Biomedical subjects
Publications and source records attributed to Steven L McKnight.
The evolutionary origins of periodic phenomena in biology, such as the circadian cycle, the hibernation cycle and the sleep-wake cycle, remain a mystery. We discuss the concept of temporal compartmentalization of metabolism that takes place during such cycles, and suggest that cyclic changes in a cell's metabolic state might be a fundamental driving force for such biological oscillations.
Because the transcription factor neuronal Per-Arnt-Sim-type signal-sensor protein-domain protein 2 (NPAS2) acts both as a sensor and an effector of intracellular energy balance, and because sleep is thought to correct an energy imbalance incurred during waking, we examined NPAS2's role in sleep homeostasis using npas2 knockout (npas2-/-) mice. We found that, under conditions of increased sleep need, i.e., at the end of the active period or after sleep deprivation (SD), NPAS2 allows for sleep to occur at times when mice are normally awake. Lack of npas2 affected electroencephalogram activity of thalamocortical origin; during non-rapid eye movement sleep (NREMS), activity in the spindle range (10-15 Hz) was reduced, and within the delta range (1-4 Hz), activity shifted toward faster frequencies. In addition, the increase in the cortical expression of the NPAS2 target gene period2 (per2) after SD was attenuated in npas2-/- mice. This implies that NPAS2 importantly contributes to the previously documented wake-dependent increase in cortical per2 expression. The data also revealed numerous sex differences in sleep; in females, sleep need accumulated at a slower rate, and REMS loss was not recovered after SD. In contrast, the rebound in NREMS time after SD was compromised only in npas2-/- males. We conclude that NPAS2 plays a role in sleep homeostasis, most likely at the level of the thalamus and cortex, where NPAS2 is abundantly expressed.
Budding yeast grown under continuous, nutrient-limited conditions exhibit robust, highly periodic cycles in the form of respiratory bursts. Microarray studies reveal that over half of the yeast genome is expressed periodically during these metabolic cycles. Genes encoding proteins having a common function exhibit similar temporal expression patterns, and genes specifying functions associated with energy and metabolism tend to be expressed with exceptionally robust periodicity. Essential cellular and metabolic events occur in synchrony with the metabolic cycle, demonstrating that key processes in a simple eukaryotic cell are compartmentalized in time.
The neuronal PAS domain protein 3 (NPAS3) gene encoding a brain-enriched transcription factor was recently found to be disrupted in a family suffering from schizophrenia. Mice harboring compound disruptions in the NPAS3 and related NPAS1 genes manifest behavioral and neuroanatomical abnormalities reminiscent of schizophrenia. Herein we demonstrate that Npas3-/- mice are deficient in expression of hippocampal FGF receptor subtype 1 mRNA, most notably in the dentate gyrus. In vivo BrdUrd-labeling shows that basal neural precursor cell proliferation in the dentate gyrus of Npas3-/- mice is reduced by 84% relative to wild-type littermates. We propose that a deficiency in adult neurogenesis may cause the behavioral and neuroanatomical abnormalities seen in Npas3-/- mice, and we speculate that impaired neurogenesis may be involved in the pathophysiology of schizophrenia.
Sprouty proteins act as intracellular inhibitors of receptor tyrosine kinase signaling. Here we show that the mammalian Sprouty2 protein contains an iron-sulfur complex that can exist in an oxidized, reduced, or nitrosylated state. Purified Sprouty2 assembles into large monodisperse spheres containing approximately 24 polypeptides per particle. Biochemical experiments indicate that the charge state of the iron within Sprouty2 particles may be insulated from ambient intracellular redox. These features offer the possibility that Sprouty2 particles are capable of receiving, maintaining, and dissipating electrical charge in a manner formally equivalent to a battery.
Laboratory mice bearing inactivating mutations in the genes encoding the NPAS1 and NPAS3 transcription factors have been shown to exhibit a spectrum of behavioral and neurochemical abnormalities. Behavioral abnormalities included diminished startle response, as measured by prepulse inhibition, and impaired social recognition. NPAS1/NPAS3-deficient mice also exhibited stereotypic darting behavior at weaning and increased locomotor activity. Immunohistochemical staining assays showed that the NPAS1 and NPAS3 proteins are expressed in inhibitory interneurons and that the viability and anatomical distribution of these neurons are unaffected by the absence of either transcription factor. Adult brain tissues from NPAS3- and NPAS1/NPAS3-deficient mice exhibited a distinct reduction in reelin, a large, secreted protein whose expression has been reported to be attenuated in the postmortem brain tissue of patients with schizophrenia. These observations raise the possibility that a regulatory program controlled in inhibitory interneurons by the NPAS1 and NPAS3 transcription factors may be either substantively or tangentially relevant to psychosis.
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Animal behavior is synchronized to the 24-hour light:dark (LD) cycle by regulatory programs that produce circadian fluctuations in gene expression throughout the body. In mammals, the transcription factor CLOCK controls circadian oscillation in the suprachiasmatic nucleus of the brain; its paralog, neuronal PAS domain protein 2 (NPAS2), performs a similar function in other forebrain sites. To investigate the role of NPAS2 in behavioral manifestations of circadian rhythm, we studied locomotor activity, sleep patterns, and adaptability to both light- and restricted food-driven entrainment in NPAS2-deficient mice. Our results indicate that NPAS2 plays a substantive role in maintaining circadian behaviors in normal LD and feeding conditions and that NPAS2 is critical for adaptability to food restriction.
Neuronal PAS domain protein 2 (NPAS2) is a mammalian transcription factor that binds DNA as an obligate dimeric partner of BMAL1 and is implicated in the regulation of circadian rhythm. Here we show that both PAS domains of NPAS2 bind heme as a prosthetic group and that the heme status controls DNA binding in vitro. NPAS2-BMAL1 heterodimers, existing in either the apo (heme-free) or holo (heme-loaded) state, bound DNA avidly under favorably reducing ratios of the reduced and oxidized forms of nicotinamide adenine dinucleotide phosphate. Low micromolar concentrations of carbon monoxide inhibited the DNA binding activity of holo-NPAS2 but not that of apo-NPAS2. Upon exposure to carbon monoxide, inactive BMAL1 homodimers were formed at the expense of NPAS2-BMAL1 heterodimers. These results indicate that the heterodimerization of NPAS2, and presumably the expression of its target genes, are regulated by a gas through the heme-based sensor described here.
PAS kinase is a serine/threonine kinase regulated in cis by a PAS domain. A genetic study of the two PAS kinase genes in budding yeast gave evidence of the involvement of these enzymes in the control of sugar metabolism and translation. Using a biochemical screen for PAS kinase substrates, three translation factors were identified as direct phosphorylation targets. PAS kinase was also found to phosphorylate UDP-glucose pyrophosphorylase and glycogen synthase, the enzymes catalyzing the two final steps in the glycogen biosynthetic pathway. Genetic, biochemical, and physiological data provide evidence that both of these enzymes are inhibited by PAS kinase-dependent phosphorylation, thereby downregulating carbohydrate storage. These studies provide evidence of a cell-autonomous signaling system that both controls and connects the balance of fuel consumption/storage to protein synthesis.
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The core apparatus that regulates circadian rhythm has been extensively studied over the past five years. A looming question remains, however, regarding how this apparatus is adjusted to maintain coordination between physiology and the changing environment. The diversity of stimuli and input pathways that gain access to the circadian clock are summarized. Cellular metabolic states could serve to link physiologic perception of the environment to the circadian oscillatory apparatus. A simple model, integrating biochemical, cellular, and physiologic data, is presented to account for the connection of cellular metabolism and circadian rhythm.