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Alzheimer’s disease (AD) is a progressive neurodegenerative disease that results in loss of neurons and synapses. The dementia associated with AD is devastating for families and poses an increasing social and economic burden as our population ages. Currently there are no drugs available that can revert or even slow disease progression. Brains from human patients with AD exhibit two defining pathological changes: 1) Accumulation of extracellular amyloid plaques which are composed of amyloid beta (Aß), and; 2) accumulation of intracellular neurofibrillary tangles which are made up of hyperphosphorylated tau (pTau) protein. These changes are thought to be toxic and contribute to the devastating neurodegeneration that occurs in AD. Recent developments in disease modeling using human induced pluripotent stem cells (hiPSCs) have allowed for modeling of Alzheimer’s disease in human neurons in a dish. We can measure Aß and pTau levels in these hiPSC derived neurons allowing us to study how the levels of these proteins become dysregulated in AD.Genetic, biochemical, pharmacological, and epidemiological data suggest a role for cholesterol in AD pathogenesis. Recent studies have shown that amyloid precursor protein (APP), the precursor to Aß contains a cholesterol binding site. We use cholesterol lowering drugs, CRISPR/CAS9 genome editing, and fluorescent complementation assays to study how cholesterol levels and mutations that abolish APP-Cholesterol binding influence Aß and pTau burden. Our data indicate that cholesterol metabolism influences levels of these toxic proteins. We also show that APP can influence cholesterol homeostasis by regulating transport of cholesterol between different cell types in the brain. Our work sheds light on a pathway which unites the variety of genetic and environmental factors that contribute to AD risk. Targeting cholesterol metabolic pathways may be a promising approach in the search for drugs which can treat or prevent AD.

Neuronal activity-responsive gene expression plays a key role in the proper development, refinement, and plasticity of neural circuits, and the dysregulation of these gene responses have been implicated in developmental and cognitive disorders. Thus, identification and molecular characterization of the cis-regulatory DNA elements driving this response in neurons has the promise to profoundly advance our understanding of neurological disease mechanisms. The transcription factor cAMP/Ca2+-responsive element binding protein (CREB) is an important stimulus-responsive factor in many cell types of the body. CREB and its transcriptional co-activators are important for gene regulation in the brain and play a critical role in cognitive development, neuronal survival, and learning and memory. In the following studies we performed mechanistic analysis in human and mouse neurons to understand genome-wide binding of CREB and its neuronal co-activator, CREB Regulated Transcriptional Co-activator 1 (CRTC1, a.k.a. TORC1) at non-coding regulatory elements in response to neuronal depolarization. In addition, we investigated post-translational modifications of CRTC1 in response to BDNF treatment to better understand the biochemical basis of this co-factor’s activation in mouse neurons. Using human induced pluripotent stem cell (iPSC)-derived neurons we hoped to explore CREB-associated regulatory element function that cannot be studied in rodent models. While transcriptional and epigenomic analysis of human post-mortem brain samples has allowed for recent advances in identifying neuronal cis-regulatory regions of the genome, these approaches cannot address the dynamic, experience-dependent nature of their function in neuronal gene expression programs. We have identified human and mouse neuronal activity-dependent regulatory elements using ChIP-seq for both histone modifications and transcription factors in human pluripotent stem cell derived neuronal cultures, and thus provide a resource map of neuronal activity responsive regulatory elements across the human and mouse genomes. We identified human neuronal stimulus-dependent binding sites for CREB and CRTC1, including those that contain SNPs associated with neurological disorders, such as the schizophrenia-associated locus of CACNB2. Other CREB-associated regulatory elements potentially control expression of primate-specific transcripts and could be important sites of genetic divergence. Further functional investigation of these sequences will allow us to understand their role in human disease and evolution.

The risks of damage to the developing nervous system of many chemicals are not known because these studies often require costly and time-consuming multi-generational animal experiments. Pluripotent stem cell-based systems can facilitate developmental neurotoxicity studies because disturbances in nervous system development can be modeled in vitro. In this study, neurons derived from embryonal carcinoma (EC) and induced pluripotent stem (iPS) cells, were first characterized to establish their suitability for developmental neurotoxicity studies. The EC stem cell line, TERA2.cl.SP-12, was differentiated into neurons that expressed voltage-gated sodium and potassium channels as well as ionotropic GABA and glutamate receptors. These cells could also fire action potentials when stimulated electronically. However spontaneous action potentials were not observed. In contrast, pre-differentiated neurons derived from iPS cells fired evoked and spontaneous action potentials. Furthermore, iPS cell-derived neurons also expressed a wide array of functional voltage- and ligand-gated ion channels. Antiepileptic drugs (AEDs) are associated with developmental neurotoxicity. These agents can cause congenital malformations, cognitive deficits and behavioral impairment in children as a result of in utero exposure. The impact of four major AEDs, namely phenobarbital, valproic acid, carbamazepine and lamotrigine, on cell viability, cell cycle and differentiation of TERA2.cl.SP-12 into neurons was studied. All AEDs tested reduced differentiating stem cell viability. Valproic acid and carbamazepine increased apoptosis and reduced cell proliferation. A brief exposure to phenobarbital, valproic acid and lamotrigine at the start of differentiation impaired the subsequent generation of neurons. Additionally, the effect of transient exposure to phenobarbital and carbamazepine on neuronal maturation of iPS-derived neurons was investigated. Exposure to both AEDs resulted in diminished membrane potentials and reduced the proportion of cells that were able to fire action potentials spontaneously in culture. The data from these studies suggest that impairments in proliferation, differentiation and maturation of neurons derived from human stem cells may be sensitive indicators of neurodevelopmental disruption by these drugs that can result from in utero exposure. Furthermore, these findings suggest that the use of human pluripotent stem cells and neurons derived from them can reduce the time, cost and the number of animals used in toxicological research.