Johannes W. Hell - US grants

[unreadable] DESCRIPTION (provided by applicant): Synapses are central players in neuronal signal transmission and prime targets for drug treatments of various neurological and mental disorders. The long-term goal of this application is to characterize the assembly and function of signaling pathways at postsynaptic sites. This proposal focuses on molecular mechanisms that target PKA together with its regulator (beta2 adrenergic receptor, beta2 AR) and its antagonist (phosphatase PP2A) to postsynaptic effector proteins, in particular the L-type Ca2+ channel Cav1.2. PKA upregulates Cav1.2 currents. Cav1.2 is clustered at postsynaptic sites. We discovered that Cav1.2 assembles the beta2 AR, the trimeric Gs protein, adenylyl cyclase, AKAP75/150, PKA and the counter- balancing phosphatase PP2A into a macromolecular signaling complex. Our single channel recordings revealed that upregulation of channel activity upon stimulation of beta2 AR is highly localized in neurons (Science 293, 98-101). This is the very first example of a complex that links a cAMP-coupled receptor to one of its ultimate targets. This localized signaling explains how the diffusible cAMP can mediate specific effects. The interaction sites of the channel with p2 AR, AKAP75/150, and PP2A, will be defined and mutated in a nifedipine-insensitive T1039Y Cav1.2 mutant and expressed in primary hippocampal cultures as will be S1928 (to alanine), the main PKA site. Localized regulation of these mutants by the beta2 AR - PKA pathway will be measured by cell-attached single channel recordings and imaging of Ca2+ transients in dendritic spines with nifedipine present to inhibit endogenous L-type channels. Complementary studies with AKAP150 KO mice and with (poly)peptides that disrupt the beta2 AR interactions with endogenous Cav1.2 will be performed. The physiological relevance of PP2A binding with the C-terminus of Cav1.2 will be analyzed in an analogous way. An increase in Cav1.2 channel activity as mediated by PKA has been implicated in depression and anxiety disorders and contributes to the etiology of Alzheimer's disease. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of Cav1.2 constitutes, therefore, a potentially effective and specific target for drugs that disrupt some of these interactions, thereby inhibiting adverse signaling events under pathological conditions. [unreadable] [unreadable]

Several factors contribute to the degeneration and ultimately loss of neurons during aging and especially during Alzheimer's Disease (AD). One potentially critical factor is an increase in neuronal Ca2+ influx through L-type Ca2+ channels. The activity of L-type channels increases with age in rats, and an inhibitor of these channels impressively improves the learning capabilities of aged rabbits when given over several weeks. These findings suggest that L-type channels may be involved in aging-related neurological changes and the etiology of AD. Our objectives are to determine molecular changes of L-type channels that may contribute to the upregulation of L-type channel activity during aging. Based on our preliminary data, we hypothesize that: (A) an increase in protein kinase A (PKA)-mediated phosphorylation of L-type channels is responsible for their upregulation during aging; (B) beta-adrenergic receptors can control the elevation in phosphorylation of L-type channels by PKA. We will test these hypotheses by determining the phosphorylation status of L-type channels in brain samples from adult rats (some treated with beta-adrenergic agonists) and aging rats as a model for senile symptoms. Brain samples from adult and aging rats will be solubilized and L-type channels immunoprecipitated for further biochemical analysis of parameters that might change during aging, e.g., phosphorylation by PKA and other kinases. To test whether the number of L-type channels present in neurons is changed during aging, relative amounts of L-type channel proteins will be determined by immunoblotting and compared to the level of various pre- and postsynaptic marker proteins. PKA is associated with L-type channels. We will measure the relative amounts of PKA subunits and PKA anchoring proteins in the L-type channel immunocomplexes to test potential changes during aging that could explain the increase in PKA-mediated phosphorylation. Finally, we will investigate whether injection of the beta-adrenergic agonist isoproterenol upregulates PKA-mediated phosphorylation of L-type channels in adult rats.

DESCRIPTION (provided by applicant): Synapses are central to neuronal signaling and key targets for drug treatments of neurological disorders. Ca2+ influx through NMDA receptors and subsequent activation of CaMKII are critical events in the induction of LTP and in learning and memory. The NMDA receptor interacts with CaMKII in a complex and highly regulated manner. This interaction places CaMKII at a strategically ideal location, where it is most effectively activated by Ca2+ influx through the NMDA receptor and where it is close to its substrates at the postsynaptic site (e.g., GluRl in LTP). Biochemical details and the functional relevance of this interaction, which is currently unproven, will be determined. Preliminary results will be scrutinized that indicate that the CaMKII, calmodulin, and a-actinin binding sites on the NR1 subunit of the NMDA receptor overlap and that Ca2+/calmodulin promotes CaMKII binding to NR1 by displacing a-actinin. Whether binding of CaMKII to the NR1 and perhaps NR2B subunit per se changes NMDA receptor activity as observed earlier for Ca2+/calmodulin binding to NR1 will be investigated with different electrophysiological methods including recording from excised inside-out patches perfused with purified CaMKII, calmodulin and a-actinin in various combinations and whole cell patch clamping using HEK293 cells transfected with NR1, NR2A, NR2B and CaMKII in different combinations. Whether CaMKII binding to the NMDA receptor is critical for recruiting CaMKII to the postsynaptic site will be tested in primary hippocampal cultures by imaging of GFP-tagged CaMKII in the absence and presence of peptides (membrane-permeable or injected) that inhibit NR1 or NR2B binding of CaMKII. The importance of the NMDA receptor - CaMKII interaction for the induction of LTP will be evaluated by intracellular recording from hippocampal slices with and without the binding-inhibiting peptides. Phosphorylation of GluR1 by CaMKII likely contributes to LTP and will be quantified in slices with biochemical methods. Overstimulation of glutamatergic synapses has been implicated in neuropathologies due to stroke, status epilepticus, and brain trauma. NMDA receptor-mediated Ca2+ influx and CaMKII activation are critical for neuronal damage caused by ischaemia. The postsynaptic anchoring of CaMKII by the NMDA receptor constitutes, therefore, a potentially very important target for drugs that can specifically disrupt this interaction and thereby alleviate neuropathologies due to overactivation of glutamate receptors.

[unreadable] DESCRIPTION (provided by applicant): The principal goal of this training grant (TG) is to promote the interdisciplinary training of graduate students in Pharmacological Sciences. Its secondary goal is to foster interactions among faculty and students from different Departments that share an interest in Pharmacological Sciences. The current TG developed out of a successful 25-year-old TG in the Department of Pharmacology, which was radically modified to attract students from a highly diverse background. The 42 trainers on this TG are drawn from 10 different PhD-granting programs, and three different Colleges, consistent with its focus on interdisciplinary training. Faculty representation is well balanced at all academic levels. All senior trainers have excellent records of training graduate students and maintain highly productive research programs. They will serve as mentors to junior faculty of great promise and potential. The curriculum provides a formal venue for both trainer and trainee interactions. Principles in Pharmacology (071:135) has been modified to accommodate trainees from different disciplines. Advanced Problem Solving in Pharmacological Sciences (071:250) has been specifically created for this TG. In this course, trainees of diverse background work as a group with mentoring by a trainer to solve a pharmacological problem and write an interdisciplinary research paper that utilizes methodology ranging from Medicinal Chemistry to Systems Pharmacology and Physiology. The Pharmacology Seminar (071:204) serves as a weekly forum to bring together the trainees and trainers. It provides trainees with experience in formal presentation of their work. Early access to research experience by trainees is emphasized, which has resulted in average times to PhD of ~5 years. It is anticipated that this TG will provide support for trainees who are among the top 25% of all eligible trainees on campus for 2 years of their graduate education. We demonstrate the existence of a sustainable and clearly-identified pool of high quality graduate students. Of this pool, 13 highly qualified students applied in 2004 and 14 in 2005. Institutional support for this TG has been particularly strong with the provision of three additional training slots during the prior award period, and a future commitment of a minority-designated slot for the duration of the next award period. The aims of the previous award, which were to further develop the curriculum and establish the existence of successful interdisciplinary program, have been met. The program is now well situated to expand upon its initial success. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): L-type Ca2+ channels regulate various functions spanning from neuronal excitability to gene transcription. The long term interest of this grant is to determine the molecular mechanisms that control activity and functional availability of the L-type channel Cav1.2. Our strong preliminary data indicate that a-actinin binds directly to the central pore-forming Cav1.2 subunit. Calmodulin binds to the same region as a-actinin and displaces it from Cav1.2 in the presence but not absence of Ca2+. Knock-down of a-actinin with siRNA or expression of two different dominant negative a-actinin fragments lead to a -50% reduction in the current density of Cav1.2 and substantially reduce peripheral Cav1.2 localization as detected by immunofluorescence. We hypothesize that a-actinin fosters surface expression of Cav1.2 by inhibiting its endocytosis. We further hypothesize that displacement of a-actinin by Ca2+/calmodulin promotes endocytosis of Cav1.2. We will define residues on Cav1.2 that are critical for a-actinin but not CaM binding and vice versa. We will inhibit the interactions between a-actinin and Cav1.2 by: a) expressing dominant negative a-actinin constructs;b) knocking down a-actinin with siRNA (we already established the siRNAs);c) expressing Cav1.2 with residues mutated that are crucial for a-actinin binding. Whether surface expression is reduced following these manipulations as compared to control conditions will be evaluated by biochemical and cell biological methods. We will investigate the role of calmodulin in dislocating a-actinin from Cav1.2 upon Ca2+ influx, thereby perhaps promoting Cav1.2 internalization, by: a) overexpressing Ca2+ binding-deficient calmodulin mutants that act as dominant negative constructs for a number of ion channels with respect to the regulation of their gating by Ca2+/calmodulin, including Cav1.2;b) expressing mutant Cav1.2 that does not bind calmodulin but still interacts with a-actinin. Ca2+-dependent internalization will be compared between control conditions (e.g., overexpression of wt calmodulin or wt Cav1.2) and test conditions (e.g., dominant negative calmodulin or mutant Cav1.2). Cav1.2 channel activity is strongly increased in aged rats. The L-type channel inhibitor nimodipine impressively improves learning capabilities of aged rodents. Hence, an increase in Cav1.2 channel activity is thought to contribute to the etiology of senile symptoms and Alzheimer's disease. Cav1.2 has also been implicated in depression and anxiety disorders. Our work on the regulation of surface expression of Cav1.2 by the interplay between a-actinin and calmodulin will fill a critical gap in our understanding of how Cav1.2 surface expression and localization is regulated. It will thereby contribute to the development of treatment of these diseases.

DESCRIPTION (provided by applicant): Synapses are central to neuronal signaling and prime targets for drug treatments of neurological and mental disorders. Norepinephrine (NE) regulates attention and alertness. The ß2 adrenergic receptor (ß2 AR) is emerging as the prevalent postsynaptic NE effector at glutamatergic synapses, where it interacts with AMPAR, NMDAR and the postsynaptic L-type Ca2+ channel Cav1.2. These complexes also contain Gs, adenylyl cyclases (ACs) and PKA, the downstream effectors of ß2 AR, for what appears to be highly localized signaling (within 100 nm) by cAMP (e.g., our work in Science 293, 98; Science 293, 2205; EMBO J 29, 482). Such spatial restriction would explain specific regulation of certain targets of the ß2 AR - Gs - AC - cAMP - PKA cascade and especially of AMPAR, NMDAR and Cav1.2. This project takes advantage of unique features of glutamatergic postsynaptic sites, which are formed by dendritic spines. AMPAR, NMDAR and Cav1.2 are localized at spine heads by a protein meshwork, the postsynaptic density (PSD), which is small (~300 nm) and can be isolated biochemically. Aim 1 is to test on a molecular level the hypothesis that specific acute or genetic disruption of the ß2 AR-AMPAR/NMDAR association affects ß2 AR-induced phosphorylation of these receptors but not of Cav1.2 that is co-localized within the very same PSDs (PSDs will be immunoprecipitated with antibodies against AMPAR, NMDAR or Cav1.2 for subsequent phospho-analysis of all 3 channels). The ß2 AR- Cav1.2 binding will be disrupted to test the reverse. Aim 2 will functionally monitor by high resolution Ca2+ imaging ß2 AR-stimulated Ca2+ influx through NMDAR and Cav1.2 within same spines with the hypothesis that disrupting ß2 AR - NMDAR binding will only inhibit ß2 AR-stimulated Ca2+ influx through NMDAR but not Cav1.2 ß2 AR (and vice versa). Aim 3 is to test on a systemic level whether ß2 AR binding to glutamate receptors, to Cav1.2, or both are important for regulation of a form of LTP induced by a tetanus of 5 Hz (endogenous theta rhythm) for 180 s that requires stimulation of the ß2 AR and Cav1.2 activity. This work will define unexplored fundamental molecular mechanisms of how NE regulates postsynaptic functions. It will thereby create a framework for understanding neurological diseases such as Alzheimer's disease, which is at least in part due to dysregulation of Cav1.2 and NMDAR by ß2 AR signaling, and stroke induced neuronal damage, which is at least in part due to upregulation of Ca2+ permeable AMPAR, which in turn are targeted to postsynaptic sites by ß2 AR signaling. NE signaling is also relevant for PTSD and depression. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of AMPAR, NMDAR and Cav1.2 constitutes a potentially effective and specific target for drugs that disrupt some of these interactions while not affecting others. Finally, this work will address the question of how localized cAMP signaling can be, which might be <100 nm given the small size of postsynaptic sites. Because ß2 ARs also associate with Cav1.2 in heart, smooth muscle and pancreas, spatially restricted cAMP signaling is of wide interest beyond its role in the brain.

DESCRIPTION (provided by applicant): AMPAR and spine dysfunction or dysregulation underlies many CNS diseases including depression, autism, PTSD, epilepsy, and stroke-induced neuronal damage. Precise postsynaptic localization of AMPARs is critical for fast synaptic transmission. It depends on PSD-95 and its interaction with auxiliary AMPAR subunits called TARPs. Despite its central role in targeting AMPARs, it is unknown how PSD-95 itself is anchored at the postsynapse. Our preliminary data suggest that A) a-actinin binds to the first 13 residues of the N-terminus of PSD-95; B) knock-down (KD) of a-actinin reduces postsynaptic PSD-95 content and mEPSCs, the latter phenocopying KD of PSD-95; C) mutating either Lys10 or Lys11 to Glu (K10E, K11E) specifically impairs PSD- 95 binding to a-actinin and postsynaptic targeting of PSD-95 and of AMPARs; D) peptide PSD95(1-13) displaces PSD-95 from a-actinin; E) injection of PSD95(1-13) decreases mEPSC amplitude. We hypothesize that a-actinin is critical for postsynaptic anchoring of PSD-95 and thereby AMPAR-TARP complexes. Proving this hypothesis will fundamentally advance our understanding of postsynaptic AMPAR localization. A), B), and D) are final data. Aims 1 and 2 will further scrutinize C) and E), i.e., whether mutating K10E and K11E or injecting PSD95(1-13) affect synaptic PSD-95 and AMPAR taregting using fluorescence microscopy and mEPSC. NMR structural analysis will identify residues in a-actinin that are important for PSD-95 binding. KD of endogenous a-actinin and replacement with either WT or mutant a-actinin will show whether mutant a-actinin is not able to rescue the KD effect on PSD-95, in contrast to our rescue with WT a-actinin. Aim 3 is to test whether NMDA-induced Ca2+ influx displaces PSD-95 from a-actinin and thereby from postsynaptic sites along with AMPARs via calmodulin (CaM). We found that Ca2+/CaM binds to the N-terminus of PSD-95 to dislodge a-actinin. Our structural NMR analysis of a complex between Ca2+/CaM and the first 71 aa of PSD-95 identified Y12 in PSD-95 as critical for CaM binding. Mutating Y12 to Glu (Y12E) prevents Ca2+/CaM binding without affecting a-actinin binding or postsynaptic localization of PSD-95. NMDA-induced Ca2+ influx displaces a portion of WT but not Y12E PSD-95 from spines. In fact, Y12E exhibits a large increase rather than decrease in spines upon Ca2+ influx. We will test whether this mutation and other manipulations unmask a mechanism that leads to postsynaptic accumulation of PSD-95 and AMPARs rather than their decrease. Such a decrease is usually seen following 5 min NMDA treatment and is referred to as chemical LTD. This exciting new direction will elucidate how Ca2+ influx can cause LTD rather than LTP. Page 1

DESCRIPTION (provided by applicant): Synapses are central to neuronal signaling and prime targets for drug treatments of neurological and mental disorders. Norepinephrine (NE) regulates attention and alertness. The ß2 adrenergic receptor (ß2 AR) is emerging as the prevalent postsynaptic NE effector at glutamatergic synapses, where it interacts with AMPAR, NMDAR and the postsynaptic L-type Ca2+ channel Cav1.2. These complexes also contain Gs, adenylyl cyclases (ACs) and PKA, the downstream effectors of ß2 AR, for what appears to be highly localized signaling (within 100 nm) by cAMP (e.g., our work in Science 293, 98; Science 293, 2205; EMBO J 29, 482). Such spatial restriction would explain specific regulation of certain targets of the ß2 AR - Gs - AC - cAMP - PKA cascade and especially of AMPAR, NMDAR and Cav1.2. This project takes advantage of unique features of glutamatergic postsynaptic sites, which are formed by dendritic spines. AMPAR, NMDAR and Cav1.2 are localized at spine heads by a protein meshwork, the postsynaptic density (PSD), which is small (~300 nm) and can be isolated biochemically. Aim 1 is to test on a molecular level the hypothesis that specific acute or genetic disruption of the ß2 AR-AMPAR/NMDAR association affects ß2 AR-induced phosphorylation of these receptors but not of Cav1.2 that is co-localized within the very same PSDs (PSDs will be immunoprecipitated with antibodies against AMPAR, NMDAR or Cav1.2 for subsequent phospho-analysis of all 3 channels). The ß2 AR- Cav1.2 binding will be disrupted to test the reverse. Aim 2 will functionally monitor by high resolution Ca2+ imaging ß2 AR-stimulated Ca2+ influx through NMDAR and Cav1.2 within same spines with the hypothesis that disrupting ß2 AR - NMDAR binding will only inhibit ß2 AR-stimulated Ca2+ influx through NMDAR but not Cav1.2 ß2 AR (and vice versa). Aim 3 is to test on a systemic level whether ß2 AR binding to glutamate receptors, to Cav1.2, or both are important for regulation of a form of LTP induced by a tetanus of 5 Hz (endogenous theta rhythm) for 180 s that requires stimulation of the ß2 AR and Cav1.2 activity. This work will define unexplored fundamental molecular mechanisms of how NE regulates postsynaptic functions. It will thereby create a framework for understanding neurological diseases such as Alzheimer's disease, which is at least in part due to dysregulation of Cav1.2 and NMDAR by ß2 AR signaling, and stroke induced neuronal damage, which is at least in part due to upregulation of Ca2+ permeable AMPAR, which in turn are targeted to postsynaptic sites by ß2 AR signaling. NE signaling is also relevant for PTSD and depression. The postsynaptic assembly of specific signaling components that control PKA-mediated phosphorylation of AMPAR, NMDAR and Cav1.2 constitutes a potentially effective and specific target for drugs that disrupt some of these interactions while not affecting others. Finally, this work will address the question of how localized cAMP signaling can be, which might be <100 nm given the small size of postsynaptic sites. Because ß2 ARs also associate with Cav1.2 in heart, smooth muscle and pancreas, spatially restricted cAMP signaling is of wide interest beyond its role in the brain.

DESCRIPTION (provided by applicant): L-type Ca2+ channels regulate gene transcription, neuronal excitability and multiple forms of synaptic plasticity. Our long term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 (e.g., Science 293, 98; Science 293, 2205; PNAS Neuron 78, 483), which is the most prevalent L-type channel in brain and heart. We recently found that ?-actinin binds directly to the IQ motif of the centra pore-forming Cav1.2 subunit ?11.2 and augments its surface localization (Neuron 78, 483). We just identified three point mutations in the IQ motif that individually impair ?-actinin binding.Our preliminary electrophysiological and surface labeling data now suggest that impairing ?-actinin binding to the IQ motif decreases surface expression and, unexpectedly, also channel open probability (Po). Aim 1 is the very first comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2 through the ER-Golgi-TGN secretory pathway and endocytic recycling and degradation pathway by surface biotinylation, N-glycosylation analysis and colocalization with respective markers like BiP and Rab5. Aim 2 is to determine and compare current density, gating currents, and single channel currents to test whether the ?-actinin binding - deficient point mutations of Cav1.2 have decreased Po. Our structural NMR analysis is guiding charge reversal experiments for unequivocal (!) assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of ?-actinin binding. We recently discovered that LTP induced by the physiological Prolonged Theta-rhythm Tetanus (PTT, here 5Hz/180s; PTT-LTP) absolutely requires Cav1.2 but not NMDAR function. Aim 3 will test our hypothesis that activity-induced alteration in postsynaptic Cav1.2 differentially affects PTT-LTP in young and old rats. Cav1.2 channel activity is strongly increased in aged rats (e.g., Science 272, 1017). The L-type channel inhibitor nimodipine impressively improves learning capabilities of aged rodents (e.g., Science 243, 809). Hence, an increase in Cav1.2 channel activity is thought to contribute to the etiology of senile symptoms and Alzheimer's disease, likely in part by impairing synaptic function. In fact we observed both, an increase in Cav1.2 surface expression and ?-actinin association in old vs. adult rat hippocampus. Thus our work on the functional interplay of Cav1.2 with ?-actinin and calmodulin is of high significance for understanding these aging-related neurological diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond.

Role of ?-actinin in Cav1.2 Function Abstract L-type Ca2+ channels are tightly coupled to gene transcription, control neuronal excitability, and mediate multiple forms of synaptic plasticity. Yet its cell biology and regulation is remarkably poorly understood. Our long term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 (e.g., Science 293, 98; Science 293, 2205; PNAS 103, 7500; Neuron 78, 483), which is the most prevalent L-type channel in brain and heart. We recently found that ?-actinin binds directly to the IQ motif of the central pore- forming Cav1.2 subunit ?11.2 and augments its surface localization (Neuron 78, 483). We now identify three point mutations in the IQ motif that individually impair ?-actinin binding. Our preliminary electrophysiological and surface labeling data suggest that impairing ?-actinin binding to the IQ motif decreases surface expression and, unexpectedly, also channel open probability (Po). Aim 1 is the very first comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2 through the ER-Golgi-TGN secretory pathway and endocytic recycling and degradation pathway by surface biotinylation, N-glycosylation analysis and colocalization with respective markers like BiP and Rab5. Aim 2 is to determine and compare current density, gating currents, and single channel currents to test whether the ?-actinin binding - deficient point mutations of Cav1.2 have decreased Po. Our structural analysis is guiding charge reversal experiments for unequivocal (!) assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of ?-actinin binding. We found that Ca2+ influx specifically through Cav1.2 leads to displacement of ?-actinin from the IQ motif by Ca2+/calmodulin and in parallel to run down of Cav1.2 via endocytosis and reduction in Po. Aim 3 is to unravel the interplay between ?-actinin and calmodulin at the IQ motif to precisely define the molecular mechanisms of how Ca2+/calmodulin displaces ?-actinin and leads to endocytosis and run down of Cav1.2. Aim 4 will test first the role of ?-actinin in neuronal Cav1.2 functions including Ca2+ influx into spines and regulation of gene expression via NFAT and then why ?-actinin association and, fittingly, surface expression of Cav1.2 is increased in rodent models of senility and Alzheimer?s disease (AD). Increased Cav1.2 channel activity contributes to senile symptoms and AD (e.g., Science 272, 1017; Science 243, 809). Cav1.2 also plays an important role in PTSD and dysregulation of Cav1.2 leads to autism spectrum disorders (Cell 119, 19-31). Thus our work on the functional interplay of Cav1.2 with ?-actinin and calmodulin is of high significance for understanding and ultimately treatment of these brain diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond including learning and memory.

PREDOCTORAL TRAINING IN PHARMACOLOGICAL SCIENCES ABSTRACT: The overarching goal of this predoctoral Pharmacological Sciences Training Program (PhTP) is to educate next generation of biomedical researchers in the concepts of drug discovery and development and to provide a clinical perspective. Trainees come mainly from 4 graduate programs (Pharmacology & Toxicology, Physiology, Biomedical Engineering and Neuroscience) developing expertise in diverse areas. This reaches from classic pharmacology and drug target identification with cutting edge methods in biochemistry, structural biology, genomics molecular and cell biology, high resolution imaging, electrophysiology and behavioral physiology, to medicinal chemistry, engineering of microfluidic and other devices, animal models of disease, novel in vivo whole animal imaging and translational therapeutics in clinical trials. The PhTP will provide focused and student-tailored small group training in the core principles of pharmacology for non-pharmacology trainees, and enmesh these students together with pharmacology students for more interdisciplinary group learning in the drug discovery and development. A second goal is to enable all trainees to communicate and collaborate across the large array of research disciplines they represent. This goal is mainly realized in a highly innovative student-driven, project-oriented course Problem Solving in Pharmacological Sciences, which reinvents itself every year based on student initiative and interest. In this way our PhTP produces experts with a variety of backgrounds that can effectively communicate and collaborate with experts from other related disciplines in the increasingly complex realm of drug development. UC Davis has an unusually strong multidisciplinary and collaborative environment related to this PhTP. UCD grants more bachelors and doctoral degrees in biological sciences than any other US university and ranks 12th in the country in extramural research funding awarded to public universities ($800 million annually). The 51 training faculty are from 22 departments in 6 colleges, where extensive collaborative interactions already exist. Trainers provide in depth expertise that ranges from identifying novel therapeutic molecular targets and development of therapeutic molecules to clinical drug and stem cell trials at the NIH-funded UCD Clinical and Translational Science Center (CTSC) and NIH-designated Cancer Center. Novel drugs for treatment of cardiovascular, neurological, and immunological diseases and cancer, the four focus areas of our PhTP, have been developed and are being brought to clinic by several faculty at UCD. The very rich and collaborative overall science environment at UCD, powerful and numerous state-of- the-art core facilities and centers will provide trainees with outstanding research opportunities spanning from Chemistry's emphasis on pharmaceutical chemistry, imaging molecules (from single molecule to in vivo), genomics, molecular/system modeling, stem cells, unique animal models (nationally recognized mouse center, Veterinary School and Primate Center) and CTSC.

Request for Research Supplement for R01 AG055357 through the PA-18-591 mechanism Abstract for Supplement of R01 AG055357 ?Biochemical and functional interactions of a-actinin and calmodulin with Cav1.2? Our long-term interest is to determine the molecular mechanisms that regulate the L-type channel Cav1.2 in health and disease (e.g., Science 293, 98; Science 293, 2205; PNAS 103, 7500; Neuron 78, 483). We found that a-actinin binds directly to the IQ motif of Cav1.2 and augments its surface localization (Neuron 78, 483). Supported by the parental R01 AG055357 grant we show through structural analysis, which informed point mutations in the IQ motif, that binding of a-actinin to the IQ motif strongly augments channel open probability (Po). Aim 1 is a comprehensive analysis of trafficking kinetics of WT and point mutated Cav1.2. Aim 2 is to determine current density, gating currents, and single channel currents for a-actinin binding - deficient point mutations of Cav1.2. Our structural analysis has guided charge reversal experiments for unequivocal assignment of deficits in surface expression and Po in the Cav1.2 mutants to loss of a-actinin binding (under full review by Neuron). We also found that Ca2+ influx through Cav1.2 leads to displacement of a-actinin from the IQ motif by Ca2+/calmodulin and in parallel to endocytosis of Cav1.2 (readied for publication). Aim 3 is to test the role of a-actinin in Ca2+ influx into spines and regulation of gene expression via NFAT, whether b- amyloid peptide 1-42 augments Po of CaV1.2 by stimulating the CaV1.2-associated b2 adrenergic receptor and thereby S1928 phosphorylation via PKA, and whether a-actinin association of Cav1.2 is increased in rodent models of senility and Alzheimer?s disease (AD). Increased Cav1.2 channel activity contributes to senile symptoms and AD (e.g., Science 272, 1017; Science 243, 809). Thus our work on the functional interplay of Cav1.2 with a-actinin and calmodulin is of high significance for understanding and ultimately development of treatments of these brain diseases. On a broader perspective it is of physiological importance as it will define important aspects that govern the functional availability of Cav1.2 with its manifold functions in neurons and beyond including learning and memory. The Aim for the request of a Supplement is to define by Cyro-electron microscopy the structure of CaV1.2 in complex with a-actinin obtained from healthy rats and compare with the CaV1.2/a-actinin structure isolated from B6.Cg-Tg(APP695)3Dbo Tg(PSEN1dE9)S9Dbo/Mmjax mice. Rationale: Consistent with our finding that surface expression of CaV1.2 increases during aging in rats (Aging Cell 13, 111-120), we found that a-actinin binding to CaV1.2 is also strongly increased in aging rats. Thus, during aging the propensity of CaV1.2 to bind to a-actinin is augmented, which must be due to conformational changes possibly because of alterations in the splicing of the channel mRNA or its phosphorylation. We will determine the structural changes that mediate increased a-actinin binding to CaV1.2.

Detection of Synaptic Proteins with Fluorescent Molecular Rotor-labeled Peptides Abstract Our goal is to establish routine methodology for development of synthetic peptides for instant and specific detection of endogenous unmodified proteins in fixed and living neurons. We will screen One Bead One Compound (OBOC) combinatorial peptide libraries invented by co-investigator (Co-I) Kit Lam (Nature 354, 82- 84) for peptides with fluorescent molecular rotors (FMRs). FMR peptides will fluoresce only when specifically bound to their target protein but not when free in solution. Initial focus will be on the key postsynaptic proteins of glutamatergic synapses (AMPARs (AMPA-type glutamate receptors), NMDARs (NMDA-type glutamate receptors), PSD-95 (anchors AMPARs and NMDARs at postsynaptic sites)). Signal to noise ratio (>2300fold) and photostability (by ~ 10fold) is superior to current xFP tags. Peptides will be made membrane permeant with the tat sequence or by myristoylation. This transformative approach will allow detection of proteins within minutes in living systems circumventing technically complicated, time consuming, and expensive genetic protein tagging. It will also dramatically accelerate (by 50fold) protein distribution in fixed cells by high and super-resolution microscop as it will not require the use of primary and secondary antibodies, not even washing steps. FMR peptides can be easily re-synthesized eliminating the variability inherent to antibody probes. Peptides will be reiteratively optimized to achive high affinity and specificity. My long-standing and overarching interest is to determine the molecular mechanisms that govern postsynaptic function (e.g., Science 293,98; Science 293,2205; Science Signaling 10, eaaf9659; Nature 411,801; Neuron 74,1023; Neuron 78,483; Neuron 81,249; Neuron 88,528; Neuron 97, 1094; Neuron 98, 783; EMBO J. 26,4879; EMBO J. 29,482; EMBO J. 31,1203; EMBO J. 33,1341; EMBO J. 35,1330; EMBO J. 36,1330; EMBO J. 37, 122). PSD-95 determines postsynaptic localization of glutamate receptors. Development of FMR-peptides that fluoresce upon specific binding to these proteins will allow live imaging of their localization and of the dynamic changes synapses with those proteins undergo over time at resting and stimulated conditions in cultured neurons and ultimately in the brain in vivo. Ultimately, I envision to develop FMR-peptides for >100 pre- and postsynaptic proteins. Others will apply our technology inside and outside the CNS in all fields of biomedical research. Page 1

Activity-dependent variation in synaptic AMPA receptor (AMPAR) content, referred to as ?synaptic plasticity?, is a mechanism whereby information is stored in neural networks that give rise to higher order cognitive skills such as learning and memory. During long-term potentiation (LTP), a widely studied form of synaptic plasticity, extrasynaptic AMPARs are recruited from nearby reserve pools, including perisynaptic regions on the cell surface and intracellular compartments, and subsequent anchored with the postsynaptic density (PSD). A large body of evidence spanning decades of investigation has established mechanisms by which AMPARs are anchored within the PSD. In contrast, the molecular mechanisms that govern AMPAR synaptic targeting to establish reserve pools of extrasynaptic receptors are largely unknown. Given that recruitment of reserve pools of extrasynaptic AMPARs underlies the rapid strengthening of synapses that occurs during LTP, the molecular mechanisms that establish such reserve pools are critical to our understanding of synaptic plasticity and represent a major gap in our knowledge. SynDIG (Synapse Differentiation Induced Gene) defines a family of four genes (SynDIG1-4) that encode brain- specific transmembrane proteins. Here we will determine the function of SynDIG4 (SD4), also known as Prrt1 (Proline-rich transmembrane protein 1) in the regulation of the reserve pool of AMPARs. Proteomic studies indicate that SD4 is a component of AMPAR complexes; however, SD4 is not enriched in the PSD, but instead colocalizes with GluA1-containing AMPARs at non-synaptic sites. Remarkably, tetanus-induced LTP, which is dependent on GluA1, is abolished in acute hippocampal slices from SD4 knockout (KO) while theta-burst stimulation LTP (TBS-LTP), which is independent of GluA1, is not impaired. Furthermore, SD4 KO mice exhibit profound deficits in two independent cognitive assays (Morris water maze, novel object recognition), demonstrating a critical role for SD4 in hippocampal-dependent learning and memory. Moreover, extrasynaptic AMPARs are reduced in SD4 KO compared with wild-type (WT) neurons. Given that reserve pools of extrasynaptic AMPARs are critical for synaptic plasticity, we hypothesize that SD4 maintains such reserve pools of extrasynaptic GluA1-containing AMPARs that are deployed during tetanus-induced LTP. In this collaborative dual-PI application we propose a comprehensive multidisciplinary approach to investigate SD4-dependent regulation of extrasynaptic GluA1-containing AMPARs with molecular, cellular, and electrophysiological methods. In Aim 1 we will define the mechanism by which SD4 maintains extrasynaptic GluA1-containing AMPARs. In Aim 2 we will determine whether synaptic targeting of reserve pools of GluA1- containing AMPARs during plasticity requires SD4. In Aim 3 we will test whether homomeric GluA1-dependent synapse plasticity mechanisms require SD4 ex vivo at multiple ages. The results of these studies will provide molecular insight into fundamental mechanisms that govern establishment and maintenance of the reserve pools of extrasynaptic AMPARs critical for synaptic plasticity and address a major gap in our knowledge.