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Kokotos, A. C.

Publications and source records attributed to Kokotos, A. C..

3 recordsLinked to original sources

A guanosine nucleotide metabolism-bioenergetics intersection drives Parkinson's disease

Several Parkinsons Disease (PD) linked mutations are known to drive deficits in a pathway that relies on an adequate supply of guanosine nucleotide triphosphate (GTP) to drive cellular neuronal processes. Similarly, there is strong evidence that a deficit in bioenergetic support for neuron function is also a major genetic driver of PD. We show here that the reliance on these two purine-based metabolites intersect at another PD susceptibility gene that encodes nucleoside diphosphate kinase (NDK) which converts ATP into GTP. We show that overexpression of NDK is strongly protective both in-vivo and in-vitro to metabolic lesions and identify mutations in NDK in humans associated both with increased risk and protection from PD. We discovered that NDK lies at the intersection of proper ATP production, de novo synthesis of guanosine diphosphate and the activity of GTP cyclohydrolase I, a consumptive pathway needed to produce the bioactive metabolite tetrahydrobiopterin (BH4) required for mitochondrial function. Loss of NDK and impairment in guanosine nucleotide synthesis exacerbate synaptic dysfunction, while boosting the GTP consuming pathway promotes bioenergetics. Additionally, analysis of genetic data taken from over 64,000 PD affected individuals and 38,000 controls from diverse ancestries reveal that several genes lying at the intersection of guanosine nucleotide metabolism and bioenergetics pose a significant risk burden for PD.

neuroscience↗

A genetic driver of epileptic encephalopathy impairs gating of synaptic glycolysis

The brain is a disproportionately large consumer of fuel, estimated to expend [~]20% of the whole-body energy budget, and therefore it is critical to adequately control brain fuel expenditures while satisfying its on-demand needs for continued function. The brain is also metabolically vulnerable as the inability to adequately fuel cellular processes that support information transfer between cells leads to rapid neurological impairment. We show here that a genetic driver of early onset epileptic encephalopathy (EOEE), SLC13A5, a Na+/citrate cotransporter (NaCT), is critical for gating the activation of local presynaptic glycolysis. We show that SLC13A5 is in part localized to a presynaptic pool of membrane-bound organelles and acts to transiently clear axonal citrate during electrical activity, in turn activating phosphofructokinase 1. We show that loss of SLC13A5 or mistargeting to the plasma membrane results in suppressed glycolytic gating, activity dependent presynaptic bioenergetic deficits and synapse dysfunction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/660213v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1f90e27org.highwire.dtl.DTLVardef@1055b7org.highwire.dtl.DTLVardef@6b9ce8org.highwire.dtl.DTLVardef@10a72bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

iATPSnFR2: a high dynamic range fluorescent sensor for monitoring intracellular ATP

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted super-folder GFP inserted between the ATP-binding helices of the{varepsilon} -subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a [~] 5-6 fold improvement in the dynamic range compared to the previous generation sensor, with excellent discrimination against other analytes and affinity variants varying from 4 M to 500 M. A chimeric version of this sensor fused to either the HaloTag protein or a suitably spectrally separated fluorescent protein, provides a ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting of the sensor to nerve terminals reveals previously uncharacterized single synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics. Significance StatementAdenosine triphosphate (ATP) is a key metabolite necessary for cellular life. Here we develop a next-generation genetically encoded ratiometric fluorescent ATP sensor that allows subcellular tracking of ATP levels in living cells. The large dynamic range makes it possible to follow the dynamics of this metabolite across cells and subcellular regions under different metabolic stressors. We expect that iATPSnFR2 will provide researchers with exciting new opportunities to study ATP dynamics with temporal and spatial resolution that has, until now, been unavailable.

biochemistry↗