Genetic studies of schizophrenia have demonstrated that more than 90% of genetic risk is confined to non-coding portions of the genome. Advances in chromatin state prediction and chromatin accessibility assays have enabled us to better characterize the genomic features making up these regions and annotate genetic risk to these elements. The focus of this dissertation is to understand the role that tissue- and cell type- specific regulatory elements and the transcription factors that bind them play in risk for schizophrenia. I hypothesized that enhancer-based transcription factor-target networks that direct neuronal development are disrupted in schizophrenia. To test this hypothesis, I used high-quality chromatin state predictions in both the developing and the adult brain to develop a framework for testing enhancer properties for association with genetic risk. Any enhancer-level annotation can be used in this type of test, including transcription factor binding counts and chromosomal contact information. I first described and validated an atlas of transcription factor binding sites across multiple human tissue, including the brain. I then used this atlas to show that neurodevelopmental transcription factors and target genes are most associated with risk for developing schizophrenia.

The parabrachial complex (PB) is a midbrain structure that is vital to survival-related functions. This region receives and integrates incoming sensory information, communicating these signals to higher brain regions that are key in shaping behavior and affect. PB responds to painful somatosensory input, however, its most compelling role is in the development and maintenance of persistent pain. Pain persisting beyond the duration of a threat or tissue injury no longer serves a physiological purpose. Therefore, this type of maladaptive pain has a severe impact on health and quality of life. Current therapies for maladaptive pain are not optimal: it is necessary to better understand the neural circuitry underlying persistent pain so that we can design more effective therapies. The work presented here aims to outline PB’s role in four rodent models of pain. I approach this by combining anatomical tracing, electrophysiology, and behavioral studies. First, I show that in a model of orofacial neuropathic pain, PB neural activity is amplified. Next, I describe a novel and direct anatomical connection between the meninges and PB, via the trigeminal ganglion; this pathway is poised to underlie migraine headache-associated pain. Finally, I conduct behavioral studies aiming to hone rodent models of pain in the context of aging and amyloid accumulation, and pain during opioid withdrawal. These findings together confirm that PB is a crucial node in maladaptive pain processing and provide direction for further work clarifying PB’s role in new behavioral contexts.

Drug-induced DNA damage, reactive oxygen species (ROS), inflammatory mediators and central neurotoxicity have all been shown to play a role in cognitive impairments after chemotherapy, after CNS injury or in neurodegenerative diseases. Intrinsic apoptosis is a regulated cell death pathway implicated in many of these conditions. This pathway proceeds sequentially through DNA damage responses, including phosphorylation of ATM, H2AX and Tumor Protein 53 (p53), transcriptional activation of pro-apoptotic BH3-only proteins, and mitochondrial outer membrane permeabilization (MOMP), activating caspase-dependent and caspase-independent apoptosis. Previous work has indicated that Sp1 may regulate p53’s transcriptional profile and ability to promote apoptosis after DNA damage. To examine the role of Sp1 in DNA-damage-induced apoptosis, my work has utilized Mithramycin, a drug that binds G-C rich DNA to compete with Sp1 chromatin binding. Our hypothesis is that Mithramycin competes with Sp1 chromatin binding and thus protects neurons from DNA damage-induced, p53-dependent intrinsic apoptosis. However, it is vital to recognize that neurons increase their resistance to cytotoxic stimuli as they mature, leading to decreased levels of apoptosis. Thus, we additionally characterized the changes in the intrinsic apoptosis pathway upon neuronal maturation. Despite significant attenuation of the intrinsic apoptosis pathway in mature neurons, we found evidence that Mithramycin can still attenuate DNA-damage dependent intrinsic apoptosis in this paradigm to the extent that it is induced by neurons. Additional experiments showed that mature animal cortices or hippocampi do not demonstrate evidence of end-stage apoptosis (caspase activity) in response to DNA damage, unlike those in immature animals. However, in vivo administration of Mithramycin was able to attenuate cortical expression of p53-dependent genes. Thus, our studies provide evidence that: 1) Mithramycin attenuates activation of the DNA damage-dependent intrinsic apoptosis pathway via indirect inhibition of p53-dependent transcription and 2) Mature neurons restrict intrinsic apoptosis pathway activation in response to DNA damage. Therefore, Sp1 is likely to play a significant role in DNA damage-dependent neuronal intrinsic apoptosis, and Mithramycin treatment is protective when this pathway is activated. However, mature neurons significantly downregulate this pathway, thus limiting the potential therapeutic effects of Mithramycin in adult animals.

Cytokines are critical mediators used by immune cells to communicate as well as protect. The IL-12 family of cytokines are made up of  and  subunits typically assembled within one cell and secreted as a heterodimer. IL-12p40 is the shared β-subunit for both IL-12 (paring with IL-12p35) and IL-23 (with IL-23p19). However, the IL-12p40 monomer is often secreted in excess during infections, but its biological role was not known. In this thesis we investigated the function of secreted IL-12p40 monomer in vivo with the central hypothesis that the monomer combines with multiple α-subunits in vivo to generate IL-12 as well as other heterodimeric cytokines. Consistent with this hypothesis, in chimeric mice containing mixtures of cells that can only express either IL-12p40 or IL-12p35, but not both together, we found that functionally active IL-12 was generated. This alternate two-cell pathway requires IL-12p40 from hematopoietic cells to extracellularly associate with IL-12p35 from radiation-resistant cells. The two-cell mechanism was sufficient to influence local T cell differentiation in sites distal to the initial infection and helped control systemic dissemination of a pathogen although not parasite burden at the site of infection. Broadly, this suggests that early secretion of IL-12p40 monomers by sentinel cells at the infection site may help prepare distal host tissues for potential pathogen arrival. In addition to this role in generating IL-12 through two-cell assembly, we found that IL-12p40 has a novel partner protein, CD5L. This novel heterodimer was present in the serum of uninfected mice, with differences in the basal levels between B6 and Balb/c animals, with Balb/c having higher amounts of p40-CD5L. Functionally, we found that treatment with p40-CD5L leads to IL-4 and IL-10 production by T cells. Taken together, this thesis offers at least two major fundamental advances in cytokine biology – one the concept of a two-cell assembled cytokine and second the identity of a novel TH2-promoting heterodimeric cytokine. The first has significance in immunotherapy and understanding immunity to tissue-specific modulation of immune responses. The second is expected to drive significant research on allergy, responses to parasites and immune deviation.