Cholesterol, mitochondria and energy metabolism:

The brain is highly dependent on mitochondrial function, and mitochondrial defects most often manifest as neurological symptoms. This reliance on mitochondria is in part due to the very high energy expenditure of neurons, coupled with a lack of energy storage. Mitochondrial respiration in the brain uses as much as 20% of the total inhaled oxygen. Many neurodegenerative diseases and physiological aging are associated with mitochondrial dysfunction. In Niemann-Pick Type C (NPC) disease, there also are indications that mitochondria are impaired, and may generate less ATP and more reactive oxygen species than normal mitochondria. We investigate the role that cholesterol plays in the development of mitochondrial dysfunction, and whether mitochondria are part of the link between imbalances in cholesterol homeostasis and neurodegeneration. We approach this question from two sides. First, we use cultured cells to manipulate cholesterol metabolism and investigate how this affects mitochondrial function and energy metabolism. Second, we investigate energy metabolism in NPC disease, using NPC as a model of neurodegeneration with a clear cholesterol defect, and to understand NPC disease pathogenesis in more detail. We use cultured primary NPC1-deficient neurons and glia, and NPC1-deficient brain to characterize alterations in energy metabolism. The connections among cholesterol, mitochondria and energy metabolism are complex, making these studies exciting and challenging at the same time. To tackle these complexities, we use a variety of techniques, including cell culture, molecular biology techniques, biochemical assays and fluorescent imaging.

Cholesterol in the synapse:

Synaptic transmission, the communication between neurons, relies on the secretion of small molecule neurotransmitters from the pre-synaptic neuron, and detection of the neurotransmitter by receptors on the post-synaptic neurons. Neurotransmitters are stored in small pre-synaptic vesicles, which fuse with the pre-synaptic plasma membrane to release the neurotransmitter. To permit sustained, rapid neurotransmitter release, vesicle exocytosis is coupled to endocytosis and reformation of synaptic vesicles, which are re-acidified and re-filled with neurotransmitters for another cycle of exo- and endocytosis. Synaptic membranes are very rich in cholesterol. Cholesterol influences membrane fluidity, permeability, curvature and transmembrane protein function, and it is an important regulator of membrane fusion and fission. In previous work on synaptic vesicle release in NPC1-deficient neurons, we found alterations in plasma membrane cholesterol and impaired synaptic vesicle release. We are now working to characterize the mechanisms regulating cholesterol homeostasis in the synapse and to understand how cholesterol influences synaptic vesicle release. A main approach to investigate synaptic vesicle kinetics uses live cell imaging by fluorescence microscopy, where fluorescent dyes or genetically-encoded fluorescent markers are monitored in live neurons during activation by electrical field stimulation. Other techniques we use include immunofluorescence or fluorescent cell staining with lipid-specific dyes, and biochemical techniques, such as subcellular fractionation, immunprecipitations, or radiotracer experiments. As in our other projects, we use a combination of different techniques to investigate the role of cholesterol in the synapse.

Cholesterol synthesis in neurons:

In the mature brain, most cholesterol is synthesized by astrocytes. Astrocytes secrete cholesterol in form of small, apolipoprotein E-containing lipoproteins, and neurons can use this cholesterol, e.g. after uptake  through lipoprotein receptors such as LRP1. However, hippocampal neurons appear to express cholesterol biosynthetic enzymes and synthesize the cholesterol precursor lanosterol even in the adult brain. To investigate whether the activity of the cholesterol biosynthetic pathway has a functional advantage for hippocampal neurons, we have developed RNA interference vectors that selectively down regulate cholesterol biosynthetic enzymes in neurons while the surrounding astrocytes remain normal. Using this approach, we can determine the functional consequences of decreased neuronal cholesterol biosynthesis.

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