Neuronal activity and energy metabolism are tightly coupled. Under normal conditions, neuronal activity controls energy metabolism and not vice versa. Visual neurons are among the metabolically most active cells in the nervous system, and their energy is derived almost exclusively from oxidative metabolism. Defective energy metabolism adversely affects visual neurons, often leading to blindness. Thus far, energy metabolism and neuronal activity have been assumed to be regulated at the molecular level by two independent mechanisms. The present proposal aims to test the hypothesis that a candidate transcription factor, nuclear respiratory factor 1 (NRF-1), co-regulates both energy metabolism and synaptic neurochemicals (glutamate AMPA receptor subunit 2 [GluR2] and/or neuronal nitric oxide synthase [nNOS]) in specific types of visual cortical neurons. NRF-1 is known to activate genes involved in mitochondrial function, including subunit genes of cytochrome oxidase (CO). Binding sites for NRF-1 have been reported on the promoters of GluR2 and nNOS genes, but it is not known if NRF-1 is necessary for their expression. Recently, NRF-1 was found to be present in mammalian visual cortical neurons and its expression was regulated by neuronal activity. Both GluR2 and nNOS are associated with glutamatergic synaptic neurotransmission and are present in mammalian visual cortex.
Specific aim 1 is to establish baseline data on the normal distribution and levels of NRF-1, GluR2, and nNOS mRNAs and proteins in visual cortical neurons in vivo and in vitro, using rat as a model.
Specific aim 2 is to suppress the expression of NRF-1 by means of small interference RNA silencing to determine if expressions of GluR2, nNOS, and CO will be severely affected in visual cortical neurons. NRF-1 knockdown neurons will be subjected to depolarizing stimulation to determine if increased activity differentially stresses GluR2-rich and nNOS-rich neurons, and if one or both of them will undergo cell death.
Specific aim 3 is to overexpress NRF-1 to ascertain if expressions of GluR2, nNOS, and CO reduced by impulse blockade can be rescued. These studies will shed light on how a transcription factor can simultaneously regulate both energy metabolism and synaptic neurochemicals in visual cortical neurons, and how genetic perturbation of this factor can lead to impaired energy production, defective expression of neurochemicals, and possible death of visual cortical neurons.
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