Investigating the importance of cholesterol homeostasis for neuronal function.

Both age-associated declines in cognitive function and neurodegenerative conditions are often associated with pathological changes in neuronal lipids. Thus, ameliorating these changes may provide a novel avenue for counteracting such conditions and for improving quality of life for the rapidly aging populations that now characterize highly developed countries, including Singapore. This is not a simple task, however, as we do not fully understand how cellular lipid compositions are established and maintained. In this proposal, we will meet this challenge by elucidating the molecular mechanisms underlying the intracellular trafficking of cholesterol, a lipid that is essential for normal brain function and whose dysregulation has been linked to a variety of neurological disorders, including neurodegenerative conditions.

Cholesterol is critical for the structural integrity of cellular membranes and for many physiological functions, including neuronal activity. Cells either synthesize cholesterol de novo in the endoplasmic reticulum (ER) – the organelle responsible for the majority of membrane lipid biosynthesis – or acquire it from external sources. In the brain, developing neurons produce cholesterol de novo. By contrast, mature neurons lose the ability to synthesizing it, and instead acquire cholesterol from glia-derived apolipoprotein. Regardless of its source, cholesterol is highly enriched in the plasma membrane (PM), where it represents approximately one half of all lipids. Thus, cells must constantly monitor the levels of cholesterol in their PM, and adjust levels of cholesterol biosynthesis or uptake to maintain PM homeostasis. This is thought to be mediated, at least in part, by communication between the PM and the ER. Despite decades of research into cholesterol metabolism, we still do not know how cells mediate crosstalk between the PM and the ER to regulate PM cholesterol homeostasis. In our preliminary studies, we found that a new family of evolutionarily conserved and ER-anchored lipid transfer proteins, the GRAMD1s (GRAMD1a, GRAMD1b, GRAMD1c), detect the amount of cholesterol available in the PM and transport cholesterol from the PM to the ER at sites of contact between these cellular structures (ER-PM contact sites). GRAMD1s therefore facilitate ER-PM crosstalk. Strikingly, recent human genetic studies, including genome-wide association studies, have identified links between one of these proteins, GRAMD1b, and both intellectual disability and schizophrenia. Our overarching hypothesis is that lipid transfer proteins, including the GRAMD1s, play critical roles in the delivery and exchange of cholesterol between the PM and other cellular compartments, and that disruption of this process causes neuronal dysfunction. Building on these exciting findings, we will: 1) characterize how GRAMD1 functions are regulated and how a GRAMD1 mutation linked to intellectual disability affects GRAMD1 function; 2) disrupt PM cholesterol homeostasis and determine the effects on brain and neuronal function (we have already generated mouse and C. elegans GRAMD1 knock-outs for these studies); and 3) determine how PM cholesterol homeostasis is regulated by other cholesterol transport systems. We will use a wide variety of innovative techniques, including in vitro reconstitution, structural biology, high-resolution microscopy, animal models, lipidomics, and CRISPR/Cas9-mediated genome editing to achieve these goals. As dysregulation of neuronal cholesterol homeostasis is linked to various neurological disorders, including neurodegenerative diseases, our studies will not only deepen our understanding of the fundamental mechanisms of cellular lipid distribution, but will also pave the way toward developing new therapeutic strategies against neurodegenerative diseases.