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.

Stiffness measures derived from MR Elastography have shown value in guiding treatment decisions and monitoring effectiveness of therapies for liver disease but it requires extra hardware, longer scan duration and is susceptible to motion and breathing artifacts. Recent studies have revealed a strong linear correlation between water diffusion and tissue stiffness, demonstrating that Diffusion Weighted MRI (DWI) can be used to estimate stiffness values in liver tissue. DWI-derived stiffness values may help evaluate treatment-induced changes in breast cancer but to our knowledge, this has not yet been tested. The purpose of my ongoing study is to calibrate DWI estimates of tissue stiffness for the breast by optimizing DWI parameters (diffusion weightings, or ‘b-values’) and  calibration coefficients (a, b), evaluating the potential of stiffness measures for monitoring response to neoadjuvant chemotherapy (NAC) in breast cancer. We collected baseline and early treatment MRI exams from 25 patients undergoing NAC in this IRB approved study along with their treatment outcomes based on pathologic response post completion of NAC. I evaluated  the stiffness values obtained from different b-value pairs (low b-values: 100/200; high b-values: 800,1500,2000 s/mm²) and calibration coefficients(a,b=-9.7,13.9:-10.8,17.5:-8.8,21.2) and compared it to the invasive breast cancer stiffness values reported in literature. I also evaluated the performance of the optimized parameters to predict treatment response. The optimal b-value pairing (b=200,1500s/mm²) and coefficients a=-9.7,b=13.9 produced stiffness values consistent with literature. Using this approach, the performance for predicting treatment outcomes between responder and non-responder groups was AUC=0.84. These preliminary findings suggest that DWI based virtual elastography could serve as a non-invasive tool to assess tumor stiffness and track treatment efficacy, potentially improving breast cancer management.

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.

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.