Psychoactive Natural Products: Endogenous Mammalian Signaling Molecules and Exogenous Neuromodulators

Abstract

Natural products constitute a principal source for pharmaceutical development due to their remarkable effectiveness in treating human disease. Plant secondary metabolites have played essential roles throughout the whole of human history, with plants serving as vital sources of food and medicine. Psychedelic-containing plants have long been regarded as sacred remedies used to achieve healing and spiritual transcendence. It is especially incredible that the human brain possesses the innate capacity to biosynthesize potent psychoactive compounds like morphine and N,N-dimethyltryptamine (DMT). While endogenous mammalian morphine and DMT are well established, the biosynthetic systems facilitating their production have yet to be fully characterized in the human central nervous system (CNS). This dissertation presents a cellular imaging approach supported by chemical biology techniques to characterize the spatial organization of psychoactive small molecule biosynthetic machinery in mammalian CNS contexts. It also reports on the ability of exogenous plant natural products to modulate neuroprotective systems. The primary methodology used in this work combines immunocytochemistry with high-content imaging to preserve and quantify spatial information about protein localization in and across CNS cell types. In Chapter 2, I characterize morphine biosynthetic machinery and morphine-like immunoreactivity in two CNS cell types. I first validated an anti-morphine-like antibody to bind morphine-like epitopes through orthogonal methodologies. I then use this antibody as a tool to explore the distribution of morphine-like immunoreactivity in mammalian neuronal and glial cell types. I also demonstrate that morphine-like immunoreactivity is functionally responsive in neuroblastoma and localizes to microglia populations in a mixed glial cell model system. In Chapter 3, I describe my efforts to elucidate the spatial organization of DMT-related biomolecules in human induced pluripotent stem cell (iPSC)-derived neurons. I first validated an antibody raised against indolethylamine-N-methyltransferase (INMT), the putative enzyme responsible for DMT biosynthesis in humans. I demonstrate that INMT is present in human iPSC-derived neurons and primarily localizes to the cytosolic face of cis-Golgi network of both developing and mature neurons. I further characterize spatial proximity between INMT and related DMT biosynthetic machinery, as well as a primary DMT molecular target: the serotonin 2A receptor (5-HT2A R). Through these efforts, I propose a working model where INMT N-methylation of tryptamine scaffolds occurs at the cis-Golgi network, and newly synthesized methylated tryptamines may diffuse across the Golgi membrane and access an intracellular 5-HT2A R population. In Chapter 4, I expand my work beyond foundational neurobiology to assess preliminary effects of two classes of exogenous plant metabolites, flavonoids and inositol phosphates, on HDAC1 modulation and neuroprotection. These data serve as preliminary evidence supporting the potential HDAC1-activating properties of both molecular classes, as well as their ability to protect against genotoxic DNA damage in neuronal systems. This work provides evidence that human neuronal systems can biosynthesize small molecules historically characterized as secondary plant metabolites and motivates future studies to explore endogenous roles of these putative neurotransmitters. Collectively, this dissertation provides the most comprehensive characterization of psychedelic tryptamine biosynthetic machinery in human neurons to date.