Vital functions of distinct subcellular NAD pools

NAD is a small molecule present in all cells. In humans, it is primarily synthesized from vitamin B3, niacin. NAD has two important cellular functions. On the one hand, it is required to convert the chemical energy of food (such as sugars and fats) to ATP, the universal energy "currency" of all cells. On the other hand, it serves important regulatory functions to control vital processes including the repair of DNA damage, transcription of genes and cell proliferation and differentiation. Various NAD-dependent bioenergetic and signaling reactions are confined to specific subcellular compartments. When used in signaling processes, NAD undergoes degradation. Therefore, the molecule needs to be permanently re-synthesized in a metabolic process called NAD biosynthesis. It is now clear that NAD biosynthesis and its regulation are important elements of central biological processes. We have established functional and structural properties as well as the subcellular distribution of NAD biosynthetic enzymes. All enzymes localize to the nucleo-cytoplasm, except for one in mitochondria. In contrast, we recently found NAD sequestered in membrane-enclosed compartments such as mitochondria, peroxisomes and even the ER and the Golgi complex, whereas the NAD concentration in the nucleo-cytoplasm is very low. This unexpected NAD distribution poses important questions which we address in this project. How is NAD maintained in these compartments, even though NAD transport is supposed to be absent from human cells? Do these pools have specific bioenergetic or signaling functions? Do they communicate with each other? Do they serve as buffer for cytosolic or nuclear NAD? We have established unique molecular tools to detect changes of organellar NAD contents. New methods will be developed to determine specific roles of biosynthetic enzymes for individual NAD pools and signaling pathways. Recently, a key role of NMNAT2 (an NAD biosynthetic enzyme) in axonal survival was revealed. Our structural analyses explain its transport to distal parts of the axon. We will therefore specifically explore the role of NAD generation in axons. Several collaborations with recognized experts consolidate the broad methodological spectrum required to successfully conduct this challenging project. Within this project period, we have focussed on the investigation of NMNAT2 in a model cell line (PC12). PC12 cells respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic for sympathetic neurons. We have established PC12 cell cultures that respond to NGF treatment and undergo phenotypical changes which resemble differentiation and formation of neurites. Quite surprisingly, overexpression of NMNAT12 in PC12 cells reduces their capacity to form lamellipodia, neurite-like extensions, upon NGF treatment. These observations point to an important role of NMNAT2 in the formation of neurites. In addition, we have established an experimental system permitting the analysis of intracellular NADH levels, an important indicator of the energetic state of cells. The method makes use of an engineered, fluorescent NADH-binding protein. When expressed in cells, fluorescence microscopy can be used to evaluate intracellular NADH levels. Using this novel tool, our experiments have revealed that NMNAT2 overexpression in PC12 cells causes a decreased NADH content. These results indicate a potential mechanism how the NAD biosynthetic enzyme NMNAT2 might influence the ability of PC12 cells to respond to NGF treatment.

Degradation of NAD, a vital redox carrier, is a key element of fundamental signaling pathways. Both bioenergetic and signaling functions of NAD are compartmentalized. We have established structural properties and the subcellular distribution of NAD biosyn thetic enzymes. All enzymes localize to the nucleo-cytoplasm, except for one in mitochondria. Accordingly, we found NAD sequestered in membrane-enclosed compartments such as mitochondria, peroxisomes, but also in the ER and the Golgi complex, whereas the NAD concentration in the nucleo-cytoplasm is very low. This unexpected NAD distribution poses important questions which we will address in this project. How is NAD maintained in these compartments, even though NAD transport is absent from human cells? Do these pools have specific bioenergetic or signaling functions? Do they communicate with each other? Do they serve as buffer for cytosolic or nuclear NAD? We have established unique molecular tools to detect changes of organellar NAD contents. New methods will be developed to determine specific roles of biosynthetic enzymes for individual NAD pools and signaling pathways. Recently, a key role of NMNAT2 (an NAD biosynthetic enzyme) in axonal survival was revealed. Our structural analyses explain its transpo rt to distal parts of the axon. We will therefore specifically explore the role of NAD generation in axons. Several collaborations with recognized experts, for example, in the neurosciences, will consolidate the broad methodological spectrum required to s uccessfully conduct this challenging project. Our most recent results have established new tools that strengthen the feasibility of our approaches. Since NAD-mediated signaling regulates fundamental processes (including life span, the circadian clock, gen e expression, DNA repair, key metabolic pathways and apoptosis) the results of this project will contribute to important research fields and have an impact on new strategies for medical applications.