Glioblastoma multiforme, a form of malignant glioma, is a central nervous system (CNS) tumor associated with the worst prognosis in both adults and children. Effective treatments for this deadly cancer have continued to be elusive to this day. Several strategies have been employed to better understand malignant gliomas including genomics, cell survival, and cell fate determination. Previously we found that a glial cell determining factor, Nuclear Factor I-A (NFIA), is expressed in gliomas and has functional roles during glioma tumorigenesis. During CNS development, NFIA expression is regulated by distinct chromatin conformations that involve the transcription factors SOX9 and BRN2. The glial cell specific chromatin architecture is also present in glioma and modulation of the architecture has profound effects on tumor growth. Interestingly, the formation of this glial loop is not mediated by SOX9 and BRN2. A candidate factor for mediating formation of this chromatin architecture is the Mediator complex subunit 12 (MED12). Here we test whether MED12 is a component of the SOX9/BRN2complex, affects NFIA expression, and begin to determine what role MED12 plays in glioma tumorigenesis. Using dual gRNAs in a CRISPR/Cas9 system to delete MED12 expression from human glioma cell lines U87 and LN229, we found that MED12 is important for tumor growth, migration, and proliferation.

Proper spinal cord development results in spatially organized motor columns that innervate muscles along the rostral-caudal body axis. This is achieved in large part through the concerted action of a combination of transcription factors. The transcription factor Nuclear Factor 1 A (NFIA) is a regulator of glial cell development where it is both necessary and sufficient to induce glial cell specification. NFIA is also expressed in developing neurons in the brain and in spinal motor neurons. Recently, differential expression of NFIA in spinal glial and motor neuron populations was found to depend on distinct chromatin architectures. However, the function of NFIA is motor neurons remains poorly understood. Here, we show that NFIA is expressed in all but one motor column in developing spinal motor neurons. We define NFIA expression in both chick and mouse spinal cords, finding that NFIA is expressed in MMC, HMC, LMCm, and LMCl columns. Using genetic manipulations in both chick and mouse spinal cord, we found that NFIA is not required for the initial specification of motor neuron populations. Rather, conditional deletion of NFIA in motor neurons leads to disorganization of the lateral medial motor neuron (LMC) columns. A reduction in expression and abhorrent migration of FOXP1 and LIM1/2 were observed with cells from the LMC-lateral column incorrectly migrating into the LMC-medial region. Thus, NFIA is important for maintain proper motor neuron columnar organization.

Glioma is the most prevalent and highly malignant form of adult brain cancer and is exceedingly difficult to manage. Neural stem cells and glial-determining factors have recently been implicated in glioma formation. Key transcriptional regulators of developmental glial genesis are expressed in glioma and have functional roles during glioma tumorigenesis. Nuak2, has been shown to be upregulated in many cancers and result in metastasis. However, the role of Nuak2 in glioma is not well defined. Using a combination of in vitro and in vivo studies, we found that loss of Nuak2 via CRISPR-mediated deletion results in decreased proliferation, migration, and tumorigenicity in glioma cell lines. Further, in a IUE mouse model of glioma CRISPR targeted deletion of Nuak2 resulting in increased survivability while over-expression of Nuak2 lead to faster tumor progression and death. Together, these studies demonstrate that Nuak2 has a vital role in glioma tumorigenicity and lays the groundwork for more in-depth mechanistic studies.

Glioblastoma is the most common of all tumors in the brain and central nervous system and has an average survival time of less than one year. The current standard of care treats these tumors with radiation and temozolomide, a DNA alkylating agent, but a vast majority of these tumors have begun to acquire temozolomide resistance, highlighting the need for new therapies. The heterogeneous nature of glioblastoma tumors as well as the difficulty of drugs to cross the blood-brain barrier leads to minimal treatment options. To test potential small molecule inhibitors as treatment for glioblastoma, we exposed glioblastoma cells to four commercially available SGK (serum and glucocorticoid-regulated kinase) inhibitors, which target downstream effectors in the PI3K/Akt pathway regulating cell survival and proliferation. We found that one of the four SGK inhibitors, GSK650394, actively inhibits glioblastoma cell viability in U87MG and selectively target SGK at concentrations below 100 μM. This inhibitor could potentially be used to treat glioblastoma but must be further tested in heterogenous glioblastoma tumors and in vivo models, especially to study if these small molecule inhibitors can cross the blood-brain barrier, and the mechanism of action in glioblastoma must be further elucidated.

Adeno-associated viral (AAV) vectors are effective gene therapy delivery candidates, and the AAV-PhP.eB capsid is effective at transducing the CNS in mice when delivered intravenously (IV). Introduction of promoter sequences to the AAV-PhP.eB genome can restrict gene expression to specific cell types. More research is needed to identify cell-specific promoter sequences that restrict gene expression to specific CNS targets. Thus, the Ple394 and Ple394-HBB sequences were examined to see if they can restrict gene expression to cortical neurons. The HB9 sequence was examined to see it could restrict gene expression to spinal motor neurons. The Ple394 promoter resulted in strong cortical neuron gene expression and comparatively less gene expression in off-target brain and spinal cord regions. The Ple394 vector did not result in significantly different gene expression in the liver compared to mice that received saline. The Ple394-HBB promoter failed to cause cortical neuron-specific gene expression. The HB9 promoter resulted in spinal motor neuron-specific gene expression with relatively little expression in the brain. The HB9 vector didn’t result in significantly different gene expression in the liver compared to mice that received saline. In conclusion, introduction of the Ple394 and HB9 promoter sequences into the AAV-PhP.eB genome were able to restrict gene expression to cortical neurons and spinal motor neurons, respectively. Identification of promoters that can drive selective gene expression in the cerebral cortex or spinal motor neurons will allow for the IV delivery of AAV gene therapies to treat diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis, respectively.

Anorexia Nervosa (AN) is an eating disorder observed primarily in girls and women. AN is characterized by a low body mass index, hypophagia, and hyperactivity. While AN has one of the highest mortality and relapse rates of all psychiatric disorders, there are no approved pharmacological treatments for AN. However, a recent GWAS study revealed eight metabolic gene loci associated with the development of AN, suggesting a metabolic origin of the development of AN. In the clinical setting, weight regain is one crucial aspect of treating AN.In contrast, obesity patients face weight loss resistance. Recent work shows that obesity alters the metabolite profile of most tissues. Such metabolic profile returns to normal upon weight loss for all tissues except adipose tissues. Such an aspect of obesity is attributed to the metabolic memory of adipose tissues. We sought to introduce metabolic memory of obese adipose tissue in Activity-Based Anorexia (ABA), an animal model of AN's hyperactivity and hypophagia aspect, by transplanting adipose tissues into ABA mice. Our previous study found that obese adipose tissue recipients lose body weight slower than control adipose tissue recipients when subjected to the ABA paradigm. We therefore hypothesized that behavioral benefits to adipose tissue transplantation shown in the ABA paradigm stemmed from its communication to the brain. Here, we show that neonatal ablation of Agouti-Related Peptide (AgRP) neurons significantly disrupts the weight loss resistance introduced by obese adipose transplantation in ABA.

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder that affects more than 10 million people worldwide. While most cases of PD are sporadic, point mutations in Leucine-Rich Repeat Kinase (LRRK2) have been determined to be the most common cause of autosomal dominant late-onset PD. One key limitation within the field of LRRK2 biology lies within our inability to visualize endogenous LRRK2 due to its low expression. Without definitive evidence in endogenous systems, there remain questions surrounding how representative conclusions drawn from overexpression systems are regarding LRRK2 kinase activity, LRRK2 protein levels, and mechanisms underlying LRRK2-driven PD. Hence, it is crucial to develop a system to better understand endogenous LRRK2. Chapter I introduces LRRK2 biology by demonstrating the progress the field has made to unravel LRRK2’s complexities within the last 20 years. Chapter II tests the ability of novel HaloTag technology CRISPR-tagged to LRRK2 to better visualize endogenous LRRK2. Chapter III encompasses the use of a CRISPRi screen to identify pathways that potentially regulate endogenous LRRK2 degradation. Notably, the CRISPRi screen revealed two pathways modulating proteasomal degradation: SUMOylation and VCP/p97 system. Through validation of specific gene targets within a knockdown system, we found that the NPL4/UFD1L complex is involved in regulating endogenous LRRK2 degradation through an unknown mechanism. Future directions would include using microscopy to further corroborate the importance of the NPL4/UFD1L complex and establish its interaction to LRRK2 in the endogenous degradation pathway.

Enhancer elements regulate cell type- and stimulus-specific gene expression and are enriched with genetic variants associated with human diseases. Studying regulatory mechanisms of enhancer activity is critical to elucidate how these variants contribute to disease, including those associated with substance use disorders (SUDs). I profiled nascent transcriptional start sites (TSSs) with capped small RNA-seq (csRNA-seq) to measure transcriptional activity of promoters and enhancers in mouse primary neurons and astrocytes in response to SUD relevant stimuli, including dopamine and forskolin in primary neurons and IL-1B in astrocytes. I identified over 100,000 regulatory elements in each cell type, most of which initiate enhancer RNAs (eRNAs). Most genomic regions with one or more TSSs, which we called transcript start regions (TSRs), were cell type-specific, and analysis of these TSRs showed different TFs are implicated in establishing cell type-specific regulatory programs in each cell-type. Neuronal stimulation with forskolin activated enhancers enriched in motifs recognized by AP1 and CREB family TFs. Astrocyte stimulation with IL-1B activated enhancers enriched in motifs recognized by NFkB, AP1, and IRF family TFs. Analysis of motif distributions relative to the TSS revealed strong positional preferences for many TFs with regulatory elements. Intriguingly, the preferred positions for motifs recognized by CREB are different for TSS that are up- versus down-regulated in enhancers. This study has generated the first comprehensive catalog of eRNA TSS in primary murine neurons and astrocytes and provides new insight into the mechanisms by which TFs regulate transcription in addiction-related pathways.

During a spinal cord injury (SCI), irreversible traumatic damage is caused to the axon tracts that travel in the white matter of the spinal cord for connection between the brain and the rest of the body, including the corticospinal tract (CST), a tract that is essential to voluntary fine motor control in humans. Like other neurons in the central nervous system, CST neurons cannotspontaneously regenerate, as intrinsic, and extrinsic factors closely regulate them. Recent studies from our lab demonstrate robust regeneration of the CST when a neural progenitor cell (NPC) graft is placed into the injury site. However, our preliminary data shows that after mid-cervical SCI, robust regeneration only occurs for hindlimb CST (H-CST) but not forelimb CST (F-CST). To promote forelimb CST regeneration, we combined both an extrinsic stimulus, such as the provision of an NPC graft, and intrinsic manipulation, such as the co-deletion of two protooncogenes, phosphatase and tensin homologue (PTEN) and suppressor of cytokine signaling 3 (SOCS3) specifically in F-CST neurons, to amplify injury-induced signals for regeneration in a mouse model. The combinatory approach promoted robust F-CST axon regeneration compared to the graft-only treated mice. Additionally, the PTEN/SOCS3 co-deletion with NPC grafting showed robust axon regeneration across the whole injury site to reach the caudal border of the graft compared to the graft-only mice, a feature required to make functional neuronal relays crossing the injury site. Indeed, co-deletion of PTEN/SOCS3 enabled graft-derived neurons to function as descending relay neurons to transmit supraspinal signals to host spinal cord neurons below the injury site. Thus, the robust regeneration of F-CST reveals the importance of intrinsic regulation factors, such as the manipulation of genes crucial for regeneration in combination with the extrinsic support of an NPC graft to promote regeneration after SCI.

Peripheral nerve injuries (PNIs) are injuries to the peripheral nervous system, typically caused by a traumatic event, and are very common in a combat and trauma setting. The nerve autograft has been regarded as the “golden standard” for PNIs, where a piece of nerve tissue, sourced from an autogenous sensory nerve, is transplanted to the site of injury. However, the nerve autograft has many shortcomings, such as pain and loss of function at the donor nerve site, neuroma formation, and risk of infection following surgery. Our lab developed a novel multichannel scaffold approach (MCS), which uses linear microstructures to restrict axonal regeneration to one direction. The MCS approach was then further strengthened by the addition of brain-derived neurotrophic factor (BDNF), which have been shown to help increase the rate of axon regeneration, and thus potentially eliminate the need for a donor nerve. In this study, the MCS approach was tested in vivo by injuring the sciatic nerve of rats and then implanting the following therapies: open tube, nerve autograft, multichannel scaffold, and the multichannel scaffold with the addition of BDNF. 12 weeks post implant we found that rats implanted with MCS, or MCS/BDNF, or nerve autograft had similar number of regenerating axons. This was significantly higher than the number of regenerating axons observed in open tube control subjects (no channels). Our findings also showed that the MCS approach, both with and without BDNF, was successful in orienting regenerating axons into the proper pathway from proximal to distal aspect of the injury compared to nerve graft and open tube implants where axons were seen deviating from this path. Our experiment was able to demonstrate that the MCS with and without BDNF therapy performs significantly better in terms of axonal regeneration and axon alignment than the open tube therapy in the sciatic nerve rat model. Thus, a multichannel scaffold can be a replacement for the standard of care, which is the nerve autograft, preventing the serious side effects of this procedure and providing an off-the-shelf alternative. The development of the MCS approach allows researchers to apply similar approaches to peripheral nerve injuries throughout the body, such as cranial nerve injuries where donor grafts are needed.