Individuals with Developmental Language Disorder (DLD; previously known as specific language impairment) have difficulty acquiring their native language with no apparent underlying cause. While this disorder is common (7% of the population, or 2 in every classroom), there is little research investigating structural differences in the brain. My mentor Dr. Cler and colleagues previously found differences related to myelination of the caudate nucleus in children with DLD. They used a specialized form of MRI, multiparameter mapping (MPM), which reveals specific aspects about the microstructure of the neural tissue. The purpose of this current research is to confirm and expand upon these findings by obtaining MPMs on adults with DLD. Twenty participants between the ages of 18 and 45 are recruited via Facebook flier, ten typically-developing and ten with DLD. Because of the lack of instruments available to assess DLD in adults, my role has been to compile a battery of tests that can accurately differentiate individuals with DLD from TD as well as from other communication disorders. The selected battery evaluates participant’s IQ, motor skills,  ability to define words, spell, and follow complex directions. Scanning is conducted at the Center for Human Neuroscience using a 3T Siemens Prisma. MPM quantitative imaging provides maps of myelin and iron content in the brain. We hypothesize that we will find reduced myelination in the caudate nucleus, a part of the basal ganglia implicated in implicit learning. The findings of the study will contribute to current neural models of DLD, helping us to identify the brain processes that are disrupted by the disorder. This information can inform future clinical interventions to help individuals with DLD in their speech and language development.

One of the great outstanding challenges in neuroscience is to understand how the human brain represents distinct objects. Studies have shown that a significant number of neurons in the visual cortex of non-human primates responded differentially to the sides of figures in 2D images even when the figures were outside of their classical receptive fields. This selectivity, termed border ownership, is believed to be responsible for the Gestalt idea of figure-ground organization, a process that allows specific regions of an image to be grouped together and given "thing-like" qualities. Many computational models have been developed to reproduce the experimental results of border ownership studies. At the same time, convolutional neural networks (CNNs), especially those specialized in image segmentation, are able to learn to solve the problem of figure-ground organization through supervised learning, all without the need for explicitly defined computational rules. We hypothesize that there is knowledge to be gained from CNNs, for they are excellent computational models for visual selectivities. Our novel 'artiphysiology' technique allows us to study the border-ownership phenomena in CNNs at a single-unit level in the same way an electrophysiologist studies the brain. The technique takes advantage of the accessibility and lack of noise of CNNs to enable high-throughput identification and analysis of circuit mechanisms. Starting from border ownership, the research aims to elucidate the mechanism by which figure-ground organization occurs at different layer depths and in different CNNs, using natural and artificial visual stimuli. The research has several applications, including improving CNN efficiency and interpretability as well as allowing for a better understanding of object recognition.

Drug addiction and abuse exacerbate many health issues globally. The nucleus accumbens (NAc), a region within the brain, contains dopamine receptor D1 and D2 medium spiny neurons (MSNs) with relevance to addiction-related behaviors. I investigated the effect of D2 MSNs on behavioral economics of cocaine-seeking through chemogenetic activation of Designer Receptors Exclusively Activated by Designer Drugs (DREADD). I used a transgenic line of rats specifically expressing the Cre-recombinase enzyme in D2 MSNs. This enzyme is responsible for selective expression of otherwise-inert viral vectors by reversing its sequence, allowing for neuron-specific genetic manipulation. I bilaterally injected either a Cre-dependent AAV containing the DREADD hM3Dq or a fluorescent marker as control into the NAc of male rats; additionally, I catheterized the rats to allow for cocaine self-administration. Activation of hM3Dq required binding to clozapine-N-oxide (CNO)—the “designer drug”—and because only D2 MSNs expressed this DREADD, they were the only neurons to activate upon administration of CNO. After recovery, rats were trained to press a lever to receive cocaine infusions; and once this self-administration task was learned, the animals were introduced to reinforcement schedules with blocks of trials with differing unit doses of cocaine. Once this phase was learned, the rats were tested in sessions that were preceded either with an injection of saline or CNO, to test how chemogenetic activation of D2 MSNs affects the cocaine demand curve. Based on studies discussing the roles of D2 MSNs, I predict that chemogenetic activation of these neurons will correlate with decreased demand for cocaine concentrations compared to those injected with vehicle or green fluorescent protein alone. This study can produce valuable insight into the role this pathway plays in the development of drug-seeking behavior, ultimately leading to a deeper understanding of the system and possible avenues for treating addiction.

Morphine and other mu-opioid receptor (MOR) ligands are commonly used treatments for pain management. Long-term administration of opiates results in receptor desensitization and tolerance, inhibiting treatment efficacy and contributing to the current opioid epidemic. Following opioid administration, peroxiredoxin 6 (PRDX6) is recruited to the opioid receptor complex through JNK (cJun N-terminal Kinase)-dependent signaling. PRDX6 activation generates reactive oxygen species (ROS), resulting in opioid receptor desensitization and one form of opioid tolerance. Recently, cannabidiol (CBD) has been implicated in decreasing acute morphine tolerance. The present study will establish a connection between morphine and CBD cotreatment with the hypothesis that cotreatment will decrease ROS production. To measure ROS, I will employ the genetically encoded ROS sensor HRM63, which fluoresces proportionally to ROS production. Because ROS is a product of JNK/PRDX6 signaling, measuring the strength of ROS-dependent fluorescence is a simple way to visualize my hypothesis using in vitro cell models. In each experiment, treatments of morphine, CBD, or combined morphine and CBD will be delivered to plated HEK293 cells stably expressing both MOR and HRM63. I will then image the cells using fluorescence microscopy to quantify ROS response to treatment. I predict that cotreatment of CBD with morphine will result in lower ROS production compared to morphine treatment alone. These results will be crucial in the ongoing characterization of CBD's role in opioid tolerance.

Small heat shock proteins (sHSPs) fall into a class of proteins known as protein chaperones, molecular tools that help prevent aggregation, an often-unhealthy phenomenon in which proteins clump together. This protection against aggregation plays a critical role in helping to prevent diseases such as cataracts, and may play a role in preventing neurological diseases associated with protein aggregation such as Alzheimer's disease or Parkinson's disease. Yet little is known about the mechanism by which sHSPs prevent misfolded proteins from aggregating. The study of this mechanism is significantly complicated by the dynamic nature of sHSPs; rather than having just one structure, these protein chaperones fluctuate between several different stable structures. The purpose of this research project was to see what insights into sHSP structure and function could be gleaned by simplifying this dynamic structure. We focused on the central region of a small heat shock protein, known as the alpha-crystallin domain (ACD). Individual ACDs associate to form dimers in an antiparallel fashion, and in doing so form a central fold known as the dimer-interface groove. In a typical wild type small heat shock protein, these ACDs would continuously slide against each other along this groove. We created three distinct cysteine mutations in the ACD of the small heat shock protein HSPB5, producing the R116C, E117C, and F118C mutants. We expected each of these mutant dimers to form a disulfide bond tethering the two ACD subunits of the dimer together at the dimer interface groove, preventing them from sliding against each other, and therefore locking the dimer's central region into one of three distinct conformations. Additionally, we expected the affinities of other proteins for the mutants' dimer-interface grooves to differ between mutants, giving us insight into how the conformational state of the ACD affects its ability to interact with other proteins.

Spinocerebellar ataxias (SCAs) are a group of hereditary diseases that are characterized by slowly progressive incoordination in gait, hand and eye movement, and cerebellum degeneration. Most forms of SCAs are caused by an expansion of short tandem repeats; however, SCA 14 is an autosomal dominant form of the disease associated with mutations in the Protein Kinase C gamma (PRKCG) gene, which encodes for the PKCγ protein, a serine/threonine kinase that plays a role in signaling and regulating cerebellar Purkinje cells' development. Our lab first reported this genetic cause in SCA 14 patients. To understand the pathogenesis of SCA 14, our lab generated mouse models of SCA 14 mutant (H101Y, F643L) and wild type (WT) PKCγ transgenic (Tg) mice using modified human-BAC constructs. By 3 months of age, both mutant Tg lines demonstrated impaired rotarod performance as compared to WT-Tg mice and showed PKCγ aggregates. Here I further examined the pathological changes by quantitatively analyzing the morphology and fluorescent intensity changes of Purkinje cells in mice cerebellum across age groups of 2-months, 6-months, and 12-months. I observed the dendritic arborization abnormality at an early age, and the abnormality is more severe in the Tg-PKCγ-F643L mice. PKCγ intensity in the cell body decreased in 2-month H101Y and F643L mice. These results revealed pathological changes and provided evidence the missense mutations caused an early development of SCA 14 cerebellar disease. It will be interesting to compare and verify these findings from the Tg mice in SCA 14 patients’ autopsy brain tissues as it could provide novel measures to characterize SCA 14 pathology.

In the retina, loss of neurons results in blindness, because the mammalian retina cannot regenerate. Although mammals cannot regenerate neurons, species such as fish and amphibians can make fully functional neurons after injury and restore their vision. Muller Glia (MG) cells in fish and amphibians are the source of this regeneration. These cells respond to injury by dedifferentiating into progenitor cells that then become neurons, replacing the dead neurons and in turn restoring sight. At the Reh Lab, we have found a way to stimulate functional regeneration in mammals through these MG cells by expressing a gene called Ascl1. This technique has limitations however, as it only causes 25% of MG to undergo neurogenesis. One of the variables potentially limiting regeneration is inflammation, as inflammation has been known to be detrimental to neurogenesis. Monocytes invade the retina after injury and potentially cause inflammation that limits retinal regeneration. To determine monocyte impact on retinal regeneration we employed a transgenic technique to ablate monocytes. I then performed our retinal regeneration paradigm and determined whether regeneration is enhanced in the absence of monocyte invasion. Using immunohistochemistry and confocal microscopy I found more regenerated neurons in retinas that lacked monocytes. These data further confirm that inflammation limits the regeneration capacity of the retina, and provides future topics to improve neural repair through modifying the immune response.

Mammalian retinas function in a more hypoxic environment than most tissues. The retina thus does not exclusively use oxygen as a terminal electron acceptor in the electron transport chain (ETC). As a result, expression of proteins involved in energy metabolism may be affected. We determined the levels of metabolic proteins of various tissues, and found that compared to eyecup (consisting of retinal pigment epithelium and choroid vasculature), kidney, and cerebellum tissue, the retina expresses higher levels of Hexokinase I. This suggests that glycolysis may occur faster in the retina than in other tissues. We also observed higher levels of the ETC proteins cytochrome c and subunit 4 of cytochrome c oxidase levels compared to both the eyecup and cerebellum. This implies an increased capacity for electron transport in the retina, despite a lower O2 tension. We also investigated post-translational protein modifications that could be affected by a hypoxic tissue microenvironment. Lysine succinylation is one such modification, and is controlled by regulators of energy metabolism such as SIRT5. I observed that succinyl-lysine intensities were higher in the retina than in the cerebellum, kidney, liver, and eyecup. Further investigation will be necessary to determine the role that lysine succinylation plays in the retina. Through these experiments we show that retina tissue is well-suited for rapid energy metabolism in spite of its hypoxic environment.

Enzymes have recently been incorporated into multiple high-value industrial syntheses, demonstrating the utility of enzymes as highly selective catalysts for practical industrial processes. However, the current scope of non-biological enzymatic reactions is narrow and new reactions and reaction pathways need to be engineered. The goal of our work in the Zalatan lab is engineering enzymes as catalysts in carbon-hydrogen bond functionalization reactions, a transformation critical for practical industrial synthesis where selective catalysis is still a major challenge. Importantly, we are interested in exploring more efficient and informed engineering approaches by establishing structure-function relationships with the enzymes that we work with. Our model system for this work is the non-heme iron(II) 2-oxoglutarate dependent oxygenase superfamily (Fe(II)-2OGs). We are using a high-throughput microfluidics based kinetic assay to determine key sites that we can target for mutagenesis and directed evolution in a candidate Fe(II)-2OG found to catalyze a new reaction. Overall, we expect that this work will enable new directions and principles for engineering Fe(II)-2OGs, and that lessons learned here can then be extended to additional industrially-relevant enzyme families.

The world’s population is rapidly aging, and we now increasingly face challenges related to the prevalence of age-related diseases. Studies from Geroscience have identified hallmarks of aging, such as the cellular senescence and mechanistic target of rapamycin (mTOR) pathways, that can be disrupted to improve health and lifespan in model organisms. Previous studies have shown that these pathways can be targeted pharmacologically by the mTOR inhibitor rapamycin or the senolytic drug combination Dasatinib + Quercetin (DQ). Our laboratory’s published data shows that rapamycin restored oral health in aged mice and reversed periodontal disease. While attempting to uncover the mechanism behind this reversal, our laboratory discovered that the age-related increase in cellular senescence was attenuated by rapamycin treatment. Thus, we hypothesized that targeting cellular senescence may provide an alternative therapeutic strategy to phenocopy the impact of rapamycin on aging oral tissues. I compared the effects of control (n=5), rapamycin (n=5), and the senolytic drug combination, DQ (n=5), in aged male mice (20-21 months old). Western Blot was performed on the mandible and salivary gland tissues to analyze the senescence marker p16INK4a. Histological assessment of H&E and lipofuscin staining was completed on the salivary glands to compare the effects of age, rapamycin, and DQ treatment. In male salivary glands, I discovered the age-related increase in p16 expression was decreased after rapamycin treatment, but not with DQ treatment. There were no significant changes in levels of p16 in the mandible with age or with the administration of rapamycin or DQ. Rapamycin treatment attenuated cellular senescence in male salivary glands, while the senolytic cocktail DQ had no impact on the aging male salivary glands. Future studies could be performed on female mice, with the addition of alternative senolytics which may target other markers of cellular senescence beyond p16.