Creatine Is Neuroprotective to Retinal Neurons in Vitro but Not in Vivo

Retinal Cell Biology Creatine is Neuroprotective to Retinal Neurons In Vitro But Not In Vivo Paul Ikgan Sia,1,2 John P. M. Wood,1,2 Glyn Chidlow,1,2 and Robert Casson1,2 1South Australian Institute of Ophthalmology, Royal Adelaide Hospital, Adelaide, South Australia, Australia 2Department of Ophthalmology and Visual Sciences, University of Adelaide, Adelaide, South Australia, Australia Correspondence: Robert Casson, PURPOSE. To investigate the neuroprotective properties of creatine in the retina using in vitro Ophthalmic Research Laboratories, and in vivo models of injury. Level 7 Adelaide Health and Medical Sciences Building, The University of METHODS. Two different rat retinal culture systems (one containing retinal ganglion cells Adelaide, North Terrace, Adelaide [RGC] and one not) were subjected to either metabolic stress, via treatments with the SA-5000, Australia; mitochondrial complex IV inhibitor sodium azide, or excitotoxic stress, via treatment with N- [email protected]. methyl-D-aspartate for 24 hours, in the presence or absence of creatine (0.5, 1.0, and 5.0 mM). Submitted: September 28, 2018 Neuronal survival was assessed by immunolabeling for cell-specific antigens. Putative Accepted: September 18, 2019 mechanisms of creatine action were investigated in vitro. Expression of creatine kinase (CK) isoenzymes in the rat retina was examined using Western blotting and immunohistochemistry. Citation: Sia PI, Wood JPM, Chidlow G, Casson R. Creatine is neuroprotective The effect of oral creatine supplementation (2%, wt/wt) on retinal and blood creatine levels to retinal neurons in vitro but not in was determined as well as RGC survival in rats treated with N-methyl-D-aspartate (NMDA; 10 vivo. Invest Ophthalmol Vis Sci. nmol) or high IOP-induced ischemia reperfusion. 2019;60:4360–4377. https://doi.org/ RESULTS. Creatine significantly prevented neuronal death induced by sodium azide and NMDA 10.1167/iovs.18-25858 in both culture systems. Creatine administration did not alter cellular adenosine triphosphate (ATP). Inhibition of CK blocked the protective effect of creatine. Retinal neurons, including RGCs, expressed predominantly mitochondrial CK isoforms, while glial cells expressed exclusively cytoplasmic CKs. In vivo, NMDA and ischemia reperfusion caused substantial loss of RGCs. Creatine supplementation led to elevated blood and retinal levels of this compound but did not significantly augment RGC survival in either model. CONCLUSIONS. Creatine increased neuronal survival in retinal cultures; however, no significant protection of RGCs was evident in vivo, despite elevated levels of this compound being present in the retina after oral supplementation. Keywords: creatine, neuroprotection, retina, creatine kinase, retinal ganglion cell, culture, ischemia-reperfusion, excitotoxicity, oxidative stress number of common blinding diseases, such as glaucoma, antioxidant and a component of the mitochondrial respiratory A diabetic retinopathy, and ischemic optic neuropathy, chain has been shown to be neuroprotective against in IOP- include bioenergetic compromise as a likely pathogenic induced retinal ischemia-reperfusion injury.9–11 Additional component. At the molecular level such an energy failure can findings of relevance to bioenergetic neuroprotection of the instigate a complex series of cellular events, including retina are that short-term hyperglycemia, as well as local membrane depolarization, Ca2þ influx, oxidative stress, mito- administration of glucose, attenuate RGC injury in models of chondrial dysfunction, excitotoxicity and, even, ultimately, cell experimental glaucoma, ischemia reperfusion, and chronic death.1–3 hypoperfusion.12–15 Bioenergetic-based neuroprotection refers to the concept Creatine, a guanidine compound that occurs naturally in of protecting injured and threatened neurons by strategies, vertebrates, plays a central role in cellular adenosine triphos- including increasing their available energy supply, conserving phate (ATP) buffering, in concert with its phosphorylated form, their fuel reserves, improving their mitochondrial function, or phosphocreatine. Creatine and phosphocreatine are intercon- augmenting their cellular energy buffering.4 Enhancement of verted by creatine kinase (CK), an enzyme family comprising cellular energy metabolism has been attempted with variable separate isoenzymes based either in mitochondria or the success in a range of neurologic disorders, such as Alzheimer’s cytoplasm. Mitochondrial CK converts creatine to phosphocre- disease,5 Huntingdon’s disease,6 and Parkinson’s disease.7 atine at the site of ATP production, with a concurrent More pertinently, this approach to neuroprotection has been production of ADP. Phosphocreatine has much greater intra- targeted to the retina in glaucoma.4 Nicotinamide, for cellular mobility than adenine nucleotides and this compound example, which acts as a substrate for Complex I in the is therefore more readily able to translocate from the respiratory chain, and also acts as a free-radical scavenger and mitochondrion to sites of energy use. Here, cytoplasmic CK an inhibitor of poly-adenosine diphosphate (ADP)-ribose catalyzes the donation of its phosphate group to ADP, thus polymerase, attenuated ischemic injury to retinal ganglion reforming ATP and creatine. This cycle is responsible for 8 cells (RGCs). Another compound, coenzyme Q10,apotent buffering of ATP/ADP levels, aiding maintenance of a cellular Copyright 2019 The Authors iovs.arvojournals.org j ISSN: 1552-5783 4360 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded from iovs.arvojournals.org on 12/11/2019 Effect of Creatine on Retinal Ganglion Cell Survival IOVS j October 2019 j Vol. 60 j No. 13 j 4361 energy reserve and allowing the easy shuttling of fuel currency tissue dissociation, cells were dispensed onto 13-mm diameter between mitochondrial sites of production and cytosolic sites borosilicate glass coverslips coated previously with poly-L- of use.16,17 lysine (10 lg/mL, 15 minutes) in 24-well culture plates for Creatine, therefore, possesses a variety of properties, which immunocytochemical, fluorescent dye-labeling, or apoptotic could contribute to a potential bioenergetic neuroprotective analyses. Mean cell density at seeding was approximately 0.5 3 action, including being able to buffer intracellular energy 106 cells/mL. Subsequently, cultures were grown at 378Cina reserves, stabilize intracellular calcium flux, inhibit mitochon- humidified incubator with 5% CO2 in growth medium (MEM drial permeability transition, and counteract oxidative stress containing 10% FBS, 91 mg/L gentamicin sulphate, 2.3 mg/L build-up.18,19 In fact, creatine supplementation has indeed amphotericin B, and 25 mM glucose). been shown to exert neuroprotective effects in both in vitro After 6 days in vitro, medium was changed and the cultures and in vivo models of major neurodegenerative conditions, were incubated for 24 hours with either creatine (concentra- including models of Huntington’s disease, Parkinson’s disease, tions of 0.5, 1, and 5 mM) or standard medium (control group). 18,20 and amyotrophic lateral sclerosis. To date, there are no To establish mitochondrial compromise, some of both the studies that have examined the direct neuroprotective efficacy creatine-treated group and the control group were subjected to of creatine in acute or chronic paradigms of RGC neurodegen- concurrent incubation for 24 hours with 1 mM NaN3 or for 1 eration. However, S-adenosyl-L-methionine, which is a biosyn- hour with 10 mM NaN3. To establish excitotoxicity, after thetic precursor of creatine, was able to restore photoreceptor creatine pretreatment, cells were exposed to 200 lM NMDA function in a rat model of retinal ischemia.21 The present study plus 10 mM CaCl2 for 24 hours. At the conclusion of the period aimed to assess the neuroprotective properties of creatine in of cell stress, cultures were fixed in 10% (wt/vol) neutral- different retinal models of neuronal compromise: retinal buffered formalin in 0.1 M phosphate buffer, pH 7.4 (NBF) for neurons in culture subjected to excitotoxic challenge or immunocytochemical analysis. In some cases, incubations metabolic impairment and rat retinas subjected to excitotoxic were carried out in the presence of the specific CK inhibitor, or ischemic insults in vivo. 2,4-dinitro-1-fluorobenzene (DNFB; 10 lM), to determine whether this enzyme was responsible for the observed actions of creatine. MATERIALS AND METHODS Experimental Design Retinal Ganglion Cell–Containing Culture System There were two phases to the overall study as follows: (1) to Retinal neuron cultures containing ganglion cells were assess the effect of creatine in culture preparations exposed to essentially established as described previously.23 Adult either energetic compromise with the mitochondrial complex Sprague-Dawley rats were euthanized and eyes were enucle- IV inhibitor, sodium azide, or excitotoxic injury with the ated and placed into PBS containing 100 U/mL penicillin/ ionotropic glutamate receptor agonist, N-methyl-D-aspartate streptomycin. Retinas were removed and chopped into small (NMDA); and (2) to assess putative neuroprotective effects of pieces with dissecting spring scissors. Tissue pieces were creatine in rat in vivo models of retinal ischemia reperfusion incubated in activated papain (2 mg/mL papain, 0.4 mg/mL DL- and NMDA-induced excitotoxicity. cysteine, 0.4 mg/mL BSA, in Neurobasal medium) at 378Cfor

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