Neuroprotection may prevent or forestall the progression of incurable eye diseases, such as retinitis pigmentosa, one of the major causes of adult blindness. Decreased cellular ATP levels may contribute to the pathology of this eye disease and other neurodegenerative diseases. Here we describe small compounds (Kyoto University Substances, KUSs) that were developed to inhibit the ATPase activity of VCP (valosin-containing protein), the most abundant soluble ATPase in the cell. Surprisingly, KUSs did not significantly impair reported cellular functions of VCP but nonetheless suppressed the VCP-dependent decrease of cellular ATP levels. Moreover, KUSs, as well as exogenous ATP or ATP-producing compounds, e.g. methylpyruvate, suppressed endoplasmic reticulum stress, and demonstrably protected various types of cultured cells from death, including several types of retinal neuronal cells. We then examined their in vivo efficacies in rd10, a mouse model of retinitis pigmentosa. KUSs prevented photoreceptor cell death and preserved visual function. These results reveal an unexpected, crucial role of ATP consumption by VCP in determining cell fate in this pathological context, and point to a promising new neuroprotective strategy for currently incurable retinitis pigmentosa.
AMP-activated protein kinase (AMPK), a master regulator of cellular metabolism, is a potential target for type 2 diabetes. Although extensive in vitro studies have revealed the complex regulation of AMPK, much remains unknown about the regulation in vivo. We therefore developed transgenic mice expressing a highly sensitive fluorescence resonance energy transfer (FRET)-based biosensor for AMPK, called AMPKAR-EV. AMPKAR-EV allowed us to readily examine the role of LKB1, a canonical stimulator of AMPK, in drug-induced activation and inactivation of AMPK in vitro. In transgenic mice expressing AMPKAR-EV, the AMP analog AICAR activated AMPK in muscle. In contrast, the antidiabetic drug metformin activated AMPK in liver, highlighting the organ-specific action of AMPK stimulators. Moreover, we found that AMPK was activated primarily in fast-twitch muscle fibers after tetanic contraction and exercise. These observations suggest that the AMPKAR-EV mouse will pave a way to understanding the heterogeneous responses of AMPK among cell types in vivo.
Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.
SummaryMilk sugar is composed of glucose and galactose. Galactose is less suitable as an energy source than glucose. Thus, it has been a puzzle as to why mammals utilize galactose as a major component of milk sugar. Here we show that in hypoglycemic conditions, the presence of a trace amount of galactose, but not glucose, is able to maintain the production of mature glycoproteins and to abolish cell-death-inducing endoplasmic reticulum stress. In severely sugar-limited conditions, both glucose and galactose enter into the glycolytic pathway, but galactose is not able to raise the phosphofructokinase 1 activity, leading to the accumulation of fructose-6-phosphate, which in turn is utilized for the maturation of glycoproteins (e.g., growth factor receptors) and allows the activation of their intracellular signaling and prevents cell death from hypoglycemic conditions. Thus trace amounts of galactose may play unexpectedly important roles in the growth of infants and their protection during starvation.
Genetically encoded biosensors utilizing the Förster resonance energy transfer (FRET) are powerful tools for live cell imaging of various cellular processes. Our group has previously developed a series of FRET-based biosensors, named "ATeam," for visualization of ATP levels inside a single living cell. ATeam not only provides a window of insight into a single cell but also allows for visualization of ATP levels in mitochondrial matrix of a single living cell. This novel tool is able to monitor alterations in cellular ATP in response to various treatments in real time. Here we present a method for the evaluation of ATP levels in mitochondria in living cells by using ATeam. At the end of this chapter, an example of experimental results is described for a better understanding of the presented procedure.
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