselves, a behavior sometimes seen in humans (although OCD behaviors comprise a much wider spectrum), and especially those with autism (ritualized behaviors, insistence on sameness in their environments). How was it possible to reverse the behavior of the OCD mouse? (HINT: is a specific brain region affected by the mutation?) Do any of the drugs used to treats humans for OCD have an affect on the behavior of the OCD mice? Does this make sense in terms of the function of the protein deleted in the mouse model? Is there any connection between these observations on the OCD mouse and human brain anatomy? (HINT: neuro-imaging).
2. Autism & Serotonin. Ed Cook (Chicago) is a pioneer in the genetics of autism related disorders. He is responsible for accurately pinpointing events on chromosome 15q11-13 that are associated with Prader-Willi and Angelman’s syndromes. Devlin et al. review the role of serotonin in autism. What four lines of evidence do they adduce for supposing that dysregulation of the serotonergic system is one of the causes of autism? What is the function of the serotonin-processing gene discussed by Cook? See MIM182138.
Using genetic samples from a large cohort of trios (affected individuals and their parents), Devlin et al. assess whether over-transmission of any alleles of HTTLPR is linked to autism. What is HTTLPR? What does it have to do with serotonin levels in the brain? What are the major alleles? What is a SNP? What is a haplotype? Are any alleles of HTTLPR over-transmitted in this cohort of families with autism? Is there any connection of this allele to OCD?
Chugani and colleagues use PET neuro-imaging to measure serotonin levels in living brains. How is this possible? (HINT: what compound did they synthesize? What is its half-life?). What are the two main observations of the relative abundance of serotonin in the brains of autistic individuals? Were they able to draw specific conclusions about autistic individuals who cannot speak? They posit a maternal influence on brain development during gestation that might cause the differences they observe in serotonin distribution. What specific model do they suggest involving cortical organization of neurons? (HINT: mini-columns).
3. Non-pharmacological Interventions for Major Depressive Disorder. Knowledge of dysfunctional brain circuits underlying depression allow the possibility of focusing electromagnetic energy in regions of hyperactivity so as to depolarize overly-active neurons. In the paper by Ressler & Mayberry three different interventions are described. Briefly describe the physical principles of operation and the targeted brain structures that seem to yield results. What specific brain structural differences are described between individuals with Major Depressive Disorders and control subjects? (HINT: Figs 1 & 2).
4. Structural analysis of a serotonin transporter homologue with bound TCA. We have seen repeatedly in this course that bacteria possess structural homologues for many of the protein domains that comprise eukaryotic proteins: AMPA receptor and β2-adrenergic receptor. It is interesting that the synthetic precursors of all of the major neurotransmitters (glutamate, GABA, glycine, serotonin, dopamine, norephineprine, adrenaline, histamine) are amino acids. Further, many of the neurotransmitter receptors are built around amino acid binding cassettes. The sodium-dependent serotonin transporter, the target of selective serotonin reuptake inhibitors (SSRI’s, antidepressants), is no exception. It turns out that the Leu transporter (Leu-T) has many of the pharmacological properties of SSRI’s. Singh et al. (Ed Gouaux’s group again) have determined the structure of Leu-T in complex with a tricyclic antidepressant (TCA).
What experiments led them to conclude that clomipramine rather than, say, impramine was the strongest inhibitor of Leu-T? (HINT: summarize the results in Fig. 1). Figure 2 presents the broad outlines of the structural results. Can you see the Chlorine in the electron density of the clomipramine structure? How do crystallographers obtain the ‘wire-cage’ – i.e. the electron density – for substrate-bound structures when they already know the structure of the apo-protein? (HINT: OMIT Maps). Describe the binding interactions between the chlorine atom and the protein. What stabilizes the position of the N2 atom of clomipramine? Can you explain why it is protonated and thus able to form this bond? What is the ‘ionic lock’ that joins the TCA in occluding the vestabule? Yes! another great arginine. What is the structural relationship between the trapped Leucine molecule, clomipramne, and this arginine (HINT: guanadino sandwich). What is the physical chemistry significance of the hydrophobic groups surrounding the salt-bridge? (HINT: dielectric constant).
Modeling: these authors did such a great job that there is little to improve upon. Please prepare a version of the guanidine sandwich for inclusion in your ‘Great Arginines of 543’ series.
5. Structural analysis of serotonin transporter homologue with bound SSRI’s. The presynaptic serotonin reuptake transporter is the selective target of drugs such as fluoxetine (PROZAC) or sertraline that increase the residence time of serotonin in the synaptic cleft following release of the neurotransmitter. The structures presented by Zhou et al. elucidate the basis for the blocking action of these drugs. The key structural feature that confers specificity is the halogen binding pocket (HAP). The binding of the halogenated drugs is principally due to this binding site. What are the hallmarks of this site? Is there a crucial arginine? Are there salt-bridges? What are the chemical forces responsible for stabilizing the electronegative halogens?
The normal function of the SERT homologue is to transport leucine into cells. What is the structural relationship between the leucine binding site and the HAP? Produce an image with SWISSPDB (or other suitable program) illustrating this connection?
Does the chlorine atom on clomipramine bind in the HAP?
Zhou et al describe binding assays carried out on the mutagenized proteins NET (norepinephrine transporter) and DAT (dopamine transporter). How did they decide which amino acids to mutate? What do their results prove?
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