Postdoc Research Symposium 2016 Location: Genentech Hall, Mission Bay, UCSF Date: Friday June 17th, 2016

Program and Abstracts

Postdoc Research Symposium 2016 Location: Genentech Hall, Mission Bay, UCSF Date: Friday June 17th, 2016

Program: 08:00 - 08:45 Registration & breakfast 08:45 - 09:00 Opening remarks by Elizabeth Watkins, UCSF Grad Dean and VC Student Academic Affairs 09:00 - 09:45 Keynote Speaker: Laura A. Schmidt The bigger picture: Getting political leverage on obesity and diabetes 09:45 - 10:45 Postdoc talks session I: Biology I 1. Alba Gonzales Junca: Densely ionizing radiation effects promotes more aggressive epithelial tumors 2. John Chen: Identification of cellular factors that control Tau aggregation and propagation 3. Alexander Kintzer: Structure of a vacuolar two-pore channel TPC1 10:45 - 11:15 Coffee break 11:15 – 12:15 Postdoc talks session II: Translational 4. Rosaura Esteve-Puig: Long noncoding RNA as a potential target in drug resistant melanomas 5. Ana Ruiz-Saenz: Novel strategy to target the undruggable HER3 in human cancers: blocking its translocation into the endoplasmic reticulum 6. Poornima Ramkumar: Tumor microenvironment dependent control of myeloma cell growth and drug response 12:15 – 12:30 12:30 – 02:00 02:00 – 02:30 02:30 – 03:30

Flash Poster talks: 3 x 3min Poster session & lunch Panelist introduction (Luke Bonser) Panel session: The future of postdoc research With: Gary McDowell (Future of Research), Katie Peer Thompson (P-value) & Dmytro Lituiev (Union) 03:30 – 04:15 Coffee break 04:15 – 05:15 Postdoc talks session III: Biology II 7. Nadege Morisot: mTORC1 in the orbitofrontal cortex drives habitual responding for alcohol 8. Tomasz Nowakowski: Developing cerebral cortex at single cell resolution: mechanisms of development and Disease Vulnerability 9. Zuzana Krejciova: Creutzfeldt-Jakob disease prion propagation in human iPS cell-derived astrocytes 05:15 – 06:00 Conversation with Paul Ekman 06:00 – 08:30 Networking reception and prizes!

Thanks to our sponsors:

Abstract list: 1- Densely ionizing radiation effects promotes more aggressive epithelial tumors Alba Gonzalez Junca, Barcellos-Hoff Lab, Radiation Oncology, Mount Zion Research Center, UCSF 2- Structure of a vacuolar two-pore channel TPC1 Alexander F Kintzer, Stroud Lab, Biochemistry and Biophysics, UCSF 3- Title t.b.d. Wen Li, Hylton, Radiology & Biomedical Imaging, UCSF 4- Inhibition of the integrated stress response restores cognition after brain injury Edel Hennessy, Susanna Rosi, Physical Therapy, UCSF 5- Counterfeit Prescription Medication Containing Fentanyl Xander van Wijk, Clinical Chemistry and Toxicology, Laboratory Medicine, ZSFG 6- Spliceosome cleavage profiling reveals the catalytic activity of the spliceosome across thousands of substrates Jordan Burke, Madhani Lab, Biochemistry and Biophysics, UCSF 7- Title t.b.d Miriam Stoeber, Mark Von Zastrow Lab, Psychiatry, UCSF 8- Long noncoding RNA as a potential target in drug resistant melanomas Rosaura Esteve-Puig, Ortiz-Urda Laboratory, Dermatology, Helen Diller Family Comprehensive Cancer Center, UCSF 9- Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation Cristina Arrigoni, Dan Minor Lab, Cardiovascular Research Institute, Cardiovascular Research Institute, UCSF 10- Title t.b.d. Shruthi Viswanath, Andrej Sali Lab, Bioengineering and Therapeutic Sciences, School of Pharmacy 11- Erastin pre-treatment sensitizes xCT positive tumor cells to radiation therapy Lara Cobler, Timmerman Lab, HDFCCC, UCSF 12- Novel strategy to target the undruggable HER3 in human cancers: blocking its translocation into the ER Ana Ruiz-Saenz, Moasser Lab, Medicine, UCSF 13- Identification of cellular factors that control Tau aggregation and propagation John Chen, Kampmann Lab, UCSF School of Medicine, Institute for Neurodegerative Diseases, UCSF 14- Genetic & microenvironment-dependent control of cancer drug response Poornima Ramkumar, Kampmann Lab, IND, UCSF 15- mTORC1 in the orbitofrontal cortex drives habitual responding for alcohol Nadege Morisot, Dorit Ron's Lab, Neurology, UCSF 16- Regulation of cell polarization by the pseudokinase Tea5 Regina Lutz, Fred Chang Lab, Cell and Tissue Biology, School of Dentistry, UCSF 17- Developing Cerebral Cortex at Single Cell Resolution Tomasz Nowakowski, Kriegstein Lab, Neurology, Stem Cell, UCSF 18- Inequity in noise pollution in the United States Joan Casey, RWJ Health & Society Scholars Program, Center for Health and Community, UCSF and UC Berkeley 19- Title t.b.d. Mark McCormick, Kennedy Lab, Buck Institute for Research on Aging 20- Increased Sensitivity to Busulfan in Artemis-deficient Mice Permits Low-dose Conditioning. Sushmita Roy, Cowan Lab, Pediatrics, UCSF 21- Creutzfeldt-Jakob disease prion propagation in human iPS cell-derived astrocytes Zuzana Krejciova, Prusiner Lab , Institute for Neurodegenerative Diseases, UCSF 22- Eggerthella lenta's Bioactivation of Polyphenolic Lignans: Unleashing the Super in Superfoods Elizabeth Bess, Peter Turnbaugh Lab, Microbiology & Immunology, UCSF 23- Control of Dynein-based transport by Bicaudal D family adaptor proteins Andrea Serra-Marques, Dumont Lab, Cell & Tissue Biology, UCSF 24- Generating a novel vector for the overexpression of genes in the rat brain T. Peter Lopez, Prusiner Lab, Neurology, Institute for Neurodegenerative Diseases, UCSF 25- Structure-Based Drug Design for Sodium-dependent Glucose Transporters Paola Bisignano, Grabe Lab, CVRI, UCSF 26- Monitoring dendrite regeneration after injury in vivo Katie Thompson-Peer, Yuh-Nung Jan Lab, Physiology, UCSF 27- The X Chromosome, Aging and Alzheimer's Disease Elena Minones Moyano, Dubal lab, Neurology, UCSF 28- A Novel Mechanism for Neurodegenerative Disease Pathogenesis: Direct Inhibition of Lysosomal Proteolysis Victoria Butler, Kao Lab, Neurology, UCSF 29- Targeting L-selectin on neutrophils as a novel treatment for spinal cord injury Dylan McCreedy, Linda Noble Lab, Neurological Surgery, UCSF

[Poster 1 / Talk 1] Densely ionizing radiation effects promotes more aggressive epithelial tumors Alba Gonzalez Junca, Barcellos-Hoff Lab, Radiation Oncology, Mount Zion Research Center, UCSF Session: BIOL Our studies suggest that tissue architecture disruption (dysplasia) is a result from TGFβ mediated epithelial to mesenchymal transition (EMT). We hypothesized that macrophages, recruited and activated in irradiated tissues, contribute to the loss of epithelial junctions and cell cycle control by TGFβ. To test this, we established an in vitro model using non-malignant human epithelial cells, MCF10A, co-cultured with human macrophages differentiated from PBMC. We irradiated MCF10A with 25 cGy of γ-radiation, or high LET radiation (Fe56 and Si36), and EMT was evaluated using quantitative immunofluorescence for loss of E-cadherin and β-catenin. Co-culture of irradiated MCF10A monolayers with differentiated macrophages, efficiently induced a TGFβ-dependent EMT and fail to display normal acinar morphology in a 3D assay. Dual staining of p-Smad2, a TGFβ signaling readout, and the proliferation marker Ki67, showed that the frequency of pSmad2-Ki67 double positive cells, which are resistant to TGFβ antiproliferative effect, concomitantly increased with EMT. When we blocked the macrophage-secreted proinflammatory cytokine, IFNγ in the co-cultures of macrophages and irradiated epithelial cells, the induction of EMT and the increased frequency of TGFβ-resistant cells were prevented, suggesting that IFNγ is a mediator of the macrophages effect. Interestingly, we only observed a complete EMT phenotype accompanied by an increase in the frequency of cells resistant to TGFβ anti-proliferative effect when epithelial cells were irradiated with high LET radiation, revealing a radiation quality dependence in the ability of macrophages to prime epithelial cells to generate dysplasia and TGFβ–resistant cells.

[Poster 2 / Talk 3] Structure of a vacuolar two-pore channel TPC1 Alexander F Kintzer, Stroud Lab, Biochemistry and Biophysics, UCSF Session: BIOL Two-pore channels (TPCs) comprise a subfamily of eukaryotic voltage- and ligand-gated cation channels that contain two non-equivalent tandem pore-forming subunits that then dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking of filoviruses, excitability, and cellular amino acid homeostasis. TPCs are activated by a decrease in transmembrane potential, an increase in cytosolic Ca2+ ions, and inhibited by luminal low pH and calcium, and regulated by phosphorylation. We report the crystal structure of TPC1 from Arabidopsis thaliana at 2.87Å resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains, and regulatory ion-binding sites. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate cytoplasmic Ca2+-activation. One of the two voltage sensing domains (VSD2) encodes voltage sensitivity and inhibition by luminal Ca2+ locks VSD2 in a resting conformation, distinct from the activated VSDs observed in structures of other voltage-gated ion channels. The structure shows how potent pharmacophore trans-Ned-19 allosterically acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection by Ebola virus and Filoviruses presumably by altering their fusion with the endolysosome, and delivery of their contents into the cytoplasm. The structure of TPC1 paves the way for understanding the complex function of these channels and may aid the development of antiviral compounds.

[Poster 3] Wen Li, Hylton Lab, Radiology & Biomedical Imaging, UCSF Session: TRAN This study was performed to investigate improvements in prediction of pathologic complete response by breast MRI for patients in a multi-center neoadjuvant clinical trial. The investigation was performed by breast cancer subtype defined by hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status. Women with locally advanced breast cancer (tumor size ≥ 3cm) were enrolled. Each patient had four dynamic contrast-enhanced MRI examinations: before NACT (MRI1), after one cycle of NACT (MRI2), between the anthracycline-based regimen and taxane (MRI3), and after NACT and prior to surgery (MRI4). Percentage change in functional tumor volume (ΔFTV2-4) was used to predict pathologic complete response (pCR). FTV was re-calculated using thresholds of: early percentage enhancement (PE) was varied from 30% to 200% in steps of 10% and signal enhancement ratio (SER) from 0 to 2 in steps of 0.2. More improvements in AUC caused by optimization were observed in subtypes than in the full cohort. The most improvement in AUC for predicting pCR occurred at various thresholds and treatment time point for each subtype: 140% / 1.4 at ΔFTV2 for HR+/HER2- resulting AUC from 0.71 (CI=0.43−0.99) to 0.83 (CI=0.68−0.99); 130% / 2.0 at ΔFTV3 for HER2+ resulting AUC from 0.61 (CI=0.42−0.80) to 0.75 (0.60−0.91); 140% / 0 at ΔFTV3 for triple negatives resulting AUC from 0.78 (CI=0.61−0.95) to 0.85 (0.72−0.99). In summary, improvements in predicting responders can be made by varying PE and SER thresholds for calculating FTV, and by analyzing the data within tumor subtypes.

[Poster 4] Inhibition of the integrated stress response restores cognition after brain injury Edel Hennessy, Susanna Rosi Lab, Physical Therapy, UCSF Session: BIOL The integrated stress response (ISR) inhibits normal mRNA translation via eIF2α phosphorylation. ISRIB is an ISR inhibitor that enhances memory in normal animals. Loss of cognitive function and sustained ISR are associated with traumatic brain injury (TBI). We investigated the efficacy of ISRIB on TBI induced cognitive deficits using two models. Controlled cortical impact (CCI) models focal contusion injury in C57BL/6 mice. Spatial learning and memory retention were measured in the radial arm water maze 28 days post-injury. ISRIB (2.5mg/kg) or vehicle were administered intraperitoneally prior to each training day. CCI animals failed to learn the location of the escape platform, where CCI-ISRIB animals learned as well as sham. Memory consolidation was measured 24 hours and 7 days post-training. CCI-ISRIB animals performed as well as sham indicating ISRIB restored the ability of CCI animals to learn and remember a new task. LTP was examined in the CA1 with CCI-vehicle animals showing a decreased LTP level, which was restored by ISRIB. Closed-head injury (CHI) models diffuse injury in C57BL/6 mice and the delayed-matching-to-place paradigm was used to assess working/episodic-like learning and memory. Fourteen days post-injury ISRIB (2.5mg/kg) or vehicle were administered intraperitoneally prior to each training day. ISRIB treated animals performed as well as controls, indicating that ISRIB reversed the CHI deficits. Dendritic spine density was examined in the CHI model; there was a hyper-density in CHI which was diminished by ISRIB. In these models targeting the ISR can completely reverse chronic loss of cognitive functions induced by head trauma.

[Poster 5] Counterfeit Prescription Medication Containing Fentanyl Xander van Wijk, Clinical Chemistry and Toxicology, Laboratory Medicine, ZSFG Session: TRAN The United States is experiencing an opioid epidemic with synthetic opioids such as fentanyl responsible for the highest rise in death rates in recent years. Fentanyl, a potent opioid receptor agonist, can cause significant central nervous system and respiratory depression. From the months of October to December 2015, eight patients were referred to the California Poison Control System San Francisco after ingesting a counterfeit Xanax product sold by street dealers, as well as one fatality. We analyzed biological samples from these patients and concluded that victims ingested large quantities of fentanyl, and in some cases etizolam, a benzodiazepine analog. Analysis of counterfeit tablets revealed the presence of a high concentration of fentanyl as well as etizolam, whereas the active ingredient of Xanax, alprazolam, was absent. Subsequently, during March and April 2016, seven cases of counterfeit Norco ingestion and intoxication were identified in San Francisco. These counterfeit Norco tablets most likely originated from Sacramento, where 52 cases and 12 fatalities were identified. Whereas Norco typically contains acetaminophen and hydrocodone, we found that these counterfeit tablets predominantly contained fentanyl and promethazine. Promethazine likely potentiates the opioid effect. Both fentanyl and promethazine were also found in all patient samples. In conclusion, the distribution of counterfeit pharmaceutical products containing fentanyl resulted in significant morbidity and mortality, and continues to represent a major public health threat.

[Poster 6] Spliceosome cleavage profiling reveals the catalytic activity of the spliceosome across thousands of substrates Jordan Burke, Madhani Lab, Biochemistry and Biophysics, UCSF Session: BIOL The faithful expression of genes requires precise recognition of exon-intron boundaries. It has long been hypothesized that the staggering complexity of the spliceosome and its dynamic remodeling reflects the evolution of proofreading mechanisms that detect and edit errors that would otherwise undermine the fidelity of gene expression. However, because the error rate of the spliceosome is unknown, it is unclear to what extent errors are edited by the spliceosome itself versus downstream RNA turnover mechanisms. Current methods for detecting spliceosomal cleavage events are generally limited to products that are stabilized by mutants in decay mechanisms (which can have secondary effects). To directly assess splicing fidelity, we have developed a method, termed spliceosome cleavage profiling, to visualize the endonucleolytic events that occur within active spliceosomes. As a model, we use Cryptococcus neoformans, an intron-rich yeast whose genome harbors 40,946 introns. To generate profiles, we immunoprecipitate endogenous spliceosomes from cell extracts while preserving the the association of substrates, intermediates and products. We elucidated the sites of cleavage by using ligation to 3’ hydroxyls of the extracted RNA and Illumina sequencing. Remarkably, in addition to sharp signals at 5’ and 3’ splice sites, we observe novel signals within exons and introns that are likely aberrant cleavages. By enabling quantitation of spliceosome-bound precursor and the products of both steps of splicing, our method provides the means for assessing splicing fidelity in vivo in an unperturbed cell and enable tests of the role of spliceosomal complexity in the fidelity of gene expression.

[Poster 7] Miriam Stoeber, Mark Von Zastrow Lab, Psychiatry, UCSF Session: BIOL Recent advances in understanding opioid receptor (OR) function have provided tools to manipulate receptors, activate ligands, and detect downstream signaling cascades but so far it has not been possible to directly sense receptor activation. Here we utilize a recently generated G protein mimetic nanobody for the μ-OR to develop a biosensor that specifically binds ORs in an active state. We establish a TIRF assay to sense OR activation and deactivation cycles in living cells and demonstrate biosensor specificity, rapid binding kinetics, and reversibility. Strikingly, our results reveal that within minutes of opiate or opioid peptide addition, internalized μ- and δ-ORs display an active conformation in endosomes as detected by a second wave of biosensor recruitment. Endosomal OR activation is ligand dependent and rapidly reversible by antagonist. We show that active δ-ORs in endosomes mediate sustained inhibition of adenylyl cyclases (AC) after agonist wash out, which suggests that all modules of Gi-protein and AC signaling cascades are localized to or cross-talk with endosomes. Our results advance the spatiotemporal understanding on cellular OR signaling and may have exciting potential for pharmacology and therapeutics.

[Poster 8 / Talk 4] Long noncoding RNA as a potential target in drug resistant melanomas Rosaura Esteve-Puig, Ortiz-Urda Lab, Dermatology, Helen Diller Family Comprehensive Cancer Center, UCSF Session: TRAN Melanoma is the most common lethal form of skin cancer. Major advances in targeted therapy for melanoma have been made and show a dramatic initial response. However, many patients develop resistance to the treatment within weeks to months of its initiation. Activating mutations in BRAF/ NRAS oncogenes account for over 80% of all melanomas and hyper-activate the MAPK pathway. Long noncoding RNAs (lncRNAs) are a new class of epigenetic regulatory molecules that have emerged as important players in cancer development and progression. Recent scientific discoveries indicate that lncRNAs can serve as potential biomarkers, prognostic indicators, predictors of therapeutics response and targets for new treatments. Our study aims to find novel targets involved in the acquisition of resistance to MAPK inhibitors. We identified a lncRNA highly differentially expressed in drug resistant melanoma samples, called MAPK Inhibitor Resistance Associated Transcript (MIRAT). If MIRAT is a widely relevant mechanism of acquired resistance it would be a potential target. We determined that MIRAT is an antisense intergenic lncRNA localized mainly in the cytoplasm and found in exosomes from resistant melanoma cells. The silencing and overexpression of MIRAT, as well as preliminary interactome results, showed that MIRAT can modulate the MAPK and PI3K signaling cascades. Our efforts are focused on the elucidation of MIRAT’s functional role, the identification of its DNA/RNA/protein binding partners and the assessment of MIRAT as a potential target in drug resistant melanomas.

[Poster 9] Unfolding of a Temperature-Sensitive Domain Controls Voltage-Gated Channel Activation Cristina Arrigoni, Dan Minor Lab, Cardiovascular Research Institute, Cardiovascular Research Institute, UCSF Session: BIOL Voltage-gated ion channels (VGICs) are outfitted with diverse cytoplasmic domains that impact function. To examine how such elements may affect VGIC behavior, we addressed how the bacterial voltagegated sodium channel (BacNaV) C-terminal cyto- plasmic domain (CTD) affects function. Our studies show that the BacNaV CTD exerts a profound influ- ence on gating through a temperature-dependent unfolding transition in a discrete cytoplasmic domain, the neck domain, proximal to the pore. Structural and functional studies establish that the BacNaV CTD comprises a bi-partite four-helix bundle that bears an unusual hydrophilic core whose integ- rity is central to the unfolding mechanism and that couples directly to the channel activation gate. Together, our findings define a general principle for how the widespread four-helix bundle cytoplasmic domain architecture can control VGIC responses, uncover a mechanism underlying the diverse BacNaV voltage dependencies, and demonstrate that a discrete domain can encode the temperature- dependent response of a channel.

[Poster 10] Shruthi Viswanath, Andrej Sali Lab, Bioengineering and Therapeutic Sciences, School of Pharmacy Session: BIOL Every division a eukaryotic cell assembles a bipolar mitotic spindle to capture and accurately segregate its duplicated genome. Errors in this process are implicated in oncogenesis, developmental errors, and cell death. The structures at the poles of spindles are an aspect of the assembly process that is poorly understood. Here we determine the molecular architecture of the yeast spindle pole body (SPB), the structural and functional equivalent of the metazoan centrosome. Data from in vivo FRET and yeast twohybrid, along with SAXS, electron tomography of purified SPBs, X-ray crystallography, and electron microscopy were integrated by a Bayesian modeling approach. This computational procedure addressed data noise, sparseness and ambiguity and allowed building a low-resolution model of the SPB. The model predicted a previously unidentified layer on the nuclear face of the SPB core, which was confirmed by high-resolution tomography data and has implications for the regulation of pole assembly.

[Poster 11] Erastin pre-treatment sensitizes xCT positive tumor cells to radiation therapy Lara Cobler, Timmerman Lab, HDFCCC, UCSF Session: BIOL Approximately 2/3 of cancer patients receive radiation therapy (RT), with use limited by deleterious effects on normal tissues. RT produces double strand DNA breaks via reactive oxygen species (ROS) generation and fast electron-DNA interactions. Endogenous peroxidases maintain cellular ROS control. Their substrate, glutathione, is normally synthesized from endogenous substrates. However, stressors such as RT or replication stress increase ROS, requiring extra-ordinary amounts of glutathione. This necessitates cystine import, via induction of xCT (SLC7A11). xCT is constitutively expressed on subsets of many solid tumors, and xCT inhibition kills or severely retards tumor growth. We hypothesized that xCT inhibitor treatment would specifically sensitize xCT+ tumors but not surrounding normal xCT- tissues to RT. Here we demonstrate that pretreatment of xCT+ triple negative breast tumor lines with the inhibitor erastin reduces intracellular glutathione levels and sensitizes to gamma irradiation in a preclinical model of radiation therapy that is most commonly used to treat primary breast cancer. Based on preliminary data, we also propose a mechanism of cooperation between xCT inhibition and gamma-radiation.

[Poster 12 Talk 5] Novel strategy to target the undruggable HER3 in human cancers: blocking its translocation into the ER Ana Ruiz-Saenz, Moasser Lab, Medicine, UCSF Session: TRAN There is increasing evidence implicating HER3 in several types of cancer, as a resilient copartner for HER2 in HER2-amplified cancers, and as a driver of resistance in many other types of cancer. However, the development of targeted therapies to inactivate HER3 function has been a challenging endeavor. Its tumor-driving functions are only sometimes driven by ligand-activation, and targeting its extracellular domain with antibodies has only limited effects. Its kinase domain functions in allostery, not catalysis, and the classical ATP-analog class of tyrosine kinase inhibitors fail to inactivate it. Here we describe a novel approach to target HER3: interfering with its cotranslational insertion into the endoplasmic reticulum (ER) eliminating HER3 protein expression and function. The small-molecule cotransin CT8 binds the Sec61 translocon channel in the ER membrane and prevents the nascent HER3 protein from initiating its cotranslational translocation into the lumen of the ER, resulting in the degradation of HER3. In addition, CT8 treatment suppresses the compensatory induction of HER3 that accompanies lapatinib treatment of HER2-amplified cancer cells, making for a durable suppression of HER2-HER3 signaling with the lapatinib-CT8 combination. Consistent with the effective inactivation of HER2-HER3 signaling, CT8 treatment synergistically enhances the apoptotic effects of lapatinib in HER2-overexpressing cancer cells. Our results thus highlight Sec61-dependent processing as a novel strategy to eliminate HER3 expression and function with considerable potential for the treatment of HER2-amplified and other types of cancers.

[Poster 13 / Talk 2] Identification of cellular factors that control Tau aggregation and propagation John Chen, Kampmann Lab, UCSF School of Medicine, Institute for Neurodegerative Diseases, UCSF Session: BIOL The pathologic aggregation of Tau in the human brain is a hallmark of Alzheimer’s disease as well as other neurodegenerative diseases collectively known as Tauopathies. Intriguingly, Tau aggregation has been shown to spread across cell-cell boundaries in a prion-like manner. While some biochemical mechanisms that influence Tau aggregation have been well characterized in vitro, the cellular mechanisms that dictate Tau aggregation and propagation in vivo remain poorly understood. This represents a significant challenge towards the development of therapeutics targeting the pathologic progression of Tau. Here, I will employ complementary approaches to identify and understand how numerous cellular pathways control the fate of Tau aggregates. I have employed a quantitative functional genomics screen to identify genes that impact Tau aggregation in a cell-based model. In our preliminary genome-wide screen, we have identified numerous trafficking and degradation factors in our top hits. To validate our hit genes, I am applying various biochemical techniques to identify how these cellular factors perturb Tau aggregation. In addition, because unique prion-like Tau strains have been identified in various numerous Tauopathies, I will analyze whether these cellular factors control the conversion of Tau aggregates into these distinct conformations. The information gained from these approaches will create new therapeutic targets for treating neurodegenerative diseases associated with Tau aggregation.

[Poster 14 / Talk 6] Genetic & microenvironment-dependent control of cancer drug response Poornima Ramkumar, Martin Kampmann Lab, IND, UCSF Session: TRAN Acquired drug resistance is a major clinical problem in a variety of cancer types. We lack an understanding of the interplay of factors in the tumor and its microenvironment that control drug sensitivity. Several published studies and my own results show that stromal cells reduce the sensitivity of myeloma cells to proteasome inhibitor, carfilzomib. Given the importance of the microenvironment in controlling cancer drug response, there is an urgent need for more complex models that simulate aspects of tumor-microenvironment biology. I have established a 3D co-culture system where the myeloma cells are growing in the presence of stromal cells in porous microcarrier beads that better recapitulates the physiological environment in patients. Our lab has co-developed a CRISPR/Cas9-based functional genomics platform that enables us to conduct genome-wide loss-of-function (CRISPRi) and gain-offunction screens (CRISPRa) to identify genes controlling cell growth and drug response. I have engineered the myeloma cells to express components of the CRISPRi system enabling us to perform genome-wide screening to identify factors that modulate sensitivity to carfilzomib in the mono- and coculture system. Currently, we are performing a pilot CRISPRi screen in cells growing as mono- and cocultures in the presence or absence of carfilzomib. The results from this screen will enable us to dissect the effects of the tumor microenvironment on myeloma drug response. In addition our research will also pave the way to the broad application of this innovative approach to develop effective therapies in other cancer types.

[Poster 15 / Talk 7] mTORC1 in the orbitofrontal cortex drives habitual responding for alcohol Nadege Morisot, Dorit Ron lab, Neurology, UCSF Session: BIOL We previously found that mTORC1 in the rodent nucleus accumbens (NAc), a key component of the brain reward system, is activated by alcohol and in turn, controls alcohol-related behaviors. Here we report that mTORC1 is specifically activated in the orbitofrontal cortex (OFC) of excessive alcohol-drinking rats. The OFC orchestrates goal-directed and habitual actions, which are key steps in the development of AUD. Thus, we hypothesized that mTORC1 in the OFC is required for goal-directed and/or habitual responding for alcohol. To model goal-directed and habit-driven behaviors, respectively, we used an operant paradigm in which rats self-administered alcohol under a random ratio (RR) or a random interval (RI) schedule of reinforcement, and tested whether mTORC1 inhibition or knockdown of raptor, a main component of mTORC1, in the OFC alters goal-directed or habitual responding. We found that intra-OFC infusion of the specific mTORC1 inhibitor, rapamycin, prior the devaluation test, did not affect goaldirected alcohol responding in RR-trained rats. In contrast, rapamycin infusion in the OFC restored the sensitivity to devaluation in RI-trained rats, revealing that mTORC1 in the OFC mediates habitual alcohol responding. We further showed that intra-OFC infusion of rapamycin did not alter habitual responding for sucrose, a natural reward, highlighting a specific role of mTORC1 in the OFC in driving habitual responding for alcohol. Finally, we observed that knockdown of raptor in the OFC restored goal-directed responding for alcohol. Together, our data suggest that alcohol-mediated activation of mTORC1 in the OFC contributes to the expression of habitual alcohol responding.

[Poster 16] Regulation of cell polarization by the pseudokinase Tea5 Regina Lutz, Fred Chang Lab, Cell and Tissue Biology, School of Dentistry, UCSF Session: BIOL Microtubules regulate polarized cell tip growth in the fission yeast Schizosaccharomyces pombe by transporting the cell polarity factors Tea1 and Tea4 on their plus ends to the cell tips. These proteins are critical for these rod-shaped cells to initiate growth at the “new” cell end in a process known as new end take off (NETO). How Tea proteins regulate cell polarization downstream of microtubules remains unclear. Here we identify a new factor Tea5 (also known as Ppk2) required for regulation of cell polarization for NETO. Tea5 is a pseudokinase as it lacks key residues required for kinase activity, and additional mutations in the kinase domain do not affect its function. Overexpression of Tea5 leads to a striking loss of polarity, causing cells to become round. Biochemical assays show that Tea5 forms a tight complex with Tea3; Tea5 localizes in clusters on the plasma membrane at cell tips together with Tea3 and Tea1. Genetic studies suggest that Tea5 functions with Tea3 downstream of Tea1 in Cdc42 activation via the Cdc42 GEF Gef1. Thus, Tea5 defines a novel connection between microtubule +TIP proteins and activation of Cdc42 for cell polarization.

[Poster 17 / Talk 8] Developing Cerebral Cortex at Single Cell Resolution: Mechanisms of Development and Disease Vulnerability Tomasz Nowakowski, Kriegstein Lab, Neurology, Stem Cell, UCSF Session: BIOL Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. By analyzing cell type classification infrences from single cell RNA sequencing data we demonstrate that shallow sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. By extending this approach to study the molecular heterogeneity of single cells from the developing human brain, we are able to discover and predict novel aspects of neural stem and progenitor cell biology, including distinctive behaviors, activation of intracellular signaling pathways, gene expression changes during cell cycle progression, and predict susceptibility to viral infections, including the Zika virus. Finally, we revisit the current knowledge of radial glia heterogeneity in the human cortical development, focusing on the cytoarchitecture of the radial glia scaffold. We propose an extended model of primate cortical neurogenesis with a re-organization of the radial glia scaffold and patterns of migration and neurogenesis during supragranular cortical layer formation.

[Poster 18] Inequity in noise pollution in the United States Joan Casey, RWJ Health & Society Scholars Program, Center for Health and Community, UCSF and UC Berkeley Session: TRAN Background: Prior research reports disparities in environmental exposures, but no nationwide studies have assessed relationships between race/ethnicity and socioeconomic status and noise pollution. Objectives: (1) To assess racial/ethnic and socioeconomic inequities in daytime and nighttime noise pollution in the continental United States and (2) to consider the modifying role of metropolitan level racial residential segregation. Methods: We used a geospatial sound model to estimate Census block group-level L50 nighttime and L50 and L10 daytime noise based on 1.5 million hours of 2000-2014 acoustical data and geospatial features. Block group demographic variables from the 2006-2010 American Community Survey included race/ethnicity, education, poverty, homeownership, and linguistically isolated households. We estimated associations using polynomial terms in spatial error models adjusted for total population and population density. We also evaluated the relationship between race/ethnicity and noise, stratified by levels of racial residential segregation, as estimated by a dissimilarity index. Results: Block groups with a higher proportion of nonwhite and lower socioeconomic status individuals had higher nighttime and daytime noise levels. For example, urban block groups with 50% Black residents had on average nighttime noise levels 3dBs higher than block groups with 0%. Block groups in more segregated metropolitan areas had higher noise levels for all race/ethnicities, but a higher proportion of White residents continued to be associated with lower noise levels. Results were consistent across urban and suburban/rural areas, for daytime and nighttime noise, and with multiple spatial weight and neighbor definitions. Conclusions: We observed consistent inequities in noise pollution. These differences in exposure may help partially explain health disparities in the United States.

[Poster 19] Mark McCormick, Kennedy Lab, Buck Institute for Research on Aging Session: BIOL In the budding yeast S. cerevisiae, yeast mother cells always show a finite replicative lifespan, averaging around 27 divisions. We performed a systematic screen for increased replicative lifespan in yeast, using a nearly complete set of 4,698 viable single-gene deletions, to ask which single-gene deletion genotypes exhibited a markedly increased lifespan. This screen identified 238 genes whose deletion extended lifespan, including 189 not previously reported. Many of these genes cluster into known overrepresented biological processes. These overrepresented groups include ribosomal proteins, the SAGA chromatin remodeling complex, mannosyltransferase genes, and genes involved in tricarboxylic acid cycle metabolism, among others. This comprehensive genome-wide view of genes that influence replicative lifespan could shed light on the fundamental mechanisms of regulation of lifespan. The 238 genes identified show a statistically significant overlap with genes that have been shown to extend organismal lifespan when deleted or knocked down in C. elegans, and this overlap suggests quantitatively that some of the genes and pathways identified in this screen are likely to have broadly conserved effects. Given that genes previously identified in S. cerevisiae and C. elegans have now led to drugs that can extend the lifespan of mice, we view each new gene and biological process identified here with this potential clearly in mind.

[Poster 20] Increased Sensitivity to Busulfan in Artemis-deficient Mice Permits Low-dose Conditioning Sushmita Roy, Allergy Immunology and Blood and Marrow Transplant Division, Dept. of Pediatrics and Benioff Children’s Hospital, UCSF Session: TRAN Currently, hematopoietic stem cell transplantation (HCT) is the only effective cure of T-B-NK+ SCID, but without conditioning with alkylating agents the majority of children with T-B-NK+ SCID fail to reconstitute B cell immunity even with an HLA matched sibling donor. Defects in DCLRE1C, encoding the DNA repair protein Artemis, cause T-B-NK+ SCID (ART-SCID), in which patients are particularly susceptible to high doses of alkylating agents and ionizing radiation. To evaluate potential conditioning approaches for gene corrective autologous HCT we used non-leaky Art-/- mice to evaluate non-alkylating agents in conjunction with very low doses of busulfan (Bu) to open marrow niches. Recipients were C57Bl/6 CD45.2 Art-/- or wild type (WT), and donors were congenic WT CD45.1 mice. The pre-conditioned recipient mice were transplanted with WT CD45.1 lin-cKit+Sca1+ (LSK) sorted cells. First, we evaluated possible synergy of anti-ckit (ACK2) and AMD3100 with non-myeloablative Bu doses. ACK2 pretreatment resulted in significant multilineage engraftment compared to sham-treated controls, while AMD3100 had no added impact on chimerism, and there was no synergy between AMD3100 or ACK2 with 10mg/kg Bu in Art-/recipients. Interestingly, 10mg/kg of busulfan resulted in significant T, B and granulocyte engraftment in all 6 Art-/- but in only 1 of 6 WT recipients, suggesting that Artemis deficiency confers increased sensitivity to this alkylating agent. Also, high donor B cell and neutrophil chimerism were observed in the 10mg/kg Bu-conditioned Art-/- recipients compared to the one engrafted WT recipient. These results show that 1) very low-dose Bu by itself provides effective conditioning in Art-/- mice, resulting in T and B cell reconstitution; and 2) Art-/- recipients are more sensitive than WT to this alkylating agent.

[Poster 21 / Talk 9] Creutzfeldt-Jakob disease prion propagation in human iPS cellderived astrocytes Zuzana Krejciova, Prusiner lab, Institute for Neurodegenerative Diseases, UCSF Session: BIOL Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD). The absence of a human cell culture model, in which human CJD prions replicate, has hampered CJD research for over four decades, and represents a major obstacle in the discovery of effective therapeutics. The mechanisms of neurodegeneration responsible for prion diseases are not completely understood, but increasingly, neuron-glia interactions are thought to be involved. One of the hallmarks of prion disorders is reactive astrogliosis, and some in vivo experiments argue that the PrPSc pathogen replicates both in astrocytes and neurons. Based on these findings, we developed a human cell system for propagating CJD prions in vitro using human iPS cells differentiated into functional astrocytes. Astrocytes were transiently exposed to CJD brain homogenates and further cultured in fresh media for several days. The accumulation of nascent human PrPSc in these cultures was measured by immunoblotting and immunocytochemistry. Our findings are consistent with the hypothesis that cellular susceptibility to prion infection is dependent on (1) an appropriate differentiated post-mitotic cellular phenotype and (2) genotypic homology of agent and host for the critical polymorphic residue at position 129 in PRNP. To our knowledge, this is the first demonstration that normal, functional and non-transformed human cells are susceptible to infection with human PrPSc prions in vitro. This work represents a fundamental advance in modelling human prion disorders by establishing a relevant in vitro model system for studying mechanistic aspects of human prion infection and readily scalable system to facilitate drug discovery.

[Poster 22] Eggerthella lenta's Bioactivation of Polyphenolic Lignans: Unleashing the Super in Superfoods Elizabeth Bess, Peter Turnbaugh Lab, Microbiology & Immunology, UCSF Session: BIOL Small molecules consumed in food and pharmaceuticals are frequently metabolized in the body, changing their function. Liver metabolism of these compounds has been extensively evaluated to develop algorithms that predict their pharmacokinetics. However, these models have largely neglected an aspect of metabolism with significant implications for drug efficacy and toxicity: the human gut microbiome. Encoding an estimated 100-times more genes than the human genome, the trillions of microbial cells in the human gastrointestinal tract are a vast source for small-molecule metabolism. We address the gap at the intersection of pharmacology and the gut microbiome by demonstrating a pipeline to identify the microbial genes responsible for chemical metabolism. The microbiome’s metabolism of dietary molecules is dependent on an individual’s unique set of gut microbial genes, which varies amongst individuals. Identifying the genes that encode small-molecule metabolism will serve as a tool to predict which chemical transformations an individual’s gut microbes can perform. This is exemplified by bacterial metabolism of dietarily abundant, plant-derived lignans. One of the most consumed lignans, pinoresinol, contains a benzyl-ether motif that is reduced by some, but not all, strains of the gut bacterium Eggerthella lenta. This chemical transformation precedes additional bacteria-mediated transformations to produce the uniquely microbial metabolite enterolactone, which is linked to decreased breast-cancer risk. We have identified a bacterial gene responsible for pinoresinol metabolism and explore how it may be used as a diagnostic tool to predict benzyl-ether metabolism and the potential to benefit from dietary-lignan consumption.

[Poster 23] Control of Dynein-based transport by Bicaudal D family adaptor proteins Andrea Serra-Marques, Dumont Lab, Cell & Tissue Biology, UCSF Session: BIOL Cargo transport along microtubules is driven by the collective function of microtubule plus- and minusend-directed motors (kinesins and dyneins). How the velocity of cargo transport is driven by opposing teams of motors is still poorly understood. Here, we combined inducible recruitment of motors and adaptors to Rab6 secretory vesicles with detailed tracking of vesicle movements to investigate how changes in the transport machinery affect vesicle motility. We find that the velocities of kinesin-based vesicle movements are slower and more homogeneous than those of dynein-based movements. We also find that Bicaudal D (BICD) adaptor proteins can regulate dynein-based vesicle motility. BICD-related protein 1 (BICDR-1) accelerates minus-end-directed vesicle movements and affects Rab6 vesicle distribution. These changes are accompanied by reduced axonal outgrowth in neurons, supporting their physiological importance. Our study suggests that adaptor proteins can modulate the velocity of dyneinbased motility and thereby control the distribution of transport carriers.

[Poster 24] Generating a novel vector for the overexpression of genes in the rat brain T. Peter Lopez, Neurology, Institute for Neurodegenerative Diseases, UCSF Session: BIOL Abstract: The gene that encodes the prion protein, Prnp, is one of the most highly expressed genes in the mammalian nervous system. Given this observation, we set out to develop a translational tool that utilizes the rat Prnp locus to drive ectopic gene expression in the rat brain. We compared the genetic structure of rat Prnp to mouse and human and identified conserved cis-regulatory elements that may be crucial for brain-specific Prnp expression. We tested the hypothesis that these conserved Prnp elements are capable of driving neuronal gene expression by generating a vector that encompasses a portion of the rat Prnp genetic locus. Importantly, the rat Prnp open reading frame (ORF) was replaced with an XhoI restriction site for cloning. To validate the in vivo neuronal specificity of this vector in rats, we generated a Prnp-LacZ/EGFP construct and generated two transgenic Tg(Prnp-LacZ/EGFP) rat lines. Both reporter rat lines exhibit ectopic LacZ activity and EGFP fluorescence in embryonic and adult brains. For functional validation, we restored the rat Prnp ORF and generated multiple lines of Tg(Prnp-ratPrP) rats. We are currently testing whether overexpression of Prnp accelerates onset of prion disease. This novel vector provides a much-needed tool for the study of multiple human neurodegenerative diseases and psychiatric disorders in rats.

[Poster 25] Structure-Based Drug Design for Sodium-dependent Glucose Transporters Paola Bisignano, Grabe Lab, CVRI, UCSF Session: BIOL Sodium-dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport sugars across the plasma membrane. In humans, there are six subtypes, SGLT16, expressed widely in the small intestine, kidney, lung, muscle and brain . Due to their role in sugar reabsorption, SGLTs are currently exploited as a drug target for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part because high resolution structures of mammalian SGLTs do not exist. However, the structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved in complex with galactose opening up the possibility for structure based drug design . vSGLT is in an inward facing conformation poised to deliver the sugar to the cytoplasmic space. Here we combine homology modeling, pharmacophore searching, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) identify inward-facing and outward-facing binders to vSGLT to better understand the mode of action of drugs that inhibit hSGLT2, and 2) identify binders to vSGLT that can be used to trap the transporter in different states for use in biophysical experiments.

[Poster 26] Monitoring dendrite regeneration after injury in vivo Katie Thompson-Peer, Yuh-Nung Jan Lab, Physiology, UCSF Session: BIOL Neurons receive information along dendrites, and send electrical signals along axons to synaptic contacts. The factors that control axon regeneration have been examined in many systems, but research into dendrite regeneration has been almost entirely neglected. Ramón y Cajal observed that dendrites in the central nervous system fail to regenerate after severing, yet dendrite sprouting is observed in the spinal cord after contusion. Traumatic brain injury, stroke, and many forms of dementia are characterized by simplifications of dendritic morphology, yet we don’t know when dendrite regeneration occurs. The extensive literature on normal dendrite patterning of class IV da sensory neurons in the Drosophila peripheral nervous system allows us to explore fundamental features of dendrite regeneration, specifically looking at the ways in which dendrite growth after injury is similar to or different from normal dendrite outgrowth. We observe that robust injury-induced growth occurs after full dendrite removal. In many ways regenerated dendrites are like uninjured dendrites, such as the trafficking of sensory ion channels and the expression of cell-type specific transcription factors. However the growth and patterning of injury-induced dendrites is significantly different from uninjured dendrites. Unlike normal dendrites, regenerating dendrites fail to avoid crossing over other branches from the same neuron. Regenerated dendrites are also pruned differently from uninjured branches. Injuring neurons as different developmental stages highlights the variable patterning of regrowing dendrites. Together our work describes essential features of dendrites grown in response to acute injury.

[Poster 27] The X Chromosome, Aging and Alzheimer's Disease Elena Minones Moyano, Dubal lab, Neurology, UCSF Session: BIOL Males die before females and this sexual dimorphism extends to neurodegenerative conditions such as Alzheimer’s disease (AD). We have shown that sex chromosomes mediate male disadvantage using human amyloid precursor protein (hAPP) mice that model aspects of AD. To dissect whether it is the presence of a Y or the lack of a second X chromosome, that underlies male disadvantage in AD-related toxicity we used XY* mice. These mice give rise to four sex genotypes roughly equivalent to: XX and XO with ovaries, and XY and XXY with testes. We crossed XY* males with hAPP females, to produce eight genotypes exhibiting varying dosages of X and Y +/-hAPP. Male and female mice with one X (XY-hAPP and XO-hAPP) died faster and showed worse cognitive deficits than those with two X’s (XX-hAPP and XXY-hAPP). The Y chromosome did not govern mortality or deficits. Next, we tested whether the second X-chromosome confers neural resilience at the cellular level. In parallel with our in vivo findings, XY and XO neurons showed increased cell death following acute Aβ toxicity, compared to XX and XXY neurons. To gain insight into X-factors that may contribute to brain resilience in humans, we evaluated expression of the X chromosome in the frontal cortex of elderly individuals from the Memory and Aging Project and Religious Orders Study. Preliminary results revealed a subset of X-encoded genes that correlate with cognitive resilience in aging humans. Our study reveals a new role for the X chromosome in counteracting mortality, cognitive deficits, and cellular toxicity related to AD. Identification and therapeutic modulation of X-factors and/or X-mediated protective mechanisms may help increase longevity and resilience to neurodegeneration in both men and women.

[Poster 28] A Novel Mechanism for Neurodegenerative Disease Pathogenesis: Direct Inhibition of Lysosomal Proteolysis Victoria Butler, Kao Lab, Neurology, UCSF Session: BIOL Neurodegenerative diseases lead to impaired protein homeostasis. The lysosome plays a key role in proteostasis through its function breaking down and recycling macromolecules. Lysosomal proteases are classified as serine, cysteine or aspartyl cathepsins based on their active site residue. Although peptide based serine and cysteine protease inhibitors have been described (serpins and cystatins, respectively), aspartyl protease inhibitors remain undefined. Progranulin is a highly conserved lysosomal protein whose deficiency results in the neurodegenerative disease frontotemporal lobar degeneration (FTLD) and whose absence causes a lysosomal storage disorder, neuronal ceroid lipofuscinosis (NCL). Despite its importance, the function of progranulin and its bioactive cleavage products, the granulins, are unknown. Here we describe a role for granulins as direct aspartyl protease inhibitors. We show that progranulin production and cleavage into granulins increases with age and stress. Granulins impair ER stress resistance in C. elegans and alter lysosome morphology. Granulin 3 co-immunoprecipitates the lysosomal aspartyl protease ASP-3 and occludes the active site of ASP-3 to inhibit protease activity.

[Poster 29] Targeting L-selectin on neutrophils as a novel treatment for spinal cord injury Dylan McCreedy, Linda Noble Lab, Neurological Surgery, UCSF Session: BIOL Spinal cord injury (SCI) results in extensive infiltration of pro-inflammatory leukocytes that promote secondary damage through the release of neurotoxic substances. We have previously shown that Lselectin, a lectin-like receptor broadly expressed on the surface of leukocytes, contributes to inflammation and damage early after spinal cord injury. Pharmacological abrogation of L-selectin via systemic delivery of diclofenac (DFA), an FDA-approved non-steroidal anti-inflammatory that activates the cleavage (shedding) of L-selectin by cell surface metalloproteases, improved long term neurological recovery following SCI. However, the specific leukocyte subtypes affected by DFA treatment remain unknown. We examined the expression of L-selectin (CD62L) in leukocyte subsets from the peripheral blood of uninjured mice by flow cytometry and found high L-selectin expression in inflammatory monocytes and neutrophils while expression of L-selectin in patrolling monocytes was low. To determine which of the leukocyte subsets are susceptible to L-selectin cleavage, we systematically administered a single bolus injection of DFA or vehicle and performed flow cytometry at 2, 8, and 24 hours post-injection to assess the onset and duration of L-selectin shedding. While there were no differences in the expression of Lselectin on patrolling or inflammatory monocytes between DFA- and vehicle-treated mice at any time point examined, DFA resulted in a modest yet significant increase in L-selectin on neutrophils at 2 hours post-injection followed by robust shedding of L-selectin on neutrophils at 8 and 24 hours post-injection. These findings reveal the preferential shedding of L-selectin on neutrophils and suggest that L-selectin expressed on neutrophils contributes to acute damage after SCI.