Autism Spectrum Disorders (ASD) are a combination of neurological and developmental abnormalities, with 1 in every 36 children diagnosed worldwide. Brain Selective Kinase 2 (BRSK2) is one of the strongest autism-associated genes, with 35 de novo mutations reported to date. Patients harboring BRSK2 variants clinically present with neurodevelopmental disorders, including speech delay, intellectual disability, motor dysfunction, and behavioral abnormalities. Despite its strong ASD association, the molecular functions of BRSK2 and the mechanisms through which it regulates neurodevelopment remain unclear. My project aims to investigate the molecular role of BRSK2 by identifying its localization in the developing hippocampal and cortical neurons. The function of a gene is reliant upon its localization within the cell. To identify the subcellular localization of BRSK2 during early neurodevelopment, I am analyzing the subcellular distribution of BRSK2 in cultured primary embryonic rat neurons at different developmental time points, using immunocytochemistry and confocal microscopy. To delineate the impact of missense mutations in BRSK2 on its localization, I am analyzing the phenotype of cultured hippocampal rat neurons with GFP-tagged engineered constructs harboring the BRSK2 mutants. My analysis found that both hippocampal and cortical neurons display mostly cytoplasmic BRSK2 localization, with a significant association with the subcellular endomembrane as well as the plasma membrane (PM). Interestingly, BRSK2 was also found at the dendritic spines at day in vitro (DIV) 12. We are currently investigating whether any of these missense mutations disrupt inter-organelle communication between the endomembranes and plasma membrane. BRSK2’s localization in the endomembranes could explain disruptions in protein processing, dendritic development, or neuronal polarity linked with the missense mutations that eventually impact neurodevelopment, leading to autism. Discovering BRSK2’s localization will help contribute toward the future development of targeted therapies for ASD caused by the dysfunction of the BRSK2 kinase.

The kappa opioid receptor (KOR) system is a promising target for substance use disorder, yet its role in long-term addiction regulation remains unclear. This project investigates how selective activation of the KOR/c-Jun N-terminal kinase (JNK) pathway activates the enzyme peroxiredoxin VI (PRDX6), triggering the release of reactive oxygen species (ROS) and resulting in long-lasting KOR inactivation distinct from its canonical Gαi pathway. I investigate whether JWT-101, a repurposed ligand, acts as a long-term KOR antagonist by inducing JNK-mediated ROS production, potentially offering new therapeutic avenues. KOR-Cre mice were injected in the prefrontal cortex with oROS-Gr, a fluorescent tag that senses ROS concentrations, for selective expression in KOR-positive neurons. Using high-resolution two-photon microscopy, I monitored ROS levels in live brain slices after 2 weeks from these mice. Bath application of JWT-101 led to increased fluorescence, indicating elevated ROS production and thus, JNK activation. To confirm JNK path specificity, I applied MJ33, an inhibitor of PRDX6. Fluorescence was reduced following MJ33 treatment, indicating that JWT-101 acts in a KOR/JNK manner. These findings suggest that JWT-101 induces KOR inactivation through ROS-mediated signaling. This research provides insights into KOR/JNK signaling in substance use disorders, with implications for developing targeted therapies for recovery and relapse prevention.

The locus coeruleus (LC) is a major neuromodulator source with widespread projections to distinct functional targets that influence arousal, anxiety, learning, and other behavioral states. Our lab has previously shown LC excitation triggers the release of norepinephrine (NE) into the basolateral amygdala (BLA). Recent studies suggest LC terminal stimulation may release DA into the dorsal hippocampus (dCA1) enhancing novelty-associated spatial learning. Our recent data show LC stimulation evokes DA release. Previously, release across regions, paradigms, and behaviors typically associated with LC have not been characterized, due to difficulty in separating DA from NE using traditional sensing methods. Due to this, the relationship between the LC and other DA systems remains unclear. To understand the mechanisms by which the LC may release DA independently of the ventral tegmental area (VTA), a major DA source, we have employed optogenetic stimulation to evoke release from neuron terminals and quantify the release dynamics of NE and DA. We used fluorescent biosensors to detect NE and DA, captured by a fiber optic cable and amplified to observe the relative dynamics of DA release. These sensors have tuned affinity and selectivity for NE and DA and use fluorescence as a proxy for neuromodulator release. In this project, we aim to elucidate how and under what conditions the LC is releasing DA across regions with different functions during aversive and appetitive behaviors. These data will enhance our understanding of the LC neuromodulator signaling that can become maladaptive and afflict anxiety, addiction, and more, and also demonstrate that the release of DA from the LC is dependent on the behavior induced.

TBC domain containing kinase (TBCK) is an understudied protein with three domains: a pseudokinase; Tre-2, Bub2, and Cdc16; and rhodanese, and is highly expressed in the brain. Homozygous mutations in TBCK cause a rare neurodegenerative disorder in children, which clinically presents as syndrome infantile encephalopathy, brain atrophy, cerebellar hypoplasia, and muscle hypotonia. Two mutations in particular, Arg126Stop and Arg511His in the pseudokinase and TBC domains respectively, are commonly found among TBCK patients. The progression of the disease is characterized by a global regression in brain development, severe intellectual disability, and premature death in acute cases. The pathogenic mechanism underlying TBCK syndrome is unclear, but past studies show that TBCK patient neurons demonstrate aberrant metabolite buildup in the lysosome likely resulting from abnormal lysosomal activity. Immunoprecipitation mass spectrometry was performed for wild type TBCK in both N terminal and C terminal tags, revealing a preliminary list of both known and unknown interactors for TBCK. To further investigate the early developmental implications of mutant TBCK, CRISPR/Cas9 directed mutagenesis is being used to generate two induced pluripotent stem cell (iPSC) lines harboring the Arg126Stop and Arg511His mutants for subsequent differentiation into neural progenitor cells (NPC) and neurons. Immunofluorescent imaging of the mutant NPCs will confirm the recapitulation of growth and lysosomal defects present in patient cells. To analyze the effect of TBCK mutation on lysosomal function/content, we will immuno-isolate lysosomes through lysosome immunoprecipitation (Lyso-IP) and identify proteomic changes through mass spectrometry. While providing a crucial in vitro cell model of two common patient mutations, these experiments will offer critical insight into cellular dysfunctions that contribute to TBCK disease states.

Kappa opioid receptor (KOR) ligands have been explored for anti-anxiolytic, anti-depressive, pain, and substance use disorder therapeutics. These therapeutic effects are partly due to biased signaling through the cJun N-terminal Kinase (JNK) pathway, which involves complex molecular interactions and downstream effects that inactivate the receptor by producing reactive oxygen species (ROS). JWT-101, a clinically approved compound, has been shown to produce therapeutic effects for these conditions. We hypothesize that its mechanisms of action are through KOR antagonism. I previously assessed KOR agonist-induced analgesia by measuring the latency of tail withdrawal from 52.5°C water after treatment with U50,488, a KOR agonist. Pretreatment with 15mg/kg JWT-101 24 hours before U50,488 injection effectively blocked KOR-induced analgesia in wild-type male mice. This effect was reversed by the short-acting, KOR-selective antagonist Aticaprant (5 mg/kg), suggesting that JWT-101’s action is mediated through KOR. Further investigation using in-vivo fiber photometry with the novel peroxide sensor AAV oROS-Gr revealed that JWT-101 significantly increases ROS production in KOR-expressing cells. Injection of 15 mg/kg of JWT-101 increases oROS fluorescence compared to control post-injection. Pretreatment with Aticaprant 15 minutes prior to JWT-101, blocks oROS fluorescence, suggesting that JWT-101’s activity is mediated by KOR. Pretreatment with MJ33 (a PRDX6 inhibitor upstream of JNK activity) 50 minutes before treatment with JWT-101 blocked oROS fluorescence, suggesting that this ROS production is through the JNK/PRDX6 pathway of KOR activation. This study provides insights into the mechanism of action of JWT-101 and examines the underlying molecular mechanisms of KOR-associated effects.

Stress has a profound impact on reward-seeking behaviors, increasing the likelihood of relapse in individuals with substance use disorder. One of the molecular mechanisms underlying this stress-induced shift in behavior involves the activation of kappa opioid receptors (KOR) and the downstream signaling pathways that influence both dopamine (DA) and serotonin (5HT) neurons. Specifically, the activation of KOR by the endogenous neuropeptide dynorphin triggers an arrestin-dependent pathway, resulting in the recruitment of p38ɑ mitogen-activated protein kinase (MAPK), which mediates the aversive effects of receptor activation. This project aims to explore the role of pharmacological and  stress-mediated KOR/p38ɑ  MAPK signaling in DA and 5HT neurons. To determine KOR/p38ɑ  MAPK signaling in both dopamine and serotonin release in the NAc during pharmacological activation of KOR or during stress (rFSS), we utilized CRISPR technology to manipulate p38ɑ MAPK signaling in the ventral tegmental area (VTA) and dorsal raphe nucleus  (DRN) respectively. To monitor both DA and 5HT tone in the NAc we used the novel GPCR-based DA and 5HT sensors (GRAB-DA and GRAB-5HT). Both serotonin and dopamine tone were decreased after KOR agonist administration compared to control in the NAc. Cocaine, our positive control robustly increases serotonin and dopamine compared to control.  During periods of swim stress,  serotonin fluorescence robustly decreases which can be blocked by p38ɑ excision from the DRN or pretreatment with the KOR antagonist norBNI. This study provides insights into the molecular mechanisms underlying stress-induced changes in reward-seeking behavior and identifies potential therapeutic targets for substance use disorder.

BK channels are potassium channels activated concomitantly by membrane depolarization and the elevation of intracellular calcium. We have previously shown that BK channels form clusters at the plasma membrane in heterologous cells and primary neurons, but the mechanism for their clustering is unknown. Our research seeks to discover important components that generate and maintain BK channel clusters. We hypothesize that membrane lipidic composition can be essential in BK clustering. Given the known role of PIP2 in increasing the activity of BK channels, we evaluated the role of PIP2 in their spatial organization. We expressed BK channels in a human cell line and assessed the organization in clusters using super-resolution microscopy and proximity ligation assay. We also measured ion channel mobility using fluorescence recovery after photobleaching. We expressed PIP5K and Ins5P to increase and decrease PIP2 levels, respectively. Preliminary experiments found that the expression of PIP5Kγ did not affect the mobility of single or cluster BK channels, but decreased density of channels at the plasma membrane.

Chronic pain affects about 20% of the adult population in the US, with more than 25% of these being pain that severely limit a person’s daily activities. In recent years, scientists in the field have been classifying pain as both a sensory response and emotional experience influenced by physiological and social factors. Newer research on pain behaviors and social behaviors have indicated that there is a positive association between the presence of cage mate in pain and the sensitivity to pain for a mouse. Although the behavioral responses are observed, the neural circuits mechanisms have yet to be examined. I will inject wild type mice with GCaMP in the medial prefrontal cortex (mPFC) and RCaMP in the basolateral amygdala (BLA). GCaMP and RCaMP are both genetically encoded Calcium indicators and are sensitive proxies for measuring excitatory transmission between brain regions. I will then implant fibers in both brain regions of all mice for fiber photometry recordings. After sensor expression time, I will check Calcium signals using a stressful stimulus known to stimulate excitatory pathways in mice then surgically induce pain in half of the mice. Mice will be split into chronic pain and pain-free groups, with their cage mate being either in pain or pain-free. I will perform a triad of behavioral pain testing simultaneously with fiber photometry recording, including tests for mechanical and thermal pain. I predict that for pain-free mice housed with a cage mate in pain, their pain threshold will decrease, as measured by all behavioral experiments. This should be accompanied by a stronger increase in BLA to mPFC Calcium signal when the mice are receiving painful stimuli. 

The ongoing opioid epidemic has made the need for alternative pain management strategies more urgent than ever. Nearly 1 in 5 Americans suffer from chronic pain, which has traditionally been treated with opioids and non-steroidal anti-inflammatory drugs (NSAIDs). However, both classes of drugs come with significant drawbacks. NSAIDs are often ineffective for managing chronic pain and can cause kidney and liver damage with prolonged use. Meanwhile, opioids lose their effectiveness over time, contributing to misuse, substance use disorders, and an increased risk of overdose. With few alternatives available that don't carry these risks, researchers are exploring new pain management options. One promising avenue is the use of cannabinoids, which are known for their anti-inflammatory and analgesic properties. In this study, I employ machine learning to create an unbiased kinematic and behavioral profile of mice experiencing chronic neuropathic pain using a custom-built linear track. Chronic pain and limb impairment are induced through partial sciatic nerve ligation, and a deep learning system analyzes videos of the mice to assess their movement patterns before and after treatment. I then compare these profiles to those of mice treated with NSAIDs, opioids, and cannabinoids, evaluating the effects of each treatment on behavioral measures like body position, which serves as a proxy for pain state and stress. We expect the mice treated with analgesics to show increased rearing and grooming behaviors. This research not only compares the analgesic effectiveness of cannabinoids to traditional pain-relief drugs but also helps reduce the stigma surrounding cannabinoid-based treatments.

The prefrontal cortex (PFC) is essential for cognitive functions such as decision-making, emotional regulation, and attention. Dysfunction in PFC circuitry is implicated in neuropsychiatric disorders, including Alzheimer’s disease, depression, and anxiety. Within the PFC, excitatory glutamatergic neurons and inhibitory GABAergic neurons coordinate activity to maintain proper network function. The excitatory-inhibitory balance is critical for cognitive processing, yet the role of the most abundant GPCR in the brain, the cannabinoid 1 receptor (CB1), in regulating these neuronal populations remains unclear. CB1 receptors are highly expressed across other cortical regions but have the most dense expression in the PFC where they are hypothesized to modulate synaptic transmission and plasticity. To investigate their cell-specific function, we utilized a CRISPR-Cas9 to locally knockout the CB1 receptor specific neuronal populations using a viral cre-dependent driver. This virus was administered in either vesicular GABA transporter (VGAT)-Cre or vesicular glutamate transporter (VGLUT)-Cre animals to select for inhibitory or excitatory neurons, respectively. We assessed CB1 receptor expression using RNAscope in situ hybridization to quantify CB1 mRNA in VGAT-expressing inhibitory neurons and VGLUT-expressing excitatory neurons. Fluorescence microscopy was used to visualize CB1 receptor distribution and determine whether its expression differs between these neuronal populations compared to controls. By mapping CB1 receptor expression and assessing its functional role in these neurons through previous behavioral experiments, this study provided insight into how the endocannabinoid system regulates PFC circuitry. Understanding CB1-mediated modulation of excitatory and inhibitory balance could have broad implications for neuropsychiatric disorders characterized by PFC dysfunction.

Copy number variations (CNVs) of the 16p11.2 (BP4-BP5) genomic locus are closely associated with neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia. Interestingly, 16p11.2 CNV deletion and duplication carriers exhibit some opposing phenotypes, with deletion associated with macrocephaly and obesity, and duplication with microcephaly and decreased body mass index. To identify the molecular mechanism underlying 16p11.2 CNVs, we differentiated patient-derived stem cells into neural progenitor cells (NPCs) as a model system for early neurodevelopment. Quantitative tandem mass tag (TMT) proteomics identified proteins that are phosphorylated differently between NPCs from carriers of a 16p11.2 CNV and NPCs from unaffected individuals. Notably, the differentially phosphorylated proteins found were enriched in primary cilia and centrosomal function, which is relevant for neurodevelopment. Through immunocytochemistry on the NPCs using a primary cilium specific antibody, the lab found that deletion and duplication had opposing effect on the cilia length. Deletion carriers had increased cilial length and duplication carriers had decreased cilial length. To identify which of the 30 known genes involved in 16p11.2 are drivers of these changes, knockdown and overexpression screens determined thousand and one kinase 2 (TAOK2) to be the most significant in cilia length. Using immunofluorescence assays, I found that intraflagellar transport protein 88 (IFT88), accumulates at the cilia tip in TAOK2 knockout NPCs, indicating disrupted transport within the cilia. IFT88 is a key regulator of Sonic hedgehog (Shh) within primary cilia and Shh is also a key regulator of neurodevelopment. Therefore, to understand the functional relevance of these findings on ciliary length, I performed quantitative PCR to measure changes in Shh activity. Since our findings so far demonstrate disrupted ciliary transport, I expect differences in Shh activity between wild-type and knockout TAOK2 NPCs. These investigations build our understanding of 16p11.2 CNVs and the mechanisms that implicate them in neurodevelopmental disorders.

Protein Kinase Inhibitors (PKIs) are a family of heat stable, high-affinity inhibitors of the catalytic subunit of Protein Kinase A (PKAc). In the presence of Mg-ATP, the three isoforms—PKIα, PKIβ, and PKIγ—bind to PKAc with very low dissociation constants: 0.758nm, 1.875nm, and 0.4142nm respectively. In vitro studies have shown that PKIs can translocate PKAc from the nucleus to the cytoplasm, suggesting a role for PKIs in terminating nuclear cAMP-driven PKA activity. Previous research, including studies from our lab, has found that dysregulated PKAc mutants play a significant role in Cushing’s syndrome, a rare and potentially fatal metabolic disorder caused by excessive cortisol production. Building on these findings, we hypothesized that increasing PKI expression could counteract the hyperactivity of PKAc mutants and reduce cortisol production. To test this, we expressed each PKI isoform in adrenal cell lines and assessed their steroidogenic capacity using biochemical assays such as western blots, RNA-seq, qPCR, and ELISA-based cortisol assays. We observed that PKIα and PKIγ led to a general suppression of steroidogenic associated proteins such as StAR, Cyp11a1 and SF1. This altered proteome was accompanied by significantly suppressed cortisol synthesis only in the PKIα and PKIγ expressing cells. The difference between PKIα/γ and PKIβ was surprising given that all PKI isoforms are postulated to potently inhibit PKAc. Thus, we questioned whether PKIα/γ effects are mediated through PKAc. To answer this, we have cloned mutant PKI isoforms that do not bind PKAc, and confirmed the mutant PKIs do not inhibit PKAc through kinase assays. Our next step is to express the mutant PKI isoforms in adrenal cells and assess their effect on steroidogenic capacity of the cells. Our findings suggest that PKIα and PKIγ play key roles in cortisol regulation and may have broader implications for gene regulation in adrenal cells.

Cardiovascular diseases are the leading cause of morbidity and mortality in the United States. Among the many contributing factors, mishandling of intracellular calcium (Ca2+) dynamics plays a crucial role in the etiology of cardiac diseases including heart failure, and arrhythmogenic disorders. Cardiac ryanodine receptor 2 (RyR2) channels play a central role in excitation-contraction coupling by regulating Ca2+ release from the sarcoplasmic reticulum (SR). Abnormal activity of the RyR2 by impairing Ca2+ release from the SR results in sudden death in many cardiac disorders. Thus, regulators of RyR2 could provide a novel therapeutic target in several heart diseases. Our initial studies implicate the role of the chloride intracellular channel, CLIC4 in modulating the activity of RyR2. We identified CLIC4 as a mitochondrial-associated endoplasmic reticulum membrane protein. The absence of CLIC4 induced faster Ca2+ release from SR, indicating abnormal RyR2 activity. Further, co-immunoprecipitation studies indicated an interaction between RyR2 and CLIC4. Moreover, we found that the absence of CLIC4 increased myocardial infarction upon ischemia-reperfusion (IR) injury in mice. Thus, based on our findings we hypothesize that CLIC4 by either stabilizing RyR2 in a closed state or by regulating the anionic gradient across SR modulates the  RyR2 activity. In this study, we will map the domain in CLIC4 specific to interaction with RyR2 and modulate its activity. We will systematically clone and express various N- and C-terminal truncated CLIC4 constructs to investigate their interaction with RyR2. Further, we will determine the effects of these constructs in modulating calcium release from RyR2. Our studies could aid in the development of a peptide-based therapeutic approach to modulate RyR2 activity in cardiac diseases.

Cannabis is the most commonly used drug in America, with 52.2 million individuals (19% of Americans) reporting use in 2021. The primary psychoactive compound, Delta-9-tetrahydrocannabinol (THC), binds to cannabinoid receptors, among the most abundant in the brain. This interaction causes mental and locomotor impairment, contributing to increased motor vehicle crashes in states with legalization. However, a comprehensive baseline for THC’s biophysical effects on behavior and motor function remains lacking. This research aims to establish such a baseline using advanced AI-driven behavioral analysis in mice. Mice received intraperitoneal injections of THC (0.1–30 mg/kg) or a vehicle solution (control). One hour post-injection, each mouse was recorded for 15 minutes in a custom Linear Track designed for dual-view (side and bottom-up) behavioral assessment. Video recordings were analyzed using an AI computer vision model tracking 29 points of interest at 100 fps. The collected data trained a THC behavioral regression AI algorithm to predict doses based on behavioral patterns. Analysis of novel videos revealed a model accuracy with a mean squared error of 0.50, successfully identifying THC-induced impairment. This approach also enabled investigations into specific brain regions mediating THC behaviors through local drug infusion. This study marks the first successful attempt to predict THC dose relative to impairment levels using AI modeling. The research aims to computerize behavioral analysis, developing a preclinical AI model capable of recognizing and predicting THC’s effects with minimal human bias and error. This technology provides a data-driven approach to characterizing subtle behavioral differences, offering potential applications in both research and clinical settings.