Timothy Aidan Ryan

Several Parkinson’s 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 (BH ₄ ) 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.

Glucose-6-phosphate (G6P) is a key intermediate in multiple energetic and anabolic pathways, and quantifying its dynamics is essential for understanding cellular physiology. We have previously developed a syndicate of intensity-based, genetically encoded sensors based on the insertion of circularly permuted GFP into a Venus-flytrap-like analyte-binding protein. Here we use the same approach to develop an intensity-based G6P Sensing Fluorescent Reporter (iG6PSnFR). We present two variants: a G6P-activated sensor that increases fluorescence and a G6P-inactivated sensor that decreases fluorescence. We validate performance across progressively more complex preparations, including purified protein in vitro, immortalized and primary neuronal cultures, isolated pancreatic islets, in an intravital liver model, and finally in vivo in C. elegans neurons. In each context, iG6PSnFR reports G6P changes consistent with expected responses to physiological perturbations.

The brain is a metabolically vulnerable organ as neurons have both high resting metabolic rates and the need for local rapid conversion of carbon sources to ATP during activity. Midbrain dopamine neurons are thought to be particularly vulnerable to metabolic perturbations, as a subset of these are the first to undergo degeneration in Parkinson’s disease, a neurodegenerative disorder long suspected to be in part driven by deficits in mid-brain bioenergetics. In skeletal muscle, energy homeostasis under varying demands is achieved in part by its ability to rely on glycogen as a fuel store, whose conversion to ATP is under hormonal regulatory control. In neurons, however, the absence of easily observable glycogen granules has cast doubt on whether this fuel store is operational, even though brain neurons express the key regulatory enzymes associated with building or burning glycogen. We show here that in primary mid-brain dopaminergic neurons, glycogen availability is under the control of dopamine autoreceptors, such that dopamine itself provides a signal to store glycogen. We find that when glycogen stores are present, they provide remarkable resilience to dopamine nerve terminal function under extreme hypometabolic conditions, but loss of this dopamine-derived signal, or impairment of access to glycogen, makes them hypersensitive to fuel deprivation. These data show that neurons can use an extracellular cue to regulate local metabolism and suggest that loss of dopamine secretion might make dopamine neurons particularly subject to neurodegeneration driven by metabolic stress.

Brain metabolism is a fascinating and exciting research topic. Many new metabolic features of the brain continue to be unveiled and much remains to be discovered. To truly enable scientific progress, it is imperative that discoveries and theories are continually challenged through scholarly discussion. Here we address an earlier editorial by Bolaños et al. 2025 to underline the importance of embracing and respectfully engaging in scientific debate. image

The brain is a metabolically vulnerable organ as neurons have both high resting metabolic rates and the need for local rapid conversion of carbon sources to ATP during activity. Midbrain dopamine neurons are thought to be particularly vulnerable to metabolic perturbations, as a subset of these are the first to undergo degeneration in Parkinson’s disease (PD), a neurodegenerative disorder long suspected to be in part driven by deficits in mid-brain bioenergetics (1). In skeletal muscle, energy homeostasis under varying demands is achieved in part by its ability to rely on glycogen as a fuel store, whose conversion to ATP is under hormonal regulatory control. In neurons however the absence of easily observable glycogen granules has cast doubt on whether this fuel store is operational, even though brain neurons express the key regulatory enzymes associated with building or burning glycogen (2). We show here that that in primary mid brain dopaminergic neurons, glycogen availability is under the control of dopamine auto receptors (D2R), such that dopamine itself provides a signal to store glycogen. We find that when glycogen stores are present, they provide remarkable resilience to dopamine nerve terminal function under extreme hypometabolic conditions, but loss of this dopamine derived signal, or impairment of access to glycogen, makes them hypersensitive to fuel deprivation. These data show that neurons can use an extracellular cue to regulate local metabolism and suggest that loss of dopamine secretion might make dopamine neurons particularly subject to neurodegeneration driven by metabolic stress. Significance Statement: This work demonstrates that a neuron’s metabolic resilience is actively shaped by local neuromodulation, providing a potential explanation for why different neuronal subtypes exhibit unequal vulnerability to metabolic stressors and why dopamine neurons become more vulnerable when they lose the capacity to release dopamine.

The current collection contains all experimental protocols associated with the study "NeuromodulatorControl of Energy Reserves in Dopaminergic Neurons".

This protocol delineates the analysis for the detection of synaptic activity within synaptic boutons, employing the genetically engineered pHluorin biosensors.

This protocol delineates the analysis of ATP dynamics within synaptic boutons, employing the last generation of the genetically engineered ATP biosensors, called iATPSnFR2, as detailed in a recent publication.

Dopaminergic neurons consist some of the most difficult cells to culture due to their scarcity, intricate arborization and intrinsic vulnerability. The culture of primary VM neurons from murine brains has advanced the understanding of neuronal function at the cellular and molecular level. This protocol presents an established method to isolate and culture dopaminergic neurons from rat midbrain brains along with a method to sparsely transfect dopaminergic neurons.