Neural tube defects (NTDs) impact 3000 pregnancies a year in the US and are caused by both genetic and environmental factors. NTDs arise from errors in neural progenitor signaling, migration, proliferation, and differentiation during embryonic development. Spina bifida (SB), a prevalent NTD, can reduce the functioning of neural pathways responsible for pain and motor function in the lower body. A recent study discovered a novel variant of the receptor GPR161 present in screened infants with SB, but absent in all screened infants without. GPR161 is a G protein-coupled receptor localized in the primary cilia known to participate in the regulation of the pathways of key stem cell differentiation ligands, sonic hedgehog (Shh) and Wnt. Our study seeks to investigate the molecular mechanisms which connect the novel GPR161 variant p.Trp202Gly to neural tube defects using an in vitro model of neural stem cell differentiation. GPR161 variant and knock-out (K/O) lines are generated using CRISPR Cas9 technology in induced pluripotent stem cells (iPSCs). iPSCs are then guided through neural differentiation and harvested for analysis at multiple key stages of neural progenitor development. Markers of neural differentiation, SB, and downstream GPR161 factors are analyzed using western blot, RT-qPCR, immunostaining, and RNAseq. We expect to see a change in Shh activity in the variant line compared to the WT.

Alzheimer's disease (AD) is the most common cause of dementia, a general term for memory loss and other cognitive abilities. Although this disease has been a major research focus since the 1980s, the pathological mechanisms are still not well understood, and therapeutic interventions have been ineffective. The most definitive method for classifying AD is through identifying accumulations of toxic amyloid-beta (Aβ) and tau proteins in post-mortem brain tissue. Dr. Su-in Lee’s lab has developed a machine learning method that integrates the pathological protein phenotypes with gene expression levels in the same brain tissue. They have highlighted 25 genes with expression level changes that correlate with the tau and Aβ protein aggregation phenotypes. For this proposal, I have integrated these human neuropathology-based phenotypes with the genetic power of Caenorhabditis elegans (C. elegans) to directly test the impact of these candidate genes on the cellular pathology. Previously, all C. elegans tau models had neuronal specific expression. However, neurons are resistant to RNAi. Therefore, I generated a novel transgenic C. elegans tau AD model that has been codon-optimized to express tau in body wall muscles instead of neurons. I measured the animal’s health with age in a series of phenotypic assays: egg-laying, growth, movement, paralysis, and lifespan analysis. This line exhibits premature paralysis and decreased crawling speeds, providing an easy to score phenotype. This new model allows for high-throughput RNAi screening to test the identified 25 genes’ effects on worm health by utilizing the automated worm-movement technology developed in the Matt Kaeberlein lab that can simultaneously determine the rate of paralysis of thousands of worms. The results of my genetic screening will lead to a better understanding of the human genes that are dysregulated in human AD brains, provide a basis for genetically-dissecting the pathways influencing tau toxicity, and suggest new therapeutic targets.

Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the formation of senile plaques and neurofibrillary tangles through the accumulation of toxic amyloid-beta and Tau protein. There is growing recognition that extracellular vesicles (EVs) can package and transport toxic peptides associated with neurodegenerative disorders – such as AD – to other cells in the brain. Researchers in the Kaeberlein lab have designed methods to isolate these type of vesicles from C. elegans nematodes, a popular invertebrate genetic model. However, current nematode EV purification methods do not permit the following of EV signals from specific tissues when they are under AD proteotoxic-stress. I have generated a transgenic C. elegans AD model that has muscle specific expression of the pathogenic human Tau protein. The protein coding sequence was designed to use optimized codons to ensure high expression of the transgene. I have also generated transgenic nematode lines that express versions of known transmembrane proteins with small affinity tags in a tissue specific manner. The small affinity tags on the proteins make it possible to specifically pull down the EVs from designated tissues through standard immunohistochemistry techniques. The abundance of tissue-specific EV protein and RNA cargos from transgenic lines with or without human Tau have then been quantified using LC-MS-MS and RNAseq analyses, and parsed and condensed into a MySQL database via a C# program. The database allows for simple searching through large amounts of MS data, making data analysis more efficient and effective. Thus the methodology and tools I develop in this project could become a promising new approach for identifying novel therapeutic gene targets and biomarkers of AD stress.

Substance abuse leads to alterations in cognition that affects processes such as impulse control and valuation. Decreased impulse control and aberrant valuation are responsible for continued drug seeking and are thought to be escalated by external stress stimuli. Stress leads to release of an endogenous opioid neuropeptide called dynorphin which binds to the Kappa Opioid Receptor (KOR). Upon KOR binding, dynorphin induces a protein signaling cascade that also promotes drug seeking behavior. In this study, we investigated the dynorphin/KOR system in the medial prefrontal cortex (mPFC) due to its critical role in cognition. We examined the properties of dynorphin release in the mPFC of C57BL/6 mice in response to different external stressors to determine if this nucleus is a potential therapeutic target for stress-induced drug seeking behaviors. Using a pharmacological approach, we first showed that systemic administration of U50,488, a KOR agonist, leads to KOR activation in the mPFC. U50,488 administration also disrupted cognition by impairing performance in a working memory behavioral task. We next tested whether different stress modalities stimulated mPFC dynorphin release and disrupted cognitive performance. Surprisingly, repeated forced swim stress did not cause dynorphin release in the mPFC and did not disrupt cognitive performance although it did activate dynorphin release in the Dorsal Raphe nucleus, as expected. In contrast, different stressors, including repeated foot shock and precipitated morphine withdrawal did effectively lead to KOR activation in the mPFC. This indicates that dynorphin release in the mPFC is dependent on the type of behavioral stress. Future experiments will utilize an in-vivo dynorphin sensor, kLight, to detect dynorphin release in real-time in response to these stressors. Exploration of the differences in dynorphin/KOR system functioning in response to different stress modalities is important for establishing how this system may be targeted to alleviate stress-induced drug seeking behaviors.

Dopamine (DA) neurons found in the ventral tegmental area (VTA) are associated with reward feedback, and dysfunction in DA circuitry is associated with disorders such as Parkinson’s, schizophrenia, bipolar, and addiction to drugs. To adequately treat these diseases, we must have a more complete understanding of how dopamine contributes to emotional processes. This research project addresses this issue by investigating neuropeptide regulation of dopamine neurons in the VTA. The bed nucleus of the stria terminalis (BNST) is a brain region that expresses many neuropeptide genes and sends strong projections to the VTA. We utilized Cre driver lines to isolate neurons that produce the peptides Neurotensin, Neurokinin B, and Corticotropin Releasing Factor. We injected a virus into the BNST that induces the expression of a light activated ion channel and allows us to stimulate axon terminals in the VTA. I then conducted behavioral experiments to assess the effects of activating these peptidergic inputs. Dopamine-dependent behaviors relating to pleasure, reward, and anxiety were measured through the behavioral tests of Real Time Place Preference, operant conditioning, and Open Field respectively. Most likely due to low expression, my behavioral analyses did not yield statistically significant results. Moving forward, it may be necessary to increase viral titer for wider expression. In the future, I intend to use CRISPR/Cas9 technology to isolate neuropeptide function from fast neurotransmitter release in these circuits. This research, by producing findings that help explain how neuropeptides modulate DA neurons, has the potential to generate advances for the understanding and treatment of dopamine-related psychiatric disorders.

Axonal guidance proteins are known to aide in neurodevelopment by directing neuronal growth cones to their target locations through a combination of attraction and repulsion cues. Many axonal guidance proteins are present in the mature brain, and evidence suggests their potential role in maintaining synaptic connectivity. The present study has sought to determine whether axonal guidance cues contribute to the balance of excitatory and inhibitory input onto dopamine neurons in the ventral tegmental area (VTA) in the adult mouse brain. With CRISPR-Cas9 gene editing, a Cre-dependent adeno-associated virus (AAV-DIO-SaCas9-sgRNA) was administered directly to the VTA of adult mice via stereotaxic injection to genetically inhibit the expression of axonal guidance proteins of interest. DAT-iCre mice, expressing Cre under the DAT promoter, received AAV-DIO-SaCas9 injections containing a short guide RNA for either Ntn1 (the gene encoding netrin-1, a secreted axonal guidance cue), Dcc (a netrin receptor) or Unc5c (a netrin receptor characterized as causing growth cone repulsion). Control mice received stereotaxic injections of AAV-DIO-YFP. After a healing period, dopamine-specific behavioral assays were performed to test for alterations in dopamine mediated behaviors. Electrophysiology was used to measure changes in inhibitory and excitatory connectivity in dopamine neurons of the VTA. Preliminary results suggest that netrin-1 and netrin receptor expression in dopamine neurons of the VTA may play a role in maintaining excitatory synaptic connectivity. These findings could shed light on the role of excitatory and inhibitory connectivity, as well as dopamine-associated psychiatric conditions such as schizophrenia and substance use disorders.

In mammals, the endogenous physiological and behavioral master clock consists of approximately 20,000 neurons, a region in the brain called the suprachiasmatic nucleus (SCN). These neurons produce molecules whose levels oscillate in a robust 24-hour rhythm. Studies show that proper functioning of the neurotransmitter GABA is necessary for the maintenance of circadian entrainment to the light-dark cycle and synchronicity between these otherwise self-sustaining, individual oscillators. Although approximately 90% of the cells in the SCN communicate via GABA transmission, the exact role of GABA in the SCN is not fully understood. Our lab has confirmed that removal of the vesicular GABA transporter (Vgat), a channel that allows entry of GABA into the vesicles of presynaptic neurons and thereby enables GABA release into the synapse, from SCN neurons results in circadian arrhythmicity in mice activity patterns under constant darkness. The goal of my project is to determine how the lack of GABAergic transmission affects the expression of other chemical signals SCN neurons use. Specifically, my project has been focused in quantifying neuronal fibers containing vasoactive intestinal peptide (VIP), a neuropeptide that communicates light information to the SCN and is critical for the maintenance of circadian rhythmicity. Using image analysis of brain sections previously immunostained against VIP, I showed that VIP fiber density is markedly reduced in animals lacking Vgat expression in the SCN. My study provides novel evidence of a relationship between GABA release, the expression of VIP within the SCN, and the overall impact this relationship has in the regulation of circadian rhythms.

Traumatic spinal cord injuries (SCI) are a catastrophic type of injury that disrupt many functions of daily life such as mobility, temperature regulation, sexual function, and bowel/bladder use. Approximately 80% of individuals who suffer from SCI develop neurogenic bladders, and subsequently require catheterization. Patients with sacral SCI have different neurogenic bladders than those with higher injuries; specifically, severing the peripheral innervation at the sacral level causes a non-contracting and low pressure bladder, without the scar tissue found commonly in higher injuries. This suggests that peripheral denervation decreases the incidence of non-compliant bladder complications. Bladders primarily have two uses - storage and voiding during urination. One common strategy to improve the storage function of neurogenic bladders is to denervate them with direct injections of Onabotulinumtoxin-A (ONA). However, this treatment is only applied after other treatments have failed, leaving time for significant damage to occur. In the current study, we propose to study the effects of direct ONA injections acutely after SCI. Our two aims are 1) to determine the treatment window for chemodenervation that best reduces bladder wall hypertrophy and fibrosis, and 2) to determine whether bladders remain compliant after chemodenervation. Using a rat model, we applied two treatments - SCI + Saline, SCI + ONA, with both saline and ONA directly injected into the bladder wall. A control group received laminectomy without injury. After treatment, we plan to measure bladder function using a urinary incontinence scale, as well as histological measurements of collagen deposition and muscle area to quantify bladder wall hypertrophy and fibrosis. We anticipate that early ONA injection (as defined in Aim 1) will be associated with less fibrous and more compliant bladders in the long term when compared with non-chemodenervated bladders. If our hypotheses are correct, we plan on getting IRB approval for a clinical trial.