mTOR, the mechanistic target of rapamycin, is a serine/threonine kinase which is an enzyme that phosphorylates the hydroxyl group on a serine or threonine side chain. mTOR kinase regulates protein synthesis, cell growth, and metabolism in response to nutrients and energy in most eukaryotes. mTOR consists of two distinct complexes, mTORC1 and mTORC2. These complexes can be further divided into three components: mTORC1 consists of mTOR, Raptor, and mLST8, and mTORC2 consists of mTOR, Rictor, and mLST8. mTORC1 is critical in metazoan development and has been implicated in aging, cancer, diabetes, cardiovascular disease, and hypoxia, a state in which tissues are deprived of oxygen and can not carry out normal metabolic activity. Previously, the Crowder lab conducted a mutant screen in C. elegans, for hypoxia resistant mutations, and has recently identified a missense (changes one amino acid to another) reduction of function mutation in the daf-15 gene, the C.elegan ortholog of Raptor. A unique feature of this mutation is that the function of Raptor can be turned on and off by varying temperature. It has normal hypoxia resistance at 20 degrees, increased resistance between 21-22, and developmentally arrests at 25 degrees. I and the other authors made use of this conditional developmental arrest phenotype to screen for genetic suppressors, mutations that suppressed developmental arrest. Using genetic mapping, sequencing, and complementation testing, we have identified multiple mutations in three different genes responsible for restoring Raptor function. Preliminary results show mutations in the gene rnf-126 (ring finger protein) suppress the Raptor mutation. Current work by myself and others is designed to answer how these genes control Raptor function and hypoxia sensitivity. Elaborating the function of these genes will define novel mechanisms whereby Raptor and mTORC1 controls metabolism, hypoxic injury, and development.

The functional connectivity of the brain evolves throughout the life of every individual. These changes, often referred to as neuroplasticity, can be impacted by a wide range of variables from diseases to eating a favorite dessert. How can these changes be modulated to treat neurological diseases and disorders such as post traumatic stress and major depressive disorder? My colleagues and I are intrigued with the prospects of neuromodulation as a therapeutic for abnormal brain connectivity and network dynamics, leading me to the question “At what rate do these connections accumulate and decay with optogenetic modulation; Optogenetics, a technique that uses light to activate or inhibit genetically targeted neurons, offers high cell-specificity and temporal resolution that allows us to zoom into the network dynamics and find more finely tuned results that can help in the development of neuromodulation therapies. I plan to use both single site and paired-pulse optogenetic inhibition to gain a clearer understanding of how functional connectivity behaves during and after repeated modulation periods followed by extended recordings of spontaneous activity with no modulation. By analyzing the pairwise coherence of the local field potentials collected using electrocorticographic recordings in non-human primates, I anticipate seeing targeted changes in functional connectivity when comparing before and after each inhibition session. By analyzing the rate of change in connectivity I plan to understand the timeline of neuroplasticity following optogenetic modulation, thus informing the development of future neuromodulation therapies. This research could have a profound impact on the future therapeutic paradigms for neurological and neuropsychiatric disorders that can accelerate recovery for individuals with these conditions.

Mitochondria are the main oxygen consumers in eukaryotic cells and as such are the primary organelles affected by oxygen deprivation, hypoxia. Hypoxia alters the size and shape of mitochondria (so called mitochondrial dynamics) but the responsible mechanisms and their role in hypoxic cell death is unknown. The Crowder lab has recently discovered that a mutation in the Mechanistic Target of Rapamycin Complex One (mTORC1) protein Raptor confers hypoxia resistance. mTORC1 is a master regulator of metabolism and is known to affect certain aspects of mitochondrial biology. In this work I show that disrupting mitochondrial dynamics with mutants in mitochondrial fission produce hypoxia resistance but that mutants with altered fusion have normal hypoxic sensitivity. I have built compound mutants containing both fission and fusion machinery mutants together with the hypoxia resistant Raptor mutant. Using these mutants, I am testing how Raptor controls fission and fusion and whether either is required for its hypoxia resistance. Our preliminary findings indicate that the hypoxia resistance of the Raptor mutant does not require FZO-1-mediated mitochondrial fusion. By exploring the interaction of mitochondrial fusion and fission with Raptor, we are beginning to understand how these important organelle and metabolic regulators combine to control hypoxic cell death.

Two-photon microscopy enables single-cell resolution recording of neural activity via the expression of proteins that change fluorescence brightness levels based on neural activity. This technology can be used in conjunction with behavioral tests in animal models to investigate the neural mechanisms underlying cognition, sensory processing, and internal states. Here we present findings to improve 2-photon data quality through a denoising algorithm, which removes random non-neural noise from data, and subsequently extract neural-behavioral relationships through deep-learning classification. In this project, I wrote custom python scripts to perform these complex analyses using open-source packages on the UW high performance computing cluster. Two-photon in vivo images of fluorescent indicators can be contaminated by varying levels of noise, related to the recording device or the environment. Such noise is prohibitive for detecting neural structures. Here, I apply a convolutional neural network (CNN)-based denoising algorithm, DeepInterpolation, to mitigate the noise present in neural activity recordings. We hypothesize that denoising will achieve a significantly higher single-pixel signal-to-noise ratio (SNR) compared to the raw data, and enable significantly more neural structures to be detected by segmentation algorithms. Deep learning techniques have shown promising results in improving the classification of video data. Data acquired from two-photon microscopy are sequences of images across time, yet most analyses focus on pixel-averaged time-series extracted from individual neurons. The relational information between space and time that may inform of underlying neural mechanisms is therefore lost in these approaches. Here, we propose the application of Deep 3-dimensional convolutional networks (3D ConvNets) to learn spatiotemporal features of two-photon imaging data and to classify local circuit interactions related to animal behavior.As a whole, the goal of this work is to provide an open-source working example for the classification and feature extraction of two-photon imaging neural activity recordings. This pipeline can be used to gain insight into spatiotemporal dynamics related to event-related behaviors in two-photon imaging datasets.

 Alzheimer’s disease (AD) is the most prevalent of neurodegenerative diseases, with over 6 million Americans suffering from the illness and cases increasing each year. TREAT-AD (TaRget Enablement to Accelerate Therapy Development for AD) is an NIH-funded, multi-institutional network that identifies and addresses new targets for AD drug development. Genetic targets of interest were identified via RNA-sequencing and proteomic analysis of post-mortem tissue from participants with AD. We tested the effects of inhibiting or overexpressing selected target genes hypothesized to increase the risk of Alzheimer’s. One such target is the CD44 gene, which regulates GABA receptor activity. To efficiently manipulate genetic expression, we used clustered regularly interspaced palindromic repeats (CRISPR) technology to enhance (CRISPRa) or repress (CRISPRi) genetic transcription in neural progenitor cells, stem cells in the process of differentiating into neurons. We used a cell line engineered to harbor CRISPRa and CRISPRi machinery. CRISPRa involves a catalytically inactive Cas9 protein fused to a transcriptional activator. In CRISPRi, the inactivated Cas9 is fused to a transcriptional repressor. This, along with the quantification of molecular pathways linked to Alzheimer’s, permits a window into the conditions that lead to earlier onset of AD, and thereby conditions that might be altered by new drug treatments. Here we report that underexpression of genes related to endosomal trafficking leads to changes in protein buildup that may be related to earlier onset of AD.

Cell division is essential to all living organisms, and microtubules are critical for this process. Microtubules in bundled kinetochore-fibers generate forces to move chromosomes by stochastically switching between assembly and disassembly states. Electron tomographs of mammalian mitotic spindles show that microtubule tips maintain a high level of coordination despite stochastic switching between states. The collective behaviors of microtubule bundles give rise to chromosome oscillation, which promotes the alignment and separation of chromosomes along the spindle equator during metaphase. To understand how stochastically-switching microtubules can produce synchronized motions in vivo, we use a mechanical model that describes the force-dependent dynamics of microtubules. The model reproduces the bistability of microtubule bundles observed in vivo and suggests that this coordination could be explained by sufficiently stiff mechanical coupling. We also propose a new way to characterize microtubule bundle state based on the substates of microtubules within the bundle. We will use this characterization to calculate microtubule bundle switch rates, which are technically difficult to distinguish otherwise, thereby laying the groundwork for future comparisons between bundle switch rates in vivo and in silico. In the future, we plan to explore the assumptions on which this model is based by analyzing the relationship between microtubule velocity and forces. We predict that this result will validate the assumed force-velocity relation of microtubule growth and contribute to a more accurate understanding of kinetochore-fiber behaviors. 

Spatial transcriptomics is a set of neuroscience methods that enables the visualization of gene expression patterns across the brain. Using Expansion-Assisted Iterative Fluorescence in situ hybridization (EASI-FISH) protocols, a cutting-edge spatial transcriptomic approach, we can label unique RNA segments with multiple colored fluorescent probes in neurons throughout an extracted ex vivo brain section. Brain sections are then volumetrically imaged using a confocal microscope and collected images undergo analyses to align images and segment cells and fluorescence expression. Current limitations of these analysis techniques include computing resource usage and the lack of parallel dataset analysis which we aim to address by adapting open source software to be compatible with cloud-based computing. Here we utilize the Hyak cluster operated by University of Washington-IT to overcome memory, CPU, and GPU limitations of desktop computers. To benchmark the analysis package, we analyzed 3D volumetric tissue from the medial preoptic area (MPOA) of the mouse brain labeled for the genes Vgat and Esr1, markers for inhibitory neurotransmitter and estrogen receptor expression respectively. Our scientific goal is to understand the enrichment of such genes during female mating behaviors. From previous transcriptomic data, we expect that around 70% of Esr1 positive cells in the MPOA will be Vgat positive and will be located in the nucleus part of the MPOA. These neurons will tend to be activated after mating in female mice. However, how these double positive neurons locate in high resolution 3D space is not known and will provide novel insights. The future goal is to apply the analysis package to whole-brain light sheet microscopy data. The broader impact of this work is the development of a high-throughput open-source analysis pipeline for the quantification of multiplexed gene expression patterns across multiple spatial scales.

The increasing availability of both prescription and illicit opioids has caused substance use disorders to skyrocket. Treatment options such as therapeutics that can inactivate Kappa opioid receptors (KOR) have been shown to reduce drug-seeking behavior through the modulation of intracellular signaling pathways. Downstream KOR activation, G-protein coupled receptor kinase 3 (GRK3)/arrestin-dependent pathway leads to activation of p38 mitogen-activated protein kinase (p38 MAPK) and feelings of dysphoria. In another pathway downstream of KOR receptor activation, a G-protein mediated response and activation of cJun kinase (JNK) leads to the generation of reactive oxygen species (ROS). Selective activation by biased ligands of the JNK mediated pathway result in the release of ROS, which leads to the eventual depalmitoylation of the G-αi/o subunit of the KOR. This results in the long-term inactivation of KOR, which is predicted to improve stress resilience and to prevent drug-seeking behavior. Drugs such as Nalfurafine and Nalmefene can selectively activate KOR such that ROS is produced. Using 2-photon microscopy to detect fluorescence that indicates the release of ROS by Nalmefene and Nalfurafine into the ventral tegmental area of transgenic mice, I can better understand the potential of these drugs for long-term inactivation of KOR. I have observed under a light microscope that when Nalfurafine was washed onto slice, an increase in ROS was observed. Nalmefene showed a similar trend to Nalfurafine but increased ROS to a lesser extent. Additionally, when either Nalfurafine or Nalmefene were added to a solution of naloxone, there was no significant increase in ROS. Using the data collected from slice and behavioral assays such as the tail-flick test, we can illustrate the positive therapeutic effects that KOR inactivators can have on substance use disorders in the long term.

Microtubules are dynamic polymers of 𝛼- and 𝛽-tubulin subunits instrumental in the organization and division of chromosomes during mitosis. There is an intrinsic structural polarity to microtubules due to the orientation of 𝛼𝛽 heterodimers in the microtubule lattice, so that there is a fast growing plus end and a slower growing minus end. Kinetochores are protein complexes that assemble on chromosome centromeres and attach to microtubules. Proper chromosome segregation relies on kinetochore attachment to the plus ends of microtubules. Kinetochores are thought to initially bind the microtubule lattice and plus end attachments are then achieved by the action of plus end directed motor proteins or microtubule disassembly. While the plus end attachment is essential for mitotic fidelity, it remains unknown if the kinetochores themselves have an intrinsic polarity preference. Using total internal reflectance fluorescence microscopy, we have found that individual kinetochores assembled on centromeric DNA have a strong preference for binding the plus ends of stabilized microtubules in the absence of motor proteins and ATP or microtubule dynamics. Furthermore, using optical trapping we are able to measure the rupture forces of kinetochores on both ends of microtubules and have found that the observed preference for plus ends is matched by a greater binding strength at plus end tips. These results together give insight into how kinetochores could efficiently form plus end tip attachments and how they likely play a part in cell cycle regulation by using tension to sense a correct attachment. A better understanding of the specific mechanisms of kinetochore microtubule binding is valuable for understanding control of mitotic progression and could potentially inform more targeted anti-cancer therapies that focus specifically on dividing cells without impacting regular cell function.