TRPV4 is a calcium-permeable nonselective cation channel mostly expressed in chondrocytes. Previous studies show the TRPV4 channel becomes up-regulated during chondrogenesis, revealing that TRPV4 channel activity is crucial to cartilage development and thus skeletal development. Activating missense mutations in the TRPV4 channel cause a spectrum of skeletal dysplasias including Spondylometaphyseal Dysplasia, Kozlowski type (SMDK), a moderate disorder with pronounced short stature, kyphoscoliosis, and short limbs, most commonly caused by the R594H mutation. Although a milder TRPV4 mutation has been studied, a viable animal model to study the more severe effects the SMDK disorder has on skeletal growth has yet to be conducted. To effectively model SMDK, we generated a conditional knock-in mouse for R594H. Postnatal induction at weaning using Acan-CreER and tamoxifen produced mice with statistically significantly shorter femora, humeri, hip bones, tibiae, tails, and multiple lumbar vertebral heights for both sexes. Midgestational induction of the mutation in chondrocytes using Col2a1-Cre resulted in mice with statistically significantly shorter femora, humeri, hip bones, tibiae, lumbar vertebral heights, body lengths, and tail lengths for males. Qualitative analysis revealed smaller epiphyses with widened metaphases at the proximal tibia. Together, these analyses provide evidence of a representative animal model for the SMDK disorder seen in patients. Furthermore, it understanding of how the R594H mutation affects skeletal development that has not been possible previously. In the future, this mouse model will also be used for testing TRPV4 inhibitors to prevent disease progression of the TRPV4 skeletal disorders.

Caenorhabditis elegans is a free-living nematode that serves as a model system for understanding sensory behaviors. While studies have shown carbon dioxide (CO2) avoidance by well-fed young adults and CO2 attraction by young adults that were starved or grown in a high-CO2 environment, the responses of older adults have not yet been examined. We tested the carbon dioxide response of C. elegans adults using a CO2 chemotaxis assay with both temperature-sensitive CF512 animals aged 1 to 3 days and floxuridine-treated N2 wild-type animals aged 1, 4 and 7 days, where day 1 is the first day of adulthood. To determine the response of older adults to carbon dioxide, we compared the CO2 responses of older adults to those of younger adults under well-fed conditions. We hypothesize that C. elegans shows a decreased response to CO2 as the worms age, due to neural deterioration and loss of signaling activity. Notably, we found that the N2 animals maintained a robust aversion to CO2 up to day 7. These findings support the conclusion that C. elegans maintains a robust and consistent response to CO2 as they age, even as other tissues undergo age-related degeneration. These findings increase our understanding of age-related changes in CO2 response in C. elegans and will enhance our understanding of the factors that drive age-related changes in other organisms, including humans.

In alternative pre-mRNA splicing, introns are spliced out while exons are joined together in various combinations to generate different mRNA isoforms from the same gene. RNA binding proteins (RBPs) can regulate alternative splicing through binding to cis-regulatory elements on the pre-mRNA to either enhance or repress the inclusion of exons. Many RBPs contain regions that allow them to condense into phase-separated structures. However, it is not clear how the self-assembly property of these proteins affects their ability to regulate alternative splicing. The RNA binding Fox (Rbfox) proteins are a highly conserved family of RBPs with important roles in the regulation of alternative splicing in neuronal development. Previous studies in our lab showed that Rbfox in the nucleus is bound to chromatin as part of a large assembly of splicing regulators (LASR) containing at least 7 other proteins. The Rbfox/LASR complex can self-assemble into still higher-order structures through Rbfox’s C-terminal domain (CTD). The Rbfox CTD contains tyrosine residues that are important for this self-assembly property. However, it is not known if there is a specific segment within Rbfox’s CTD that mediates its self-assembly. In this project, we will use co-immunoprecipitation and phase-separated droplet assays of recombinant proteins to determine the residues within Rbfox’s CTD that allow it to self-assemble. This study will further our understanding of protein domains that form condensate-like structures and their role in the regulation of alternative splicing.

The protein DHX9 serves diverse roles in human and murine cells and is implicated via proteomics data in regulating X chromosome inactivation (XCI) during embryonic stem cell (ESC) differentiation. To understand the importance of DHX9 during differentiation and the XCI process, mouse ESCs (mESCs) were utilized to observe the effects of its depletion on ESC proliferation and self-renewal as well as transcript levels and localization of the long-noncoding RNA Xist, master regulator of XCI. To rapidly deplete DHX9, the degradation TAG system (dTAG) was used, marking DHX9 for ubiquitination upon addition of the molecule dTAG-13. mESCs were treated with dTAG-13 or a control dTAG molecule for various times and counted every 24 hours to assess proliferation. Coupled with colony formation assays to assess self-renewal and colony formation ability, these experiments were repeated on differentiating mESCs to determine the importance of DHX9 during differentiation. Furthermore, IF and FISH quantified the effects of DHX9 depletion on Xist levels and cloud formation. We found that DXH9 depleted mESCs exhibited decreased potential for colony formation, proliferation, and self-renewal. Cells treated with dTAG-13 exhibited less growth and more cell death than their control counterparts; furthermore, IF/FISH imaging showed the localization of Xist was disrupted in the absence of DHX9. To elucidate during which stage in the cell cycle differentiating cells are affected upon, DHX9 depletion, FACS cell sorting and western blot analysis of DHX9 interacting proteins will be done. Overall, our data define novel functions for DHX9 in stem cells and early development.

Neural antibody testing has been used as a primary biomarker for the diagnosis of immune-mediated epilepsy. Neural antibodies have been implicated in a variety of central nervous system (CNS)-specific immune disorders, and even in asymptomatic patients, casting doubt on its effectiveness for tracking the progression of the disease. As part of the initial diagnostic workup for patients with a suspected immune-related etiology, electroencephalography (EEG) and magnetic resonance imaging (MRI) are used to reveal structural and electrophysiological abnormalities. Distinct subtypes of immune-mediated epilepsies have been shown to exhibit an array of clinical symptoms that may vary based on the severity of the condition. It is necessary to identify antibody-specific patterns of EEG and MRI features that may help clinicians diagnose and track the development of the disease, as well as differentiate between related neural antibody-related conditions. We predict that for each subtype of immune-related epilepsy, there will be a distinct pattern of MRI and EEG features. We performed a case-controlled, retrospective analysis of patients who received a serum or cerebrospinal fluid (CSF) neural antibody test. Age of seizure onset, seizure frequency, the result of antibody testing, and CSF molecular and blood cell composition were also collected by accessing the UCLA Health System. By using the MATLAB computing platform, we will perform a quantitative analysis of the MRI and EEG data to extract potential relationships that may exist with the clinical presentations of each antibody-specific epilepsy.