Summary The rapid advances in stem cell technology have offered the hope for neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease. Transgene- or chemical-assisted dedifferentiation and transdifferentiation of somatic cells produce versatile neuronal stem cells or precursor cells, which can be further differentiated to neurons. However, getting mature neurons and functional synapses remains to be a lengthy and inefficient process, especially for dopaminergic neurons. New evidence showed the vital influence of extracellular microenvironment in neurodevelopment and synaptogenesis. Cholesterol is a unique and essential lipid in the eukaryotic plasma membrane. In addition to its biophysical role in membrane organization, cholesterol also actively regulates transmembrane proteins including cytokine receptors and adhesion proteins by direct binding or promoting lipid rafts. Hence, it plays an important role in regulating neuronal stem cells. In fact, ample evidence has shown that membrane cholesterol is required in neuronal differentiation, neurite growth, synaptogenesis and plasticity. However, there are few tools including statins, cyclodextrins and genetic mutations (e.g. NPC1) to reduce cholesterol. Notably, they either cause cell- wide cholesterol shortage or nonspecifically reduce other steroid lipids. More limited is the way to actively increase cholesterol, which is only possible via cyclodextrin-cholesterol complex. This is particularly problematic for neurons or neuronal precursor cells since they already have the highest membrane cholesterol concentration than almost all other types of cells in the human body. Recently, we have discovered that graphene, a novel carbon nanomaterial, selectively interacts with and enriches cholesterol in the plasma membrane of various cell types including neurons. By so doing, graphene potentiates synaptic transmission and receptor activity in a cholesterol-dependent manner. Consistently, it was reported by several groups that graphene promoted neural stem cell proliferation and differentiation with little toxicity. Therefore, we propose to study how graphene can be used to manipulate membrane cholesterol and to promote neuronal differentiation as well as synaptic maturation. First, we will further study molecular mechanism underlying graphene’s interaction with membrane cholesterol and its contribution to membrane organization. Second, we will test graphene’s effect on promoting neurogenesis and maturation from stem cells. Third, we will explore the potential use of graphene nanoflakes to selectively enhance synaptogenesis and dopamine release in stem-cell derived dopaminergic neurons. By so doing, we hope to illustrate a practical path of utilizing graphene in neuronal stem cell research and neuroregeneration, especially in the context of treating currently incurable neurodegenerative diseases like Parkinson’s disease.

Narrative Here, we propose to explore the significance of membrane cholesterol in regulating neuronal stem cell differentiation and synaptogenesis using graphene, a novel carbon nanomaterial. As an interdisciplinary and explorative project, this work will bring fresh ideas and new methodologies to optimize neuronal stem cell integration in the central nervous system, which bears great clinical implication in regenerative medicine for neurodegenerative diseases. In addition, the outcome of this project will also outline new biomedical application of graphene in cell and tissue engineering.