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Nan Zhang, Houston Methodist Research Institute

"Investigation of Toxic RNA-mediated Disease Mechanism in Spinocerebellar Ataxia Type 10"

Abstract:

Spinocerebellar ataxia type 10 (SCA10) is a neurodegenerative disease caused by pathological ATTCT repeat expansion in intron 9 of the ATXN10 gene. A toxic RNA gain-of-function is central to the disease etiology in that the expanded RNA repeats form nuclear aggregates (foci) that sequester various RNA binding proteins from their normal functions. Recent studies also suggest that, in several neurodegenerative diseases, once the repeat RNAs are exported to the cytoplasm, they undergo repeat-associated non-AUG (RAN) translation to yield toxic homopolypeptides in affected brain regions. In the case of SCA10, patients that express pure ATTCT repeat expansions only show mild ataxia, while patients with large ATTCC or ATCCC repeat interruptions exhibit severe ataxia and epilepsy. The molecular mechanism of different repeat types with respect to phenotypic stratification is currently unknown. This study set to investigate the contribution of each SCA10 repeat type to the toxic RNA gain-of-function. By performing fluorescence in situ hybridization (FISH) with multiple locked nucleic acid (LNA)-based probes, we demonstrate that different SCA10 repeats can form nuclear RNA foci in two patient-derived fibroblast cell lines and SCA10 mouse brain. Different repeat foci can colocalize in patient cells, suggesting that they come from the same intronic RNA transcript and offering potential diagnostic value for forecasting patient phenotype. By exploiting a “Biobrick cloning” technology in combination with Type IIS restriction enzymes, we generated a range of ATTCT, ATTCC and ATCCC repeat lengths (from 19 to 108) for in vitro and cell line studies. These constructs were cloned into designer vectors so that homopolymeric RAN translation products from each reading frame can be identified using unique tags on immunoblot. As many repeat expansions are bi-directionally transcribed, the antisense repeats may further contribute to RAN toxicity in terms of foci formation and RAN translation. By performing strand-specific cDNA synthesis, we were able to detect antisense SCA10 repeats in three patient-derived cell lines, which could potentially increase the toxic RNA load and sequestration of different RNA binding proteins. Taken together, the current study suggests that an RNA gain-of-function is occurring in all the SCA10 repeat types tested and that their impacts on protein sequestration and RAN translation in cell and animal models requires further investigation.

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Noah Whiteman, Department of Integrative Biology, University of California Berkeley

"Navigability of the Monarch Adaptive Landscape for Cardiac-glycoside Resistance"

Abstract:

The independent origin of the same adaptations throughout the history of life indicates that evolution is a predictable process 13. Novel adaptions often arise by stepwise changes in a lineage. An adaptive landscape is a helpful way to consider the collection of all possible paths that lead to a novel adaptive trait 12. Maynard Smith’s “Natural Selection and the Concept of Protein Space” asserts that, due to natural selection, intermediate steps in a path must either increase or maintain the level of fitness conferred by the previous step. The implication is that navigability within an adaptive landscape can be predictable, and therefore evolutionary paths can be predictable. Studies in the adaptation of drug resistance affecting highly conserved proteins in single celled organisms (e.g. E. coli and P. falciparum) have demonstrated emerging rules about navigability, however, such rules have not yet been well-studied in multicellular organisms 5, 9.

I was a co-author on a recent publication that studied the relative costs and benefits of recurrent mutations in a textbook example of convergence. In that case, we reasoned cardiac glycoside resistance in insects adapted to toxic cardiac glycoside-producing plants (e.g., foxglove and milkweeds) is a well suited trait to study adaptive landscape navigability. Numerous cardiac glycoside-adapted insects have convergently evolved the same sets of candidate resistance substitutions in the α subunit (ATPα) of the sodium-potassium pump, the only molecular target of cardiac glycosides. We previously found mutational paths to resistance involving substitutions at three amino acid positions, 111, 119, and 122, within the ATPα protein 5. We found that all mutational paths experienced amino acid replacements at position 119 prior to changes at position 122 - a pattern unlikely to be due to chance. We retraced the path to resistance in the monarch butterfly (Danaus plexippus) lineage by engineering Drosophila knock-in lines with the observed combinations of mutations at positions 111, 119, and 122 using CRISPR-Cas9 mediated homology directed repair (HDR). The mutational path of the monarch butterfly increases resistance to the cardiac glycoside, ouabain, at each mutational step 5, 9. In addition, the mutation to a serine from an alanine at position 119 increased resistance though epistasis and decreased a pleiotropic fitness cost (seizures following physical shaking) 5.

In this thesis I will study whether navigability within the monarch adaptive landscape is predictable. We define the monarch adaptive landscape as all possible mutational paths that start from the non-resistant ATPαQAN protein and end at the resistant ATPαVSH protein, separated by several intermediate step-wise mutations (Figure 1). In our previous study, we generated seven out of twelve D. melanogaster knock-in lines representing steps in the monarch adaptive landscape 5. Here, we have generated the ATPαLSH knock-in line, representing an alternative key intermediate step in the monarch adaptive landscape because (i) ATPαLSH represents an alternative end point in the adaptive landscape, as high resistance is expected for this combination of amino acid replacements 5 , (ii) half of all possible pathways within the monarch adaptive landscape include ATPαLSH as an intermediate step (iii) ATPαLSH has not been observed in nature, and therefore might restrict navigability within the monarch adaptive landscape.

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Dr. Elliott Sherr, University of California, San Francisco

"Gene and variant level differences in Coffin Siris Syndrome"

Abstract:

Previous clinical studies have identified Coffin Siris Syndrome (CSS) as a congenital disorder that presents when genes of the SWItch/Sucrose NonFermenting (SWI/SNF) complex are mutated. CSS is characterized by hypotonia, coarse facial features, intellectual and developmental delay, hypoplastic fifth fingernails and/or toenails, and growth deficiency. In collaboration with other lab members and a national clinical registry of CSS patients, I recruited a cohort of 111 patients to examine their clinical and imaging data in the context of specific genes and variant types. I designated eight SWI/SNF genes to explore including ARID1A, ARID1B, ARID2, DPF2, SMARCA4, SMARCB1, SMARCE1, and SOX11. In combination with whole exome sequencing (WES) performed by the Broad Institute and results analyzed by the Sherr Lab, I obtained clinical and imaging data to assess the phenotypic frequency of brain malformations, and the severity of developmental delay, growth delay, and neurological abnormalities. Preliminary data indicates that the presence or absence of a corpus callosum malformation is not uniform across genes, nor does it carry the same impact. Furthermore, different brain imaging groupings may possibly confer different developmental and clinical phenotypes. Ongoing analysis of these listed genes will enable us to demonstrate a correlation between specific mutations, brain malformation phenotypes, and their association with clinical findings such as coarse facies, growth delay, and additional affected organ systems. I hope to outline gene and variant level differences in the phenotypic features and possible severity of the CSS disorder. With a better understanding of these differences and correlations, these data can improve knowledge about the spectrum of Coffin Siris Syndrome.

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Daniel Feldman, PhD, Department of Molecular & Cell Biology

"Viral Expression of the Optogenetic Inactivator Halorhodopsin 3.0 in Whisker Somatosensory Cortex"

Abstract:

Optogenetics is a technique that utilizes light to control neurons that have been modified to express light-sensitive ion channels. To attain precise control in behavioral and physiology experiments, viral vectors are used to express the inhibitory optogenetic reagents either in specific cell classes within a cortical area or in cells that project to that cortical area. To do this, a virus is injected into a particular brain region and targeted for the cell type of interest, causing those cells to express an optogenetic construct, halorhodopsin. Once expressed, these channels are activated by 593nm light, resulting in the inactivation of that subset of cells. To investigate two different uses of halorhodopsin constructs, Drd3-Cre mice were combined with either a Cre-dependent halorhodopsin virus to drive local expression in L2/3 pyramidal cells in S1 or with a retrograde virus to drive expression in other cortical areas that project to S1. As a first step in adopting halorhodopsin 3.0 (eNpHR3.0) for use in whisker somatosensory cortex, I used histology methods to visualize the expression of eNpHR3.0 fused to EYFP. To do this, I perfused the mice, sectioned the brain using a microtome, applied a DAPI stain to identify and label all cell bodies, and imaged the brain tissue sections using an epifluorescent microscope, slide scanner, and a confocal. Results showed strong EYFP fluorescence in L2/3 of S1 cortex. Exclusion from L4 and the existence of labeled axons in the corpus callosum and contralateral hemisphere indicate that eNpHR3.0 was expressed in L2/3 pyramidal neurons. In the second part of my project I used similar methods to test a retrograde virus, as well as the Handy Automated Neuroexplorer MATLAB program to map an atlas projection onto the retrograde virus brain sections to quantify the number of cells in each anatomical region that project to wS1. The retrograde viral construct displayed projections from many higher-level areas of the brain to wS1 including some expected and some novel regions. The verification of expression from both parts of this project will allow for future optogenetic experiments to test the function of these cell types and circuits.

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Kaoru Saijo, Department of Molecular & Cell Biology

"Investigating the role of ERβ in inflammatory response during treatment with PolyI:C"

Abstract:

Prenatal immune activation has been implicated in increased risk of neurodevelopmental disorders in humans, including autism spectrum disorder (ASD). Maternal immune activation(MIA)-induced ASD can be modeled in mice by treating pregnant mice with viral memetic polyinosinic-polycytidylic acid (PolyI:C) during specific time points in embryonic development. PolyI:C induces inflammatory responses mediated by toll-like receptor 3 (TLR3) similar to maternal immune activation during viral infection. It results in offspring with features of ASD, including deficits in social communication and interaction, and stereotyped and repetitive behavior. In both human populations and MIA animal models, there is a strong male bias in ASD, with evidence that sex chromosomal genes and/or sex hormones may modulate the presentation of autistic phenotypes. Previous data that I generated in collaboration with lab members indicate that estrogen receptor beta (ERβ) in Cx3cr1-expressing cells (microglia, myeloid) is important for regulating the adult behavior response after E12.5 treatment with PolyI:C in a sex-specific way. Cx3cr1+ cell-specific ERβ in females seems to prevent increased marble burying and anxiety, while Cx3cr1+ cell-specific ERβ in males seems necessary to fully induce increased marble burying and anxiety. In the current research, I investigated the role of ERβ in the inflammatory response during treatment with PolyI:C in vitro by RNA expression of key inflammatory marker genes in microglial cell lines. I developed a method to knockdown ERβ in SIM-A9 and ERβ-overexpressing SIM-A9 cells using shRNA, and generated female and male immortalized microglia cell lines from brain tissue from P0 embryos. I investigated RNA expression of inflammatory marker genes and ERβ in embryonic day 12.5 whole brain homogenates of male and female mice after treatment with PolyI:C.

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Hilde Schjerven, University of California, San Francisco

"The Function of Wild-type Transcription Factor Ikaros in BCR-ABL1+ Pre-B ALL"

Abstract:

Precursor B cell subtype of acute lymphoblastic leukemia (pre-B ALL) is a cancer resulting from developing progenitor B cells in the bone marrow. Mutations in Ikaros, a crucial transcriptional factor in hematopoiesis, has been proven to be associated with loss of Ikaros function and poor prognosis in BCR-ABL1+ (i.e., Ph+) pre-B ALL cells, where Ikaros is theorized to function as a tumor suppressor. However, it is also observed that some BCR-ABL1+ leukemia cells express WT Ikaros (e.g. MXP2 cell line), and they display differences in phenotype as compared to Ikaros-mutant BCR-ABL1+ leukemia cells. The project aims to investigate the question if Ikaros is required to maintain the developmental phenotype we currently observe in Ikaros WT cells, by testing if CRISPRi-mediated repression of Ikaros will result in changes in cell growth and the expression of surface markers.

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Jamie H.D. Cate, Innovative Genomics Institute

"Testing the role of TCRA and TCRB mRNA 3’-UTR sequences on the function of the FMC63 chimeric antigen receptor"

Abstract:

A central component of the translation initiation machinery–the multi-subunit eukaryotic initiation factor 3 (eIF3)–is rapidly turned on when quiescent T cells are stimulated. Activation of T cells requires a great increase in cellular protein synthesis, along with a simultaneous increase in translation initiation.

One of the novel cancer treatment methods involves the programming of T cells to synthetically express a Chimeric Antigen Receptor (CAR) to target antigens on tumors. One of the most commonly used CARs is programmed to detect CD19 on malignant B cells. Anti-CD19 CARs have shown clinically successful results in patients who are chemotherapy-resistant and show relapsed hematological malignancies. These CARs are typically transduced into T cells using viral vectors that result in random integrations of the CAR into the T cell genome. However, recent studies have shown that introducing a CAR at the TRAC location results in uniform CAR expression as well as enhanced T-cell potency.

Recently, the Cate lab discovered that eIF3 crosslinked to both TCRA and TCRB mRNA across the entire length of the transcript, from the beginning of the 5’-UTR through the 3’-UTR. CRISPR-Cas9 genome editing to delete the eIF3 interaction sites in each of the 3’-UTRs of TCRA and TCRB showed that 3’-UTR eIF3 interactions drive the temporal expression of TCR proteins. Introducing these sequences to Nano-luciferase reporters showed that 3’-UTR eIF3 interactions are sufficient to drive a temporal receptor expression. These results highlight a new role for eIF3 in regulating the translation dynamics of the TCR and provide insights that can guide the engineering of T cells used in cell immunotherapy applications.

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Linda Wilbrecht, Department of Psychology, Hellen Wills Neuroscience Institute, University of California, Berkeley

"Measuring exploratory behavior across development in a wild-derived species: Can we study adolescent dispersal in the lab?"

Abstract:

Adolescence is a time of increased risk taking, novelty seeking, and exploration. These behaviors have an adaptive function to facilitate dispersal from the natal nest but may also have relevance to addiction and morbidity in adolescence. Our goal is to use animal models to understand the biological basis of dispersal in adolescence. Some published studies suggest adolescent rodents are more exploratory than younger and older rodents, but these studies were done in inbred lab strains that may lack more natural behaviors that support dispersal. They also were performed using artificial tasks used to measure anxiety that may not elicit dispersal related behavior. My project seeks to advance this field by focusing on Mus spicilegus, a wild derived mouse species and an ethologically inspired dispersal task. I am currently testing how exploratory behavior changes across age in M. spicilegus, and in a common strain of lab mice C57 Bl/6. To measure exploratory behavior across development I use the open field, a standard behavioral test, a novel object approach test and a custom designed apparatus to simulate a burrow leading to an open area that I built to mimic dispersal context. I am also measuring pubertal development via peripheral hormone measurements from fecal samples. So far, I have observed a significant age effect on both M. spicilegus and C57 Bl/6 exploratory behavior in open field based tasks with increases in distance traveled and time in center from P20-P40 to P50-P70. The data show both wild and lab rodents both show a significant increase in open field based exploratory behaviors during adolescence, but that M. spiciligeus is more exploratory than C57 Bl/6. Data from the custom built dispersal apparatus are still under collection, and the time course of behavior in this apparatus will be compared to behavior in the open field. Additional species comparison, sex comparison, seasonal manipulations, and pubertal results will be presented and discussed.

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Mazen Kheirbek, Department of Psychiatry, University of California, San Francisco

"Identifying the Role of Insular Cortex During Drug-Induced Place Preference"

Abstract:

Recent studies on addiction have shown that a primary driver of relapse is exposure to contextual cues associated with the drug experience. Retrieval of these associations can induce drug craving, defined as the intense motivation to pursue drugs, even in the face of long-term negative consequences. With human and rodent research showing that the anterior insular cortex (aIC) is involved in drug cravings induced by drug-related contextual cues the aIC has the potential to become an important therapeutic target in mitigating addiction and relapse. To investigate the contribution of the aIC to the expression of the conditioned drug-context association, we trained mice on a fentanyl-induced conditioned place preference (CPP) paradigm and tested the magnitude of place preference during inactivation of the aIC and under control conditions. We did not find any significant effect of inhibition of the aIC on the expression of fentanyl-induced CPP in acute test sessions 24 hours after conditioning or after a period of abstinence. These results suggest that aIC circuitry may not be necessary for the expression of the drug-context association.

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Matthew Traxler, Department of Plant and Microbial Biology, University of California, Berkeley, Koshland Hall

"Microbial Chemical Ecology in the Root Nodule Microbiome"

Abstract:

The root nodule is home to nitrogen-fixing rhizobia, but little is known about the in planta role of the accessory community that cohabit the nodule. Via successive passaging of an inoculum derived from field grown Medicago sativa nodules, the Traxler lab had selected for an accessory nodule community composed of four members, Brevibacillus brevis, Paenibacillus sp., Pantoea agglomerans, and Pseudomonas sp. We found the relative abundances recovered from the root nodules, as well as host plant phenotypes, depend on which members are present in the accessory community. In planta interactions between members of the accessory community showed cooperation and competition that occasionally varied from in vitro interactions. These findings suggest that the accessory community of the root nodule could be producing metabolites to regulate these interactions and that the nodule is a potential source of metabolite production in the plant.

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Mark D'Esposito, Department of Psychology, University of California, Berkeley

"Relating Processes of Executive Function in Women with Different COMT Polymorphisms"

Abstract:

A key question surrounding women’s health is how reproductive hormones impact cognition. Estradiol is thought to influence working memory through its relationship with the dopamine system. Baseline dopamine level is strongly related to a genetic polymorphism that dictates the genetic expression of Catechol-O-methyltransferase (COMT), an enzyme that breaks down dopamine. Previous research from our group demonstrated that estradiol differentially impacts working memory performance, as measured by working memory accuracy, as a function of COMT polymorphism (Jacobs and D’Esposito, 2011). However, working memory comprises many processes, only some of which were indexed by the task used in that study. Moreover, working memory processes overlap with cognitive control processes, which are also thought to be dependent on dopamine. In order to tease apart these processes and their relationships with baseline dopamine, we assessed performance on N-back, Eriksen Flanker, and complex span tasks in a group of all-female participants, and obtained their COMT genetic profiles. Accuracy on N-back lure trials correlated with the Flanker congruency sequence effect and with complex span performance. However, despite COMT’s previously reported interaction with estradiol and N-back lure accuracy, we found no direct effect of COMT genotype on the correlation between lure accuracy and its related measures. Together, these results strongly suggest a set of shared cognitive processes mediating our correlated measures, but baseline dopamine levels are not sufficient to explain their relationship. This may be due to additional differences in the influence of estradiol on performance, which will be the subject of future research.

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Ross Wilson, Department of Molecular & Cell Biology, Innovative Genomics Institute

"Validating assembly of engineered components for CRISPR-Cas9 delivery"

Abstract:

A current, unsolved problem in gene editing is the ability to specifically target and deliver Cas9 to a patient's cells that need altering. For most organs and tissues, in vivo delivery and distribution of Cas9 has been a tremendous challenge. AAV's and other nanoparticle carriers of Cas9 tend not to distribute well throughout the body following intravenous administration. Thus, there is a substantial need for new technologies for in vivo delivery of genome editing reagents.

The Wilson lab has been designing an adaptable Cas9 ribonucleoprotein (RNP) enzyme platform for in vivo delivery. This strategy involves coupling the RNP enzyme with targeting molecules that can promote preferential uptake into specific cell types, as well as surface-tethering a precise number of endosomolytic peptides (ELPs) that facilitate transit of the enzyme across the cell membranes. Previous work in the lab has engineered the guide RNA (gRNA) molecule to contain specific stem loop sequences that can be tightly bound to a small protein adaptor, called U1A. This adaptor can be decorated with various numbers of ELPs via conjugation chemistry. Once assembled, these components give the Cas9 RNP a virus-like ability to enter cells, ultimately enabling self-delivery for in vivo administration.

U1A adaptor-mediated recruitment of ELPs to the Cas9 RNP has not yet been biochemically validated. The goal of my project is to assemble these adaptors onto the gRNA stem loop sequence, creating a RNP capable of self-delivery to target tissue. This project employs biochemical and biophysical techniques to verify assembly of the ELP-RNP complex, and troubleshoot the assembly process. I employ fluorescence polarization (FP) binding assays to observe the binding interactions and affinity of fluorophore-tagged U1A peptides to RNPs. Additionally, I evaluate the on-rate, off-rate, and half-life of key interactions.

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Lydia Sohn, University of California, Berkeley

"Prognostic potential of extracellular vesicles: noninvasive monitoring of chemotherapeutic resistance development"

Abstract:

Chemotherapy remains the most common player in the treatment of cancer, used both independently and in combination with other systemic or localized therapies. It has been shown that patient response to chemotherapeutics is a potent predictor of prognosis and over 90% of cancer patient mortalities are related to drug resistance.1, 2 Chemotherapeutic resistance is often developed during the course of treatment when tumor cells become less sensitive to given drugs. This acquired form of resistance is particularly dangerous to patient survival due to inability and inefficiency in its detection. As such, it has become essential to develop a mechanism for monitoring potential changes in patient response to treatment. This research demonstrates the potential of extracellular vesicles (EVs) in rapid, noninvasive monitoring of tumor response to chemotherapeutics. A model system was devised to mimic interactions between tumor cells, chemotherapeutic(s), and alteration in genes conferring resistance. EVs were isolated from MCF7/wt and MCF7/ADR cell media to model EVs derived from doxorubicin-sensitive and resistant tumors, respectively. RNA was then extracted from EVs and expression of target gene top2a was quantified. Downregulation of top2a confers resistance to doxorubicin and was observed in MCF7/ADR (doxorubicin resistant) EVs compared to MCF7/wt EVs. Our findings establish the feasibility of using mRNA in tumor-derived EVs to assess drug sensitivity of the tumor via liquid biopsy.

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Mark D'Esposito, Department of Psychology, University of California, Berkeley

"Effects of orbitofrontal cortex lesions on hippocampal connectivity in humans"

Abstract:

Decision making integrates various types of information, from past experiences to social and emotional cues to physical factors of the environment. In this process, the orbitofrontal cortex (OFC) plays a key role. Previous studies have shown that the medial orbitofrontal cortex encodes the values of options, such as smaller rewards now or larger rewards later. Patients with lesions to this area have a reduced ability to integrate information to guide their decisions and regulate their behavior. The OFC is also anatomically situated to join memory, emotional, and sensory information. As such, the OFC is reciprocally connected with areas such as the hippocampus in the medial temporal lobe and the limbic and sensory regions. The interactions between these areas is thought to be regulated by the OFC. In this study, we analyzed resting state fMRI data from OFC lesion patients. These patients have reduced hippocampal connectivity with the sensory and association cortices, supporting the idea that the OFC mediates the interactions between these areas. However, OFC lesion patients have increased hippocampal connectivity with the dorsal caudate and ventral midbrain, which are subcortical regions involved in reward processing. Previous studies show that OFC lesion patients are more impulsive during intertemporal choice tasks, but patients who have stronger hippocampal connectivity with the subcortical regions are less impulsive. Together, the evidence suggests that OFC lesion patients undergo compensatory strengthening of existing hippocampal subcortical pathways to mitigate impulsivity.

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Richard Harland, Department of Molecular & Cell Biology

"Characterizing behavioral control circuits in the decentralized nervous system of the upside-down jellyfish Cassiopea"

Abstract:

The upside-down jellyfish Cassiopea is the first animal without a centralized nervous system (CNS) to have a characterized sleep-like state. Little is known about the behavioral and neurological circuitry involved in its sleep and wake periods and the effect of sleep deprivation (SD). Pulsing activity is primarily controlled by radially spaced ganglia, called rhopalia, which constitute its decentralized nervous system (DNS) and serve as pacemakers for muscle contraction. Here, we studied the local activity of the rhopalium and their influence on global behavior by analyzing their long-term behavior. By characterizing the sleep-wake state based on the animal’s inter-pulse interval (IPI) through latency to arousal experiments, we are able to characterize sleep on a pulse-by-pulse basis, allowing for data discretization, and the discovery that sleep in Cassiopea often occurs, day or night, in bursts, which correlate with changes in ganglia usage. Further, over the course of several days, ganglia change the wake/sleep profile homeostatically, connecting ganglia activity and sleep to broader theories of why animals need sleep. Along this line, we are now quantifying different molecular markers under baseline and sleep deprived conditions and will be able to determine if there is a correlation between pacemaker activity levels and certain markers. With these results, we are able to more specifically characterize the sleep-like state Cassiopea on a ganglion-specific level, and our findings support a neurological homeostatic function of sleep in an ancient model of sleep.

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Notes: Explore our per base coverage data for the SYNGAP1 gene, generated from 176 human dorsolateral prefrontal cortex samples from the BrainVar Cohort, by clicking the link below and entering “chr6:33,419,157-33,458,746” into the search box. Make sure that the Custom Tracks are set to “full.” See how coverage for this gene differs between developmental stages.

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Stephan J. Sanders, University of California, San Francisco

"Exon-level expression of SYNGAP1 in the developing human cortex"

Abstract:

De novo loss-of-function mutations in the SYNGAP1 gene, which encodes a synaptic Ras-GTPase-activating protein, are associated with autism spectrum disorder, developmental delay, and epileptic encephalopathy. SYNGAP1 haploinsufficiency is thought to cause increased excitability of excitatory neurons, contributing to seizures and impaired learning and memory (Kilinc, et al. 2018). Disease severity has been found to vary significantly depending on the location of the mutation within the gene (Vlaskamp, et al. 2019). Clinical sequencing has identified few cases with mutations within the first four exons and the individuals affected displayed a milder phenotype, whereas mutations were more frequently observed in the subsequent exons and were often associated with severe developmental delay and epileptic encephalopathy. Multiple splicing isoforms are reported for SYNGAP1, differing in their N’ and C’ terminals, with three possible N’ isoforms (named A1, A2, and B) and four possible C’ isoforms (named α1, α2, β, and 𝛾). Of note, the shorter B isoform does not include the first three exons with few reported mutations and milder symptoms, suggesting that splicing isoforms may explain some of the relationships between genotype and phenotype. Studies in rats found Syngap1 isoforms to have opposing effects on synaptic strength, with the shorter B isoform increasing synaptic strength and the longer A isoform decreasing synaptic strength and that isoform expression levels varied across development, with a significant peak in B isoform expression at P14 (McMahon, et al. 2012). In line with this, we hypothesized that the B isoform might provide some protection against mutations in the first three exons that are unique to isoform A. I aimed to compare the expression of SYNGAP1 isoforms, interpreted from exon-level expression, across the span of human cortical development in a novel dataset of RNA-seq data from 176 developing human dorsolateral prefrontal cortex samples (Werling, et al. 2020).

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Maria Luisa Gorno-Tempini, University of California, San Francisco

"The Roles of Speech Production Subnetworks in Non-Fluent/Agrammatic Variant Primary Progressive Aphasia"

Abstract:

Non-fluent/agrammatic variant primary progressive aphasia (nfvPPA) is a neurodegenerative disorder characterized by grammar and motor speech impairment, such as apraxia of speech and dysarthria. Human in vivo neuroimaging studies have identified a brain network, denoted the speech production network (SPN), which shows a consistent pattern of structural and functional changes in nfvPPA patients, suggesting that the speech deficits associated with nfvPPA arise from the regions underlying the SPN network. Furthermore, it has been shown that healthy functional and structural connectivity can predict longitudinal grey and white matter changes in nfvPPA patients. By using graph theoretical metrics, the SPN was divided into three distinct subnetworks. These results suggest that neuroimaging metrics may be used to predict the progression of nfvPPA, though this disease is complex in nature and has variable and overlapping presentations of symptoms.

This study synthesizes previous work to determine if subnetworks of the SPN are uniquely associated with different aspects of speech production. To investigate this question, a graph theoretical model was used to quantify the functional segregation of the three SPN subnetworks. In addition, speech metrics were extracted from patient performance on the Western Aphasia Battery exam to quantify different aspects of speech performance. No significant differences were found in the considered graph metrics between nfvPPA patients and healthy controls, and few significant correlations were found between graph theoretical and speech metrics. By contrast, functional connectivity showed significant associations between the fronto-insular subnetwork with a composite score of speech motor production, while the temporoparietal and subcortical subnetworks showed significant correlation with speech errors of grammar and syntactic index. These results support the hypothesis that different aspects of speech production are doubly dissociated between different subnetworks of the SPN, and that these associations are detectable using the functional connectivity of the regions in these subnetworks.

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Andrew Dillin, Department of Molecular and Cell Biology

"A Multi-Study Analysis to Find Unknown Regulators of the Unfolded Protein Response of the Endoplasmic Reticulum"

Abstract:

The Unfolded Protein Response of the Endoplasmic Reticulum (UPRER) maintains proteostasis within a cell during both endogenous and exogenous stress. Normally, these stressors cause misfolded protein buildup, and the UPRER increases ER chaperone expression or triggers autophagy or apoptosis to prevent aberrant expression and phenotypes. Misregulation of the UPRER can lead to the development of various diseases, including Alzheimer’s and Parkinson’s, and greatly decrease the lifespan of an organism. The UPRER is conserved across species, from yeast to mammals. The model organism C. elegans allows for an easily manipulatable model system of this complex cellular response. Previously, genome-wide CRISPR-Cas9 knockout screens have been conducted in mammalian cell culture by Adamson et. al and Schinzel et. al to further elucidate the UPRER. Where these studies differ is in the functional models used: Adamson et. al continued observation in cell culture while Schinzel et. al constructed transgenic C. elegans. This study compares these two datasets to find previously unknown regulators of this cellular stress response. Using a novel python script to analyze snRNA scores, as well as my own RNAi screen of 806 genes done in C. elegans, 498 genes were identified with conserved homology between C. elegans and humans that, when knocked out by CRISPR-Cas9, either enhance or deplete the UPRER. These genes can be categorized into 11 main regulatory groups, and, most notably, connects the UPRER to the mitochondrial UPR (UPRMT) for the first time. This study provides information on and future directions for identification of non-canonical regulators of the UPRER, and provides insight into previously unknown connections between the UPRER and the mitochondrial unfolded protein response.

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Gian Garriga, Department of Molecular & Cell Biology, University of California, Berkeley

"The role of BAM-2 in FMI-1 mediated axon pathfinding in Caenorhabditis elegans"

Abstract:

The Caenorhabditis elegans Flamingo homolog FMI-1 is an atypical cadherin that regulates axonal pathfinding. Studies in the fmi-1 gene have shown that:

- Mutant phenotypes are characterized by overextended HSN motor neuron axon branches that cross the midline

- Expressed in the VC and HSN neurons

Additionally, studies in the neurexin-like bam-2 gene show:

- bam-2 mutants have branch defects that are similar to the fmi-1 mutants

- Expressed in vulval cells to regulate axon branching

Since the axons of both VC and HSN neuron types branch at the vulva and innervate the vulval muscles, it is unclear whether the branching defects in the bam-2 mutants reflect defects in the HSN, VC or both axons.

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Richard H. Kramer, Department of Molecular & Cell Biology

"Histological Evaluation of Dark Rearing’s Impact on the Progression of Photoreceptor Degeneration in rd10 mice"

Abstract:

Retinitis pigmentosa is a genetic eye disease causing the gradual loss of photoreceptors resulting in progressive vision loss. Dark rearing has been shown to have a neuroprotective effect in rd10 mice, delaying the progression of photoreceptor loss, however, the mechanism by which this occurs is not fully understood. This study aims to compare the degree of photoreceptor loss in rd10 mice reared under a normal light-dark cycle with rd10 mice reared in 24-hour darkness by quantifying the ONL thickness. In addition, we seek to explore the possibility that photoreceptor degeneration can be arrested or delayed by housing the mice on a different light cycle at specific times during the progression of degeneration. Dark rearing was confirmed to have a neuroprotective effect in the rd10 mouse model of retinitis pigmentosa, resulting in a delayed degeneration of the ONL. Dark reared mice that were exposed to light had high levels of degeneration, while it is unclear whether dark rearing after light exposure can limit degeneration.

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Michael Yartsev, Department of Bioengineering and Helen Wills Neuroscience Institute, University of California, Berkeley

"Quantifying the sparsity of monosynaptic projections from motor cortex to laryngeal motoneurons in a vocal learning mammal - the Egyptian fruit bat"

Abstract:

Vocal learning, the ability to acquire and modulate vocalizations by learning from conspecifics, is a rare phenomenon in the animal kingdom. Egyptian fruit bats (Rousettus aegyptiacus) are one of the few mammals that have been shown to have a robust vocal repertoire and exhibit vocal plasticity from childhood to adulthood. However, it is not yet understood what anatomical circuitry underlies this ability. One hypothesis is that vocal learners possess a direct monosynaptic projection from the laryngeal motor cortex (LMC) onto motoneurons in the nucleus ambiguus (NA) in the brainstem, which then project to the cricothyroid muscle to control the vocal cords. Using three different fiber tracing techniques in combination with immunohistochemical staining, confocal imaging, and automatic cell/fiber/bouton counting, we identified a monosynaptic connection from LMC onto laryngeal motoneurons in NA. Further, we examined the density of synapses in the brainstem and other brain regions to demonstrate the existence of this highly specialized circuit in the nucleus ambiguus compared to other areas. These findings may shed light on the organization of vocal production circuits underlying complex language abilities and bear consequence on clinical research into vocal motor disorders.

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Riley Bove, Department of Neurology UCSF Weill Institute for Neurosciences, University of California, San Francisco

"MS Dreams: Sleep Quality in Menopausal Women with Multiple Sclerosis: Relationship with Disability and Neuroimaging Measures"

Abstract:

Background: Sleep dysfunction is a feature of many neurological diseases including multiple sclerosis (MS). Sleep also worsens among women at menopause. Sleep disorders, menopause and MS likely have a complicated and reciprocal relationship. Even though a majority of women develop MS before they experience menopause, for many years, MS care was solely focused on managing functional impairments that arose from the immune-mediated demyelination and secondary axonal loss.2 More recently, given preservation of neurologic function enabled in the era of disease modifying treatment (DMT), there has been increasing attention paid to the experiences of menopausal women with MS. In fact, both compromised sleep and menopause are linked with worsening clinical function in MS. However, little is known about how the three are interrelated. Understanding mechanisms linking reproductive aging, sleep and MS disease course could help guide future therapies.

Goals: Here, we aim to examine associations between measures of sleep, MS function, and menopausal (including vasomotor) symptoms.

Methods: I performed exploratory secondary analyses of data collected as part a study evaluating the impact of a hormonal therapy (Duavee ®) on menopausal symptoms in women with MS. All analyses were adjusted for age.

Results: Altogether, to date we examined data on 24 women with MS. In patient-reported outcomes (MS Quality of Life Survey), the number of hot flashes were the only feature significantly correlated with health distress, making it the most notable source of discomfort for patients. Additionally, worse sleep was negatively associated with the most measures of QOL encompassing both physical and mental composite scores.

On MRI analyses, both worse hot flashes and worse EDSS (a global measure of MS related disability) were correlated with MS lesion count and volume. In fact, our data corroborated the findings from earlier studies looking at MRI volumetric features. Lower brain volume in the superior frontal gyrus and anterior cingulate was associated with both worse sleep and hot flashes. Interestingly, worse EDSS and worse hot flashes showed opposing correlations with many of the evaluated structures.

Conclusions: Overall, our ongoing analyses suggest that the observed associations between menopausal symptoms (hot flashes), sleep quality, and MS symptoms require further exploration.

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Stephan Lammel, Department of Molecular & Cell Biology, University of California, Berkeley

"Activity Dynamics of Serotonin Neurons in Modulating Social Behavior in Mice"

Abstract:

Patients of neuropsychiatric illnesses such as depression, schizophrenia, and autism spectrum disorder commonly manifest symptoms of abnormal social behavior or altered sociability motivation. Despite direct associations between serotonin and social behavior, the specific circuitry and mechanisms of the serotonin system underlying social interactions remain unexplored. Using a multidisciplinary approach to measure real-time activity dynamics of Dorsal Raphe (DR) serotonin neurons during social interactions of opposite-sex mice, I show that both sexes exhibit an increase and decrease in neural activation corresponding respectively to the initiation and the end of general and nose-nose interaction. However, during nose-anterogenital interaction, a behavior almost exclusively initiated by male mice, only the male mice exhibits increasing while female mice experiences decrease in serotonin neural activity. Application of Designer Receptors Exclusively Activated by Designer Drugs (DREADD) to broadly manipulate serotonin neurons in SERT-Cre mice produced no significant trends in alterations of social behavior. Anatomical tracing of defined forebrain-projecting dorsal raphe neurons confirmed the cell subpopulations as distinct, suggesting complexity in the heterogeneity of serotonin neurons that requires further refined characterization. By advancing our understanding of specific circuits in the brain, discoveries from these experiments will yield insights critical to the development of more effective therapies for neuropsychiatric disorders.

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Ehud Isacoff, Department of Molecular & Cell Biology

"Advancing Data Analysis for Retinal Electrophysiology in MATLAB"

Abstract:

Retinal degenerative diseases cause progressive vision loss due to the death of photoreceptor cells, while often leaving the downstream retinal ganglion cells (RGCs) intact. Optogenetics presents a promising method for treatment. Previous work has shown that by expressing medium-wavelength cone opsin (MW-opsin) in the RGCs of blind mice, light-evoked retinal responses and visually-guided behaviors of these mice can be restored (Berry et al., 2019). These results can be improved by expressing multiple opsin types to more closely reflect the different photoreceptor types in the intact mammalian retina. Ongoing research using multiple opsin expression in a mouse model of retinitis pigmentosa uses multi-electrode arrays (MEAs) to collect electrophysiological data that report on the retinal response to flashes of light. These experiments, conducted by another lab member, generate large, complex datasets that greatly differ from the data generated by previous single-opsin experiments. The single-opsin experiments found that cells have a largely homogenous response to light, whereas the multiple-opsin experiments show greater diversity in response type within one retina. This change in complexity requires a nontrivial improvement in data analysis methods. To address this challenge, I have developed a data analysis script in MATLAB specifically designed to handle these large and complex datasets. The new script consists of data visualization, processing, and quantification of several relevant parameters such as latency, duration of response, amplitude, to characterize the diverse retinal responses. This will allow for an in-depth characterization of the wide variety of responses observed in multiple-opsin expressing retinas and provides an important advancement in the analysis of large retinal electrophysiology datasets.

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Kristin Scott, Department of Molecular & Cell Biology

"A Quantitative Analysis of Drosophila Proboscis Extension Phenotypes Reveals Behavioral Subprograms"

Abstract:

Feeding behavior is a critical component of animal survival. Due to its comparatively simple neural circuitry, the fruit fly Drosophila melanogaster is a powerful model to study feeding decisions; however, few neurons that control feeding are well characterized. Members of the Scott Lab conducted a screen for neurons sufficient for a feeding behavior, proboscis extension, and identified 30 different neural classes that influence this behavior. In my research, I used high-resolution video monitoring and machine learning-based approaches to provide a quantitative description of behaviors associated with activation of these 30 neural classes. I recorded over 1,250 videos of proboscis extension phenotypes upon optogenetic activation of these neuronal cell types. Next, to analyze these videos, I built an analysis pipeline that derived attributes such as the distance and angles between proboscis subparts and performed statistical analysis and clustering through PCA and heat maps. These studies provide an analysis of proboscis extension that allowed us to more accurately characterize and classify these neurons. The phenotypic differences found argue that proboscis extension can be compartmentalized into different behavioral subprograms. One key finding in my research is that bitter sensory neuron activation is capable of causing a distinct proboscis extension subprogram, despite lack of proboscis extension to natural bitter stimulation. Furthermore, the activation of another previously identified neural class was similar to the bitter activation phenotype, leading to the hypothesis that these neurons participate in bitter processing. Indeed, using behavioral assays, I found that activation of these neurons inhibited consumption, similar to activation of bitter neurons. Through immunostaining, I also found that these neurons likely contain a population of known second-order sugar neurons in addition to neurons that inhibit consumption.