Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve

Molecular Neurobiology https://doi.org/10.1007/s12035-019-1576-4 Higher Reliance on Glycolysis Limits Glycolytic Responsiveness in Degenerating Glaucomatous Optic Nerve Assraa Hassan Jassim 1 & Lucy Coughlin 1 & Mohammad Harun-Or-Rashid 1 & Patrick T. Kang 2 & Yeong-Renn Chen 2 & Denise M. Inman 1 Received: 11 December 2018 / Accepted: 20 March 2019 # The Author(s) 2019 Abstract Metabolic dysfunction accompanies neurodegenerative disease and aging. An important step for therapeutic development is a more sophisticated understanding of the source of metabolic dysfunction, as well as to distinguish disease-associated changes from aging effects. We examined mitochondrial function in ex vivo aging and glaucomatous optic nerve using a novel approach, the Seahorse Analyzer. Optic nerves (ON) from the DBA/2J mouse model of glaucoma and the DBA/2-Gpnmb+ control strain were isolated, and oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), the discharge of protons from lactate release or byproducts of substrate oxidation, were measured. The glial-specific aconitase inhibitor fluorocitrate was used to limit the contribution of glial mitochondria to OCR and ECAR. We observed significant decreases in maximal respiration, ATP production, and spare capacity with aging. In the presence of fluorocitrate, OCR was higher, with more ATP produced, in glaucoma compared to aged ON. However, glaucoma ON showed lower maximal respiration. In the presence of fluorocitrate and challenged with ATPase inhibition, glaucoma ON was incapable of further upregulation of glycolysis to compensate for the loss of oxidative phosphorylation. Inclusion of 2-deoxyglucose as a substrate during ATPase inhibition indicated a significantly higher proportion of ECAR was derived from TCA cycle substrate oxidation than glycolysis in glaucoma ON. These data indicate that glaucoma axons have limited ability to respond to increased energy demand given their lower maximal respiration and inability to upregulate glycolysis when challenged. The higher ATP output from axonal mitochondria in glaucoma optic nerve compensates for this lack of resiliency but is ultimately inadequate for continued function. Keywords Seahorse analyzer . Fluorocitrate . DBA/2J . Glaucoma . Mitochondria . Optic nerve Abbreviations AA antimycin A AD Alzheimer’s disease AMPK adenosine monophosphate activated protein kinase Assraa Hassan Jassim and Lucy Coughlin are co-first authors. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-019-1576-4) contains supplementary material, which is available to authorized users. * Denise M. Inman 1 Department of Pharmaceutical Sciences, Northeast Ohio Medical University, 4209 State Route 44, Rootstown, OH 44272, USA 2 Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, OH, USA ATP CNS CTB D2 D2G DMEM ECAR FC FCCP GLUT1 IOP MCT2 OCR ON PBS RBPMS RGC TCA adenosine triphosphate central nervous system cholera toxin-B subunit DBA/2J mouse DBA/2J-Gpnmb+ Dulbecco’s modified essential medium extracellular acidification rate fluorocitrate carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone glucose transporter 1 intraocular pressure monocarboxylate transporter 2 oxygen consumption rate optic nerve phosphate buffered saline RNA binding protein, multiple splicing retinal ganglion cell tricarboxylic acid. Mol Neurobiol Introduction Neurons, astrocytes, and blood vessels form a metabolic unit in the CNS. Glucose, lactate, and other metabolic intermediates obtained from the circulation are provided to neurons primarily through astrocytes, and enable glycolysis or oxidative phosphorylation to generate ATP. Neurotransmitters released from neurons can bind metabotropic glutamate receptors on astrocytes that increase intracellular calcium concentration, leading to generation of prostaglandins from arachidonic acid in astrocytes, and ultimately vasodilation to enable increased glucose uptake [1]. The glutamate-glutamine cycle, the astrocytic uptake of glutamate, and release of glutamine for neuronal uptake, can also provide carbons for the TCA cycle. These interactions not only enable flexibility but also engender dependence among the cells of the metabolic unit. Our understanding of these interactions and how they are altered by aging or disease are essential to our ability to manage neurodegenerative disease. Evidence is emerging to support the critical role of energy management in axon degeneration observed in neurodegenerative disease, including glaucoma [2]. The glial-specific glucose transporter GLUT1 and the neuronal-specific monocarboxylate transporter MCT2 were significantly decreased in optic nerve prior to glaucoma-related degeneration [3]. Astrocytes compromised in their uptake of glucose paired with axons incapable of transporting lactate for fuel would preclude function unless the axons could obtain glucose directly. Neurons are capable of taking up glucose [4], but whether they can do so in a way that would sustain them under such conditions remains to be determined. The DBA/2J mouse, a widely used model of glaucoma, undergoes a progressive optic neuropathy that results in asynchronous retinal ganglion cell death commencing between 10 and 12 months of age [5]. Deficits in physiological signaling prior to axon transport loss or axon degeneration was observed in the DBA/2J model of glaucoma [6]. Investigation into the cause of the signaling deficit suggested two sources— the mitochondria or the ways by which mitochondria obtain their energy substrate. We determined that the latter contributes to glaucomatous optic neuropathy by showing that critical glucose and monocarboxylate transporters are decreased prior to optic nerve degeneration [3]. The mitochondria, however, also show signs of compromise. Significantly lower mitochondria volume per volume of axon exists in glaucomatous optic nerve [7]. With intraocular pressure elevation, mitochondria in optic nerve of DBA/2J mice exhibited increased fission [8], and mitochondrial cristae loss [9]. It is likely these fragmented mitochondria have inefficient or dysfunctional oxidative phosphorylation, or increased reactive oxygen species production, potentially compromising the high metabolic demand of axons. In support of this, efforts to alter the energy balance (ketogenic diet, vitamin supplementation) toward greater substrate or cofactor availability to support oxidative phosphorylation have demonstrated significant improvement in retinal ganglion cell survival and function [3, 10]. Hence, there are large gains in neural function made possible with providing mitochondria with energy substrate. An outstanding question is the nature of the axonal mitochondria deficit in the optic nerve. It is not possible to isolate axonal mitochondria from the optic nerve (ON). Therefore, it is necessary to try to physiologically isolate axonal mitochondria and determ (...truncated)