Aspects of Retinal Energy Metabolism

Supervised by Professor Robert J. Casson & Dr. John Wood

Guoge Han

Discipline of Ophthalmology, University of Adelaide

December 2014

I

DECLARATION OF ORIGINALITY AND COPYRIGHT

This work contains no material which has been accepted for the award of any other degree in any

university or other tertiary institution and, to the best of my knowledge and belief, contains no

material previously published or written by another person except where due reference is made in the

text.

I give consent that this copy of my thesis, when deposited in the University Library, be available for

loan and photocopying, subject to the provision of Copyright Act 1968.

I also give permission for the digital version of my thesis to be made available on the websites, via the

library catalogue, the Australian Digital Thesis Program unless permission has been granted by the

University of Adelaide to restrict access for a period of time.

Guoge Han

Adelaide, Dec 2014

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

Invest Ophthalmol Vis Sci. 2013 Nov 15; 54 (12):7567-77. doi: 10.1167/iovs.13-12200.

Copyright holder The Association for Research in Vision and Ophthalmology (ARVO)

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Phone +1.240.221.2900 Fax +1.240.221.0370

Glucose-induced temporary visual recovery in primary open-angle glaucoma: a double-blind,

randomized study. Ophthalmology. 2014 Jun; 121 (6):1203-11.

Copyright holder Ophthalmology Editorial Office

Mayo Clinic, East 4, Mayo Building

Rochester, Minnesota 55905 USA

Phone: 415-447-0261 Fax: 415-727-4600

The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation

of GSK3β in Cultured Rat Retinal Cells. Invest Ophthalmol Vis Sci. 2014.

Copyright holder The Association for Research in Vision and Ophthalmology (ARVO)

1801 Rockville Pike, Suite 400

Rockville, MD 20852-5622

Phone +1.240.221.2900 Fax +1.240.221.0370

An explanation for the Warburg effect in the adult mammalian retina. Clin Experiment Ophthalmol. 2013

Jul; 41(5):517

Copyright holder John Wiley & Sons, Ltd.

The Atrium Southern Gate Chichester UK Phone: 44 (0)1865 778315

II

Papers included in this thesis

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

Han G, Wood JP, Chidlow G, Mammone T, Casson RJ.

Invest Ophthalmol Vis Sci. 2013 Nov 15; 54 (12):7567-77

The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation

of GSK3β in Cultured Rat Retinal Cells.

Han G, Casson RJ, Chidlow G, Wood JP

Invest Ophthalmol Vis Sci. 2014. (Accepted, in press)

Expression of Pyruvate Kinase (PKM) in the mice retina

Guoge Han, John PM Wood, Glyn Chidlow, Robert J Casson. (In Preparation)

Glucose-induced temporary visual recovery in primary open-angle glaucoma: a double-blind,

randomized study.

Casson RJ, Han G, Ebneter A, Chidlow G, Glihotra J, Newland H, Wood JP.

Ophthalmology. 2014 Jun; 121 (6):1203-11.

An explanation for the Warburg effect in the adult mammalian retina.

Casson RJ, Chidlow G, Han G, Wood JP.

Clin Experiment Ophthalmol. 2013 Jul; 41(5):517.

Paper Presentations

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

RANZCO Annual General Meeting and Scientific Congress 2011 –Canberra

Posters

The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation

of GSK3β in Cultured Rat Retinal Cells. ARVO 2014 Orlando USA

Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition.

ARVO 2012 Florida USA

Awards

2014 ARVO international travel grant

III

Abstract

Energy failure is a possible pathogenic component of a number of common blinding disorders,

including vascular retinopathies, glaucoma, and age-related macular degeneration. An overarching

premise of the bioenergetics-based research from the research group in which I conducted my studies

is that if energy failure constitutes a pathogenic component in ocular disease, then provision of energy

or the means of the diseased tissue to create its own additional energy may well presented a valid and

viable therapeutic solution. One such approach has been to provide an additional supply of glucose to

a tissue since this monosaccharide is used by the majority of cells as the primary fuel source for the

generation of cellular energy in the form of adenosine triphosphate (ATP).

Previous in vivo research from the group has demonstrated that elevated vitreal glucose levels

afforded robust neuroprotection to the retina and optic nerve in experimental model of acute and sub-

acute ischaemic retinal injury, and in a rat model of laser-induced glaucoma. The current research

focussed on aspects of retinal energy metabolism, and in particular, the mechanisms by which glucose

can act as a neuroprotectant under conditions of compromised energy production in the retina.

Another aim was to translate this research to the clinic and assess the effect of elevated vitreal glucose

levels on visual function in glaucoma patients.

The current thesis comprises four original papers and one perspectives paper. The first paper

characterised an in vitro model of metabolic impairment to rat retinal cultures, using the

mitochondrial complex I inhibitor, rotenone. Subsequently, the protective effect to retina cells of

glucose was investigated in this model, and the effects compared with other known energy substrates

(pyruvate and lactate). A variety of methods, including immunocytochemistry, Western blot and

TUNEL staining were used to determine neuronal and glia cell viability. Cellular energy levels were

determined by luminescent ATP assays and reduction and oxidation (REDOX) power was assessed by

nicotinamide adenine dinucleotide phosphate (NADPH) assay. Metabolic pathways were modified

with specific inhibitors. The findings from this series of experiments supported the hypothesis that the

mechanism by which glucose protects the retina in the presence of mitochondrial impairment is

IV

principally via glycolysis- generated ATP. Moreover, the glucose- stimulated anti-oxidant production

via the pentose phosphate pathway also contributed to the neuroprotection.

In the second paper, we investigated neuronal and glial death and damage mechanism in response to

rotenone treatment, with a particular focus on endoplasmic reticulum (ER) stress, and the signalling

pathways involving calcium-activated neutral protease-µ (calpain-µ) and glycogen synthase kinase 3β

(GSK3β). We found that retinal cultures were modulated via different mechanisms according to the

cell-type and the degree of reduction of ATP. Overall, retinal neurons that were subjected to rapid

ATP depletion in response to rotenone, underwent non-apoptotic death involving generation of

reactive oxygen species (ROS) and activation of calpain-µ. In contrast, glial cells, which are relatively

resistant to mitochondrial damage, were damaged via a combination of endoplasmic reticulum (ER)

stress and deactivation of GSK3β.

The appendix paper provided a brief perspective on a possible explanation for the existence of aerobic

glycolysis (the Warburg effect) in the mammalian retina. It was hypothesized that the rhodopsin

turnover drives the Warburg effect in a similar manner to a proliferating tissue. Recently, a specific

pyruvate kinase isoenzyme (PKM2) has been suggested to be a key mediator of the Warburg effect in

cancer. The third paper (manuscript in preparation) characterizes the distribution of PK isoenzymes in

the rodent retina and brain. PKM2 was distributed in the outer retina, particularly at the level of the

photoreceptor inner segments. Minimal PKM2 was detected in the inner retina of rodents, mirroring

the pattern in the brain; however, the outer retina labelling was remarkably similar to cancerous tissue.

The current body of work culminates in the fourth paper, which translated the laboratory finding to a

clinical trial. In a preliminary study on patients with epiretinal membranes scheduled for routine

vitrectomy, we demonstrated that concentrated 50% topical glucose treatment significantly increased

the vitreous glucose concentration in pseudophakic patients. We then conducted a randomized, double

blind, crossover trial on 29 eyes of 16 pseudophakic patients with severe primary open-angle

glaucoma. We assessed the effect of intensive topical glucose on visual psychophysical parameters.

Saline (0.9%) was used as a control in an initial study and a follow-up study used osmotically-

matched (8%) saline as a control. Glucose significantly improved the mean contrast sensitivity at 12

V

cycles/degree compared to 0.9% saline by 0.26 log units (95% confidence interval [CI]: 0.13 – 0.38; P

< 0.001); and in the follow–up study by 0.40 log units (95% CI: 0.17 – 0.60; P < 0.001). Neither the

intraocular pressure (IOP), refraction, nor the central corneal thickness were affected by glucose; age

was not a significant predictor of the response.

In conclusion, these studies add valuable information to the literature concerned with bioenergetic-

based protection to ocular and, in particular, retinal cells. Furthermore, the clinical study presented in

the final chapter actually demonstrates a confirmation of the “proof-of-principle” for bioenergetic

neurprotection as a treatment strategy for retinal and optic nerve diseases. Similar strategies could

now conceivably be applied to other retinal diseases. Finally, a better understanding of the unusual

retinal energy metabolism, as discussed in the preceding chapters, is likely to shed light on disease

pathogenesis and provide information that could potentially be translated to the clinic.

VI

Acknowledgement

This study was supported by funds from the NMHRC and the China Council Scholarship.

First and foremost, I would like to sincerely thank my principal supervisor Professor Robert Casson

for admitting me to undertake this PhD in the Discipline of Ophthalmology, Adelaide University. I am

very grateful to Bob, an outstanding world class scientist and supervisor in experimental and clinical

ophthalmic research; thanks for his generous help and great support throughout my PhD.

To Dr John Wood, I sincerely appreciate your understanding, kindness, and expert supervision every

step of the PhD. Thank you for understanding my different academic background and helping me get

on the right track during the hardest time at the outset. Your constant support, honest and constructive

criticism were very important for my doctoral research work. I am grateful for your valuable

knowledge and advice with the experimental techniques, and especially for your friendship. I wish

you every success in the future!

To Dr Glyn Chidlow: thank you for teaching me useful scientific skills and sharing your experience

and knowledge with me, especially in critically interpreting data.

Thank you to Teresa Mammone and Mark Daymon. Teresa helped me settle down in our lab and

taught me quite a few useful experiments during my study. You always create a happy environment in

the lab and I wish you best of luck with your Master degree and beyond. Mark, thanks for your expert

help with the immunostaining and histological techniques.

To Dr Jolly and Kylie, I will never forget your help when I had eye trauma here. Thank you!

To my new friends in the Neuropathology lab: Jim, Kathy, and Sophie. Thanks for sharing your

interesting stories over morning tea and for brightening up my days.

I am very grateful to Shan for the helpful, worldly wisdom and invaluable support from the first day I

landed in this country.

To S, we crossed paths in difficult times; thanks for your warm help.

VII

To my Chinese friends Jimin, Shurong, Li Yun: Thank you for enriching my weekends and holidays

and enjoying my life in Australia.

Last but certainly not least, I wish to say thank you to my parents for your support and always being

there during the long and hard PhD road.

Content

DECLARATION OF ORIGINALITY AND COPYRIGHT ............................................... I

Abstract .................................................................................................................................. III

Acknowledgement ................................................................................................................. VI

1.1 Introduction ........................................................................................................................ 1

1.2 Oxygen distribution and consumption in the retina ....................................................... 2

1.2.1 Histological mitochondria distribution .......................................................................... 2

1.2.2 Oxygen consumption in the outer retina ....................................................................... 2

1.2.3 Oxygen in the inner retina ............................................................................................. 3

1.3 Retinal glucose metabolism ............................................................................................... 4

1.3.1 Basic Cellular Energy Metabolism ............................................................................... 4

1.3.2 The Pasteur effect .......................................................................................................... 4

1.3.3 The Warburg Effect ....................................................................................................... 4

1.4 Metabolic energy coupling in the retina .......................................................................... 8

1.4.1 Astrocyte neuronal lactate shuttle hypothesis ............................................................... 8

1.4.2 Metabolic coupling between photoreceptors and Müller glia cells ............................... 8

1.5. Glucose metabolism in retinal neuron cells .................................................................. 10

1.5.1 Photoreceptors ............................................................................................................. 10

1.5.2 Retinal Ganglion Cells ................................................................................................ 12

1.5.2.1 Glucose metabolism in retinal ganglion cells ....................................................... 12

1.5.2.2 Role of ATP underlying retinal ganglion cell death ............................................. 14

1.6. Glucose metabolism in retinal glia cells ........................................................................ 16

1.6. 1 Glucose metabolism in retinal Müller cells ................................................................ 16

1.6.2 Glucose metabolism in retinal pigment epithelium ..................................................... 17

1.7. Glucose neuroprotection in the retina........................................................................... 19

1.7.1 Bioenergetic neuroprotection ...................................................................................... 19

1.7.2 Glucose concentration in the vitreous ......................................................................... 19

1.7.3 Glucose neuroprotection of the retina ......................................................................... 20

1.7.4 Possible mechanisms by which glucose protects the retina ........................................ 21

1.7.4.1 Role of glycolysis-derived ATP underlying glucose neuroprotection in the retina

.......................................................................................................................................... 21

1.7.4.2 Does the pentose phosphate pathway play a role? ............................................... 21

1.8. Conclusion ....................................................................................................................... 23

2. Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to

respiratory inhibition............................................................................................................. 25

3. Rotenone induced calpain-µ activation in neurons and induction of ER stress and

GSK3β in glial cells ................................................................................................................ 41

4. Emerging aspects of glucose metabolism in the retina: The expression of pyruvate

kinase in rodent retina ........................................................................................................... 65

5. Glucose-Induced Temporary Visual Recovery in Primary Open-Angle Glaucoma ... 97

6. Conclusions and future research directions .................................................................. 107

6.1 General conclusions and relationship of the thesis to the literature ......................... 107

6.2 Limitations of the studies presented in this thesis ...................................................... 109

6.2.1 In vitro study ............................................................................................................. 109

6.2.2 Validation of the antibodies ...................................................................................... 110

6.2.3 Clinical testing study ................................................................................................. 112

6.3 Other possible speculations on the neuroprotective mechanism of glucose ............. 113

6.3.1 Age and the protective effect of glucose ................................................................... 113

6.3.2 Mechanical stress and the protective effect of glucose ............................................. 114

6.3.3 PKM2 role in glucose metabolism in cancer and retina ............................................ 114

6.4 Future directions on bioenergetics therapies .............................................................. 117

7. References ......................................................................................................................... 118

8. Appendix ........................................................................................................................... 132

1

1.1 Introduction

The retina has one of the highest energy demands of any tissue in the human body.1, 2

Although the retina is anatomically an out-pouching of the brain, its energy metabolism is

more akin to that of a cancerous tumour.3, 4

This is possibly due to its high energy demands

and local oxygen tension. Glucose, which is the preferred energy substrate of the retina,

produces adenosine triphosphate (ATP) and generates metabolic intermediates for

biosynthesis.5, 6

Deficiencies in energy metabolism are certainly, or probably, part of the

pathogenesis, of a number of common blinding eye diseases, including retinal ischemia

disease, diabetic retinopathy, glaucoma, and dry age-related macular degeneration (ARMD).7,

8 These diseases currently have limited treatment options and are responsible for a large

portion of visual impairment worldwide. This chapter focuses on the unique metabolic

pattern of the retina and provides insight into retinal glucose metabolism at the mechanistic

level. The use of glucose as a neuroprotectant and its possible mechanism is also discussed.

2

1.2 Oxygen distribution and consumption in the retina

Under normal conditions, oxygen tension which is monitored at a molecular level by hypoxia

induced factor-1α (HIF-1α), is a limiting factor which regulates retinal glucose metabolism.

Thus, the distribution and consumption of oxygen in the retina is important in understanding

the metabolic pattern across different retinal layers.

1.2.1 Histological mitochondria distribution

The distribution of mitochondria within the retina reflects the consumption of oxygen at the

tissue and cellular level. Histochemical studies have identified that 60-65% of the retinal

mitochondria are located in the inner segments of photoreceptors.9 Cones have more

mitochondria than rods, due to their higher metabolic needs.10

In addition, in the inner retina,

the inner plexiform layer is noted to have more mitochondria than the inner nuclear layer.11

1.2.2 Oxygen consumption in the outer retina

Intraretinal oxygen tension has been profiled in several species, including cat12

, pig13

and

macaque monkey.14

In mammals, the choroidal circulation supplies the majority of the

oxygen demand for the photoreceptors.15

This demand is largely driven by maintaining

cellular depolarisation in the dark: the so-called “dark current”,16, 17

and the energy demands

of neurotransmission.17

In the cat and rat, photoreceptor oxygen consumption under photopic

conditions is 60% of that in the dark.18

Hence, light adaptation can increase PO2 in the outer

retina.18

Photoreceptor inner segments consume approximately five times as much O2 as the outer

segments. Hence, mitochondria are located almost entirely within the inner segments.

3

1.2.3 Oxygen in the inner retina

Both oxygen tension and consumption are reduced in the inner retina compared with the outer

retina. Oxygen is supplied to the inner retina via diffusion from the retinal arterioles, in

contrast to the photoreceptors, which receive their oxygen and nutrient demands from the

choroidal circulation. The oxygen tension within the retina depends on the distance to the

nearest vessel, being higher (40 mmHg on average) adjacent to the arteriolar wall and

remaining constant (20mmHg on average) in intravascular regions of pig retinas.14

Light adaptation has no influence on oxygen consumption in the inner retina. Moreover, in

response to a flashing light source, the inner rabbit retina demonstrates increased lactate

production (via glycolysis), but no significant oxygen consumption change.17

It is likely that

the retinal ganglion cell accounts for this response19

since it has higher electrical firing rates

in presence of a flickering light source.

Oxygen tension within the inner retina remains relatively stable in hyperoxia or hypoxia; in

contrast, the oxygen tension of the outer retina is influenced to a great extent by the oxygen

availability.14, 17

The relative contribution of glycolytic versus oxidative phosphorylation

within the different retinal layers is an interesting area for further study.

4

1.3 Retinal glucose metabolism

1.3.1 Basic Cellular Energy Metabolism

Cellular ATP is produced by two related processes: cytoplasmic glycolysis and mitochondrial

oxidative phosphorylation (OXPHOS). In glycolysis, glucose is converted to pyruvate,

forming 2 ATP molecules. In the presence of O2, pyruvate enters mitochondria where it is

usually converted to acetyl Co-enzyme A (acetyl Co-A), which then enters the Krebs cycle,

forming electron donors for OXPHOS, and leading to the generation of ~32-34 ATP

molecules. When O2 is scarce, pyruvate is converted to lactate via lactate dehydrogenase.

1.3.2 The Pasteur effect

The upregulation of glycolytic lactate production when O2 tension is decreased is known as

the Pasteur effect.20

This occurs during hypoxia and is considered to be an adaptive

response.20, 21, 22, 23

Pasteur’s original observation described the reciprocal effect, namely

inhibition of glycolysis by O2.24

1.3.3 The Warburg Effect

In 1924, Otto Warburg and his colleagues first demonstrated that malignant tumours tended

to consume glucose and produce lactate despite normal oxygen tensions. Warburg believed

that this was a pathological breach of the Pasteur effect and the phenomenon was termed

“aerobic glycolysis”, later becoming known eponymously as the Warburg effect.25

Warburg

believed that the switch from “normal” OXPHOS to “aerobic glycolysis” was not just

associated with cancer, but that it was causal.26

His team also noted that normal mammalian

retinal explants displayed aerobic glycolysis.27

This finding did not fit neatly with Warburg’s

beliefs about cancer pathogenesis and they attributed it to experimental artefact. However,

others groups later confirmed that the mammalian retina displays a strong Warburg effect.28,

29

5

In recent years, the Warburg effect has become an explosive area of study within the cancer

research community, with many publications in the world’s leading scientific journals,30-33

resulting in a deeper understanding of the Warburg effect at the molecular level. Several

possible mechanisms for this phenomenon have been considered: (1) pathological

stabilization of HIF-1α, (2) oncogene activation and loss of tumor suppressor genes, (3)

mitochondrial dysfunction in cancer cells.34

However, the fact that the retina also displays the

Warburg effect is rarely mentioned, and whether or not it shares the same molecular

mechanisms as cancer is unknown.

Vander Heiden et al. publishing in Science,33

recently summarized a widely-accepted

teleological explanation for the existence of the Warburg effect in cancer. In proliferating

cells, glucose not only produces ATP, but also provides metabolic intermediates for

biosynthesis. Proliferating cells have the ability to increase glycolytic ATP production under

hypoxic conditions, but provided glucose is abundant, in normoxia, they direct metabolic

pathways away from OXPHOS towards biomass synthesis. The ability to oscillate between

biosynthesis and energy requirements provides proliferating tissue with a powerful metabolic

strategy known as the “metabolic budget system”,35

a phenomenon which goes hand-in-hand

with the Warburg effect. The metabolic budget system can be viewed as the presence of the

Warburg effect in a tissue using glucose for biosynthesis.35

To our knowledge, a metabolic

budget system has not been investigated in a non-proliferating tissue, such as the retina.

Casson et al. have recently published a teleological explanation for the presence of the

Warburg effect in the mammalian retina: the mammalian retina shares similar biosynthesis

requirements to neoplastic tissue due to the prodigious turnover of the opsin protein in the

disc membranes of the outer segments.36

Hence, rhodopsin turnover drives aerobic glycolysis

in the retina. Thus, the reason that the Warburg effect has evolved in the mammalian retina is

because it has similar metabolic requirements to a proliferating tissue. This finding is

6

supported by the fact that the rhodopsin turnover parallels the degree of aerobic glycolysis

found in different species.37

Furthermore, the relatively low rate of photoreceptor turnover in

lower vertebrates is temperature dependent, increasing at higher temperatures,37

reflecting the

temperature-dependent Warburg effect.27

The final step of the glycolytic pathway requires pyruvate kinase (PK)38

to catalyze the

conversion of phosphoenolpyruvate39

to pyruvate, which then enters the Krebs cycle.40

PK

exists as four isoforms, which are coded by two genes: PKLR and PKM. The expression of

the R and L forms of PK is controlled by an upstream promoter containing either CAAT or

TATA in red blood cells and liver tissue, respectively.41

The M1 and M2 isoforms of PK are

alternatively spliced forms of the PKM gene.42

PKM2 is the predominant form in cancerous

tissue.43-45

Christofk et al. 31

also reported that the switch in expression of PKM1 to PKM2 in

cancer cells is responsible for the Warburg effect. This conclusion was later undermined by

evidence for the PKM2 isoform in a number of normal tissues. However, to the best of our

knowledge, PK and its possible regulation of the Warburg effect has not been investigated in

the retina.

Therefore, in chapter 4, we studied the distribution pattern of PKM1 and PKM2 in the rodent

retina and compared this to the distribution in brain.

Lactate dehydrogenase (LDH) is another possible mediator of the Warburg effect in the

retina. LDH is a tetrameric enzyme comprising two major subunits A and/or B, (encoded by

the Ldh-A and Ldh-B genes) resulting in five isoenzymes (A4, A3B1, A2B2, A1B3, and B4)

that catalyze the forward and backward conversion of pyruvate to lactate. LDHA (LDH-5, M-

LDH, or A4), which is the predominant form in skeletal muscle, kinetically favors the

conversion of pyruvate to lactate. LDHB (LDH-1, H-LDH, or B4), which is found in heart

muscle, converts lactate to pyruvate that is further oxidized in mitochondria. Cancers also

7

utilize the LDHA form, (even when oxygen is abundant) and it has become a routine clinical

surrogate marker of the Warburg effect, providing diagnostic and prognostic clinical

information.46

Like tumours, the mammalian retina also exhibits a high dependence on aerobic glycolysis,

mediated by LDHA.25

The brain, which principally relies on the OXPHOS pathway, exhibits

twenty times lower LDHA activity than retina.25

In addition, Graymore, publishing in Nature

in 1964, noted that the expression of the LDHA isoenzyme in the retina was reduced in rats

with inherited “retinitis pigmentosa”, characterized pathologically by loss of the

photoreceptors.47

This observation indicated that the photoreceptors were principally

responsible for the retinal lactate production. This is consistent with earlier evidence that the

photoreceptors were particularly susceptible to glycolytic inhibition.28

Human Müller cells in vitro also demonstrate a robust Warburg effect, more prominent than

some cancer cell lines.19, 21

Whether this occurs in vivo is unclear, and may be peculiar to the

cultured Müller system. However, production of lactate by Müller cells is believed to be an

essential element of metabolic coupling in the retina (see 1.4). Hence, the explanation for the

Warburg effect in glial cells may not be related to biosynthesis requirements, but may a

prerequisite for meeting neuronal energy demands, if lactate is the preferred substrate. This

remains controversial (discussed 1.4). Whether the same molecular mechanisms mediate the

Warburg effect in Müller cells is completely unknown.

Overall, although glucose has been long considered as the primary neuronal energy substrate

in the retina, an alternative hypothesis that neurons use glial-derived lactate as their primary

energy source (glial-neuronal metabolic coupling) has supportive evidence and is discussed

in Chapter 1.4

8

1.4 Metabolic energy coupling in the retina

1.4.1 Astrocyte neuronal lactate shuttle hypothesis

The traditional notion that central nervous system (CNS) neurons utilise glucose as their

major energy substrate was challenged in 1994 by Pellerin and Magistretti et al. They

provided evidence that astrocytes, not neurons, metabolize glucose, and transport lactate to

the neuron, which is converted to pyruvate, enters the Kreb’s cycle thereby fuelling the

energy demands of neurotransmission.48

They further asserted that astrocytic lactate

production and delivery is calibrated by neuronal glutamate production, providing a feedback

between energy supply and neurotransmission demands. This concept became known as the

astrocyte neuron lactate shuttle hypothesis (ANLSH). However, this concept has become

highly controversial, with a number of studies supporting it and others denouncing it.49, 50

In

support, MR spectroscopy studies performed by Serres et al. have suggested that net lactate

transfer between neuron and astrocytes takes place in the brain.51

Furthermore, the ANLSH

has been reinforced by quantitative mathematical models.52, 53

However, opponents also have

convincing evidence that CNS neurons preferentially utilize glucose over lactate.50, 54

Although there is controversy regarding this issue, it has never been claimed that glucose is

not a neuronal substrate, or that neurons uptake exclusively either glucose or lactate in the

brain.55

1.4.2 Metabolic coupling between photoreceptors and Müller glia cells

In 1987, Tsacopoulos et al. found that glucose was not directly required for photoreceptor

function in drone bees, but retinal glial cells did uptake glucose.56

The drone bee was selected

to study the compartmentalization of retinal metabolism because its retina comprises only

photoreceptors and glial cells. Another well-cited experiment supporting the notion of

metabolic coupling between photoreceptors and glial cells in the retina was performed by

9

Poitry-Yamate et al. using guinea pig retina.57

They used a preparation of Müller cells

attached to photoreceptors and measured radiolabelled metabolites from both cell types. They

proposed that “the net production and release of lactate by Müller cells serves to maintain

their glycolysis elevated and to fuel mitochondrial oxidative metabolism and glutamate

resynthesis in photoreceptors”. Furthermore, studies characterizing the distribution of

monocarboxylate transporters which allow passage of lactate and pyruvate through

membranes are also consistent with the above suggestion.58-60

Finally, the existence of

GLUT1 receptors on retinal neurons61

is not easily explained by ANLSH proponents.

However, the conclusions of Poitry-Yamate et al. have been criticized.8 The nature of their

preparation leads to overestimation of lactate consumption by neurons.50

In addition, studies

have indicated that although metabolic coupling may exist in mixed retinal cultures, neurons

preferentially metabolize glucose over lactate.5 Furthermore, intraocular lactate delivery was

not protective against ischaemic retinal injury and the inhibition of lactate transport did not

exacerbate ischaemic retinal injury in vivo.62, 63

Finally it is know that in vivo, photoreceptors

metabolize do glucose to lactate aerobically.64

Autoradiographic studies by Winkler et al.

also showed that glucose is the preferred energy substrate of retinal neurons.65

In conclusion, the notion of metabolic coupling in the brain and retina remains highly

controversial. It is remarkable that such a fundamental aspect of CNS biology with important

clinical ramifications remains unclear and is an area for further study.

10

1.5. Glucose metabolism in retinal neuron cells

1.5.1 Photoreceptors

Photoreceptors, comprising cone and rod cells, are highly specialized neurons with large

energy demands. They principally depend upon glucose to generate enough ATP for light

transduction or maintaining the dark current.38

The inner segment of the photoreceptor, which

contains a high density of mitochondria, uses approximately 50% of the available ATP to

pump out intracellular Na+ via cGMP-gated channels to maintain the dark current.

66 In light,

cones consume more energy than rods due to the high rates of cGMP turnover.67

Although the pathways by which the photoreceptors meet their energy demands in vivo

remain incompletely understood, there is evidence that the mammalian outer segments utilize

ATP produced from OXPHOS in the inner segment and via glycolysis in the outer segment.68

Glucose metabolism in the photoreceptor rod outer segment produces both ATP (GTP) and

NADPH to support phototransduction.68

After glucose crosses the blood retinal barriers, it is transported into photoreceptors via

GLUT1 receptors on the outer segments.61, 68

ATP production via glycolysis alone is

sufficient to sustain the dark current in bovine rods and in isolated rat retinas.68, 20

ATP

supply is supported by a creatine shuttle transporting OXPHOS-derived ATP from the inner

segment.68, 69, 70

In addition, ectopic mitochondrial proteins have been identified in the outer

segment suggesting that OXPHOS-derived ATP may be produced in the outer segment. This

concept is supported by mitochondrial enzyme histochemial studies.71

Indeed, Panfoli et al.

identified OXPHOS respiratory chain complexes I-IV as well as ATP synthase (Complex V )

expression in the vertebrate retinal rod outer segment.39,72

A number of important studies have demonstrated the reliance of photoreceptors on

glycolysis. In 1951, Noell showed that the mammalian photoreceptors were highly sensitive

11

to the glycolytic inhibitor, iodoacetate. The degree of sensitivity was far greater than noted in

the brain and was particularly evident in the photoreceptors, which were also relatively

resistant to anoxic injury. It is worth noting Noell’s conclusions: “Apparently, in these higher

vertebrates an attempt has been made to separate glycolysis and respiration with respect to

the physico-chemical processes involved in the mechanism of retinal excitation, so that a

given process (but not necessarily the same in all species) is supported predominantly by the

one or the other means.”73

Graymore, publishing in Nature in 1964, noted that the expression of the lactate

dehydrogenase A isoenzyme in the retina was reduced in rats with inherited “retinitis

pigmentosa”, characterized pathologically by loss of the photoreceptors.47

This observation

indicated that the photoreceptors were principally responsible for the retinal lactate

production. This supported earlier evidence that the photoreceptors were particularly

susceptible to glycolytic inhibition.73

And this suggestion is consistent with the responses

from electroretinographic metabolic studies on rat retinal explants.20

Winkler et al. also

determined that photoreceptors produce lactate aerobically and anaerobically in the presence

of ambient glucose and were susceptible to glycolytic inhibition. The study was also repeated

in a cone-rich mammal, replicating the findings.19

This unique metabolic nature of photoreceptors resembles rapidly proliferating or dividing

cells. In retinal explants, glycolysis generates 50% of the ATP in the photoreceptors and 80%

of the glucose taken up is metabolized to lactic acid under aerobic conditions, a manifestation

of the Warburg effect.74

In addition to providing ATP, glycolysis also provides metabolic

intermediates for amino acid biosynthesis.

In the cones, opsin is a G protein-coupled receptor comprising 348 amino acids, with a rich

glycine and serine component. In addition, turnover of the opsin protein in the disc

12

membranes of the outer segments consumes large amounts of protein. Glucose can also be

converted to GlcNAc, which is required for rhodopsin glycosylation and other cell surface

signaling trafficking.71, 75

This data this led to the speculation that the retina uses glycolysis

aerobically not only for energy production but also for biosynthesis.76

Glucose is needed in rod outer segments for production of cytosolic NADPH, via the pentose

phosphate pathway. This molecule serves a number of fundamental cellular functions:

transforming trans-retinal to all-trans-retinol by retinol dehydrogenase, inhibiting caspase-

mediated apoptosis and maintaining the redox state of the cell.77, 78

A recent study showed

that isocitrate and malate, both metabolic fuel sources derived from mitochondria also

contribute to the synthesis of NADPH in rod outer segment in the presence of transient

nutrient shortages.79

1.5.2 Retinal Ganglion Cells

1.5.2.1 Glucose metabolism in retinal ganglion cells

Retinal ganglion cells, (RGCs) which are located in the inner retina, are responsible for visual

transduction from the optic nerve to the CNS. Owing to the long distance of signal delivery,

RGCs need large amounts of ATP to support nerve transmission and electrical conduction.80,

81 The RGC axons are myelinated outside the eye which greatly reduces the energy

requirements for neurotransmission; however, for optical reasons the intraocular axons are

unmyleinated, placing heavy energy demands on these cells. Mitochondria are localized

along the unmyelinated axons of RGCs, generating ATP via OXPHOS. Mitochondria are

particularly abundant in optic nerve head, indicating heavy energy demands in this region.82

In a classic study (involving self-experimentation), Noell noted that the RGCs were both

highly susceptible to anoxia and were the “weakest link in the chain” of visual perception.83

13

Mitochondria are particularly susceptible to ischaemia/hypoxia and are one of the earliest

organelles to fail under these conditions.84

Furthermore, OXPHOS malfunction results in

electron leakage and the generation of ROS from mitochondrial complexes I and III. Hence, a

positive feedback can be established where excessive ROS production in RGC cells, in turn,

can further damage OXPHOS function.85, 86

In addition, oxidative stress induced by the ROS generation influences the mitochondrial

inner membrane potential and calcium homeostasis and finally leads to RGC cells death: both

apoptosis and necrosis.87

Chronic energy failure, associated with age-related mitochondrial

dysfunction renders the RGCs susceptible to the ROS application and predisposed the

neurons to cell apoptosis.88, 89

In this process, proapoptotic factors such as cytochrome c,

Smac/Diablo and Bcl-2 trigger RGC cell apoptosis via caspase-independent mechanism.90-92

In contrast, a drastic reduction in ATP in the presence of oxidative stress is characteristic of

necrotic cell death.93, 94

Although the molecular mechanisms underpinning ROS-induced

RGC death are not fully understood, the apoptosis is involved in this process.95-98

Although it is clear that RGCs are sensitive to hypoxia, it remains unclear whether they can

also utilize glucose to produce ATP when O2 is scarce i.e. whether or not they display a

Pasteur effect. Winkler et al. attempted to address this question using cultured RGCs and by

measuring lactate and ATP production under hypoxic conditions. They concluded that RGCs

do display a Pasteur effect.99

Unfortunately their methodology was flawed because they used

an RGC-5 cell line, which was later recognized to be likely of photoreceptor origin.100, 101

However, the anaerobic type of lactate dehydrogenase (LDHA) is located in the ganglion cell

layers, suggesting that glycolysis is important.102

In addition, research from our group has

provided indirect evidence for this concept, with experiments demonstrating that the delivery

of glucose to the retina under ischaemic conditions is protective against RGC loss.62

14

1.5.2.2 Role of ATP underlying retinal ganglion cell death

1.5.2.2.1 Cell death: apoptosis and necrosis

A number of distinct modes of cell death are recognized pathologically. Classical RGC death

is categorized into two distinct models: apoptosis and necrosis. Although these death modes

are considered to lie on a spectrum, they have distinct differences, particularly related to

cellular energy supply. Apoptosis is an active, programed process that removes unnecessary

or damaged cells. This ATP dependent process, maintains organelle integrity and relatively

high intracellular energy levels until the final stages of cell death.103, 104

In contrast, necrosis

is considered to be predominantly a passive cellular demise, occurring under extreme stress.

It is characterized by swelling of the mitochondria and loss of the cell plasma membrane. A

number of studies have shown that the level of the available intracellular ATP is a

determinant of the cell death mode.89

For instance, cells with a relatively high level of ATP

tend to undergo apoptosis when subjected to lethal stress; whereas cells with a lower or no

energy supply tend to necrose.94

1.5.2.2.2 Mitochondrial associated apoptosis and retinal ganglion cell death

Mitochondrial dysfunction plays a central role in RGC apoptotic death in glaucoma and in

experimental retinal ischemia.105

With the onset and progression of the mitochondrial

impairment, the mitochondrial membrane permeability increases and cytochrome c is

released into the cytosol to form an apoptosome, which recruits procaspase-9 and caspase-3

to initiate caspase-dependent apoptosis.106

Experimental studies have shown that intravetreal

injection of caspase-3 or capase-9 inhibitors can rescue the RGC from axotomy-induced

apoptosis.107, 108

Other mitochondria derived proteins which are released to the cytoplasm

during apoptosis, such as the second mitochondria-derived activator of caspases (SMAC)

and apoptosis-inducing factor 109

, have also been involved in this process.110

So far, the

specific role of these mitochondrial toxic proteins are still unclear.

15

The optic nerve head, where the nerve fibres contain a high density of mitochondrial

organelles pass through the lamina cribrosa, is a neural region which is highly vulnerable to

metabolic insufficiency Any alteration in blood flow dynamics might reduce the glucose and

oxygen supply, resulting in impairment of OXPHOS with accumulating of ROS and

ultimately RGC death.111

In a glaucoma model, the rate of ATP production decreases,

disabling the Na+/K

+ pump, resulting in the blockade of the axonal transduction and energy

diffusion.17, 112

In addition, the generation of ROS from inefficient OXPHOS is pathogenic.113

1.5.2.2.3 Involvement of necrosis in retinal ganglion cell death

Under strong stresses, such as long-term glucose and/or oxygen deprivation, cells undergo

necrosis due to the low intracellular ATP levels. This is illustrated by a study using ATP-

liposomes to which protect RGCs from necrosis in an ischemia-reperfusion mouse model.114

Possible mechanisms underpinning necrosis have been proposed in retinal neurons, such as

the hyperactivation of the nucleus enzyme PARP1 (poly [ADPribose] polymerase-1), which

uses NAD+ as a substrates.

115 This process, in turn, will lead to the depletion of ATP since the

replenishment of the NAD+ pool is energy-dependent.

116 The loss of ATP subsequently

results in the loss of membrane selectivity, cell swelling and rupture of the plasma

membrane.117

However, specific blockade of PARP1 only afforded partially neuroprotection

of RGCs indicating alternative pathogenic pathways existed.117

16

1.6. Glucose metabolism in retinal glia cells

1.6. 1 Glucose metabolism in retinal Müller cells

Müller cells, which are the principal glia cell type in the vertebrate retina, extend from the

surface of the inner limiting membrane to the pigment epithelium and are vertically oriented

with respect to the retinal layers. This intimate apposition to every retinal neuron type allows

potential metabolic interaction between all retinal neurons and glia.1

Müller cells mainly rely on glycolysis even in the presence of ambient oxygen.21, 118-121, 122, 57

Winkler et al. showed that human Mülller cells display the strongest Warburg effect of any

reported cell type, with 99% of the available glucose converted to lactate under aerobic

conditions.21

They further demonstrated that human Müller cells can survive in the presence

of mitochondrial inhibition, but are sensitive to inhibition of glycolysis, leading to severe

ATP depletion.21

In addition, other studies have shown that Müller cells are resistant to long-

lasting anoxia and ischemia. For example, Hughes et al. noted that in a rat retina ischemia

model, which caused death of neurons in the inner nuclear layer, that there was sparing of

Müller cells.123

A similar resistance to ischemia has been reported in rabbit and monkeys.124,

125

When free glucose is unavailable, endogenous glycogen could serve as an emergency retinal

energy reservoir.126

This idea is supported by histochemical evidence that glucose-6-

phosphate, glycogen and glycogen phosphorylase are localized in the cytoplasm of retinal

Müller cells.127

In addition, it has also been suggested that changes in illumination could also

lead to glycogen mobilization from Müller cells to photoreceptors.128

Other energy substrates,

such as pyruvate, can protect glucose deprived Müller cells from oxidative stress by

scavenging the excess free radicals.129

And the production of lactate from retinal Müller cells

may be reutilized in the TCA cycle in neurons.130

17

The notion of “metabolic symbiosis” was raised by Bringmann et al. in 2006 in reference to

the metabolic interaction between retinal Müller cells and neurons.126

This included the

possible uptake of Müller cell-derived lactate by the neurons, buffering of neuronal pH, and

Müller cell-facilitated transfer of CO2 to the vitreous or blood vessels.12

The strong Warburg effect in the Müller cells remains, however, unexplained. The production

of lactateas a potential energy source for neurons would fit with a retinal equivalent of the

ANLSH (as previously discussed). Furthermore, glycolytic metabolism in glia may spare O2

for surrounding neurons. Recent evidence indicates that Müller cell ablation in a transgenic

model causes photoreceptor degeneration related to loss of neurotropic or other support of the

latter cells by the former; this could potentially be by lactate transfer.131

1.6.2 Glucose metabolism in retinal pigment epithelium

The pigment epithelium serves essential roles in the maintenance of normal retinal

physiological function. Located between the endothelium of the choriocapillaris and

photoreceptor layer, RPE cells are regarded as part of the blood retinal barrier, regulating bi-

directional flow of metabolic intermediates, and outer segment waste products.132

Thus, any

alterations in the blood retinal barrier, such as energy comprise could lead to the dysfunction

of the RPE cell layer and this may ultimately impair photoreceptor function. Conversely,

energy metabolism-associated gene mutations expressed in the photoreceptors, could lead to

RPE degeneration.133

RPE cells reply upon glucose as their primary energy substrate.134

In order to facilitate

glucose transport from the blood to photoreceptors, the RPE expresses both GLUT1 and

GLUT3 transporters in their apical and basolateral membranes. GLUT3 is responsible for

basic glucose transport whereas GLUT1 selectively transports glucose from blood to retinal

neuronal layers. 135

When glucose is unavailable, monocarboxylates such as lactate can be

18

used as an alternative energy source by RPE cells, as demonstrated in cultures studies. This is

also supported by immunohistochemical results which show that the monocarboxylate

transporter subtype, MCT1, which is responsible for lactate influx, is present on the apical

(photoreceptor) side of the cells, whereas MCT3, which undertakes lactate efflux, is located

on the basolateral face of the cells and is therefore responsible for transport of lactate into the

choroidal circulation. Although RPE preparations were shown to transport lactate,136

it still

remains uncertain whether lactate can be metabolized to pyruvate and then oxidized via

oxidative phosphorylation in these cells in vivo. It would be of great interest, for example, to

determine the lactate concentration difference across the RPE in animal models.

Lastly, lactate transport in RPE cells is not only linked to energy metabolism, but is also

coupled with the co-transport of H+, thereby regulating cell volume and retinal attachment.

137

In a MCT3 gene knockout murine model, disturbance of RPE lactate transport leads to a

decrease of pH in the subretinal space.138

Rat and human cultures of RPE cells have high levels of lactate production and high oxygen

consumption.135

139

Lactate production in rat RPE cells depends on the glucose concentration

in the medium. RPE contain substantial mitochondria (especially on the apical side),

indicating ATP production via OXPHOS as well as glycolysis.140

Recent studies have shown

that RPE cells are sensitive to mitochondrial inhibition and oxidative stress.141

In addition,

ATP generated by RPE can be released into the subretinal space and subsequently activate

photoreceptor-based P2X2 receptors, which result in the influx of calcium into the outer

segments. A body of research also indicates that the pentose phosphate pathway (PPP)

contributes to the maintenance of RPE physiological functioning.142

By magnetic resonance

spectroscopy, it has determined that 20% of glucose is metabolized through the PPP in

human RPE cell cultures.143

This shunt is thought to provide reducing equivalents such as

NADPH or GSH during photochemical stress.143, 144

19

1.7. Glucose neuroprotection in the retina

1.7.1 Bioenergetic neuroprotection

The conception of bioenergetic neuroprotection is based on the notion that if energy failure is

part of the pathogenesis, then energy delivery may be a potential therapy. Energy failure is,

by definition, a critical component of ischaemic-related diseases, and there is considerable

evidence that mitochondrial dysfunction and subsequent ATP depletion act as important

pathogenic components in other common neurological diseases.145, 146

Although some

potential bioenergetic compounds have had laboratory-based success in animal models, this

strategy has had limited success in human clinical trials.147

Glucose is the major energy substrate of the brain and retina, and research from our

laboratory has demonstrated that elevated glucose levels provide robust neuroprotection

against acute148

and chronic 3ischaemic retinal injury, and against experimental glaucoma.

149

These laboratory-based findings have recently been extended to the clinic. We have recently

shown that topical glucose delivery temporarily improves contrast sensitivity and acuity in

patients with severe primary open-angle glaucoma. Here, we discuss the development of this

approach.

1.7.2 Glucose concentration in the vitreous

Remarkably, there is little data concerning the biochemical composition of the living human

vitreous.150, 151

Almost all data are derived from animal studies, with species differences, and

from post-mortem human data.152

Reddy and Kinsey’s 1960 study of the biochemical

composition of the aqueous and vitreous in rabbits is often cited and reproduced in text

books.153

They reported a vitreous glucose concentration of 3.0µM/g, with a corresponding

plasma concentration of 5.7µM/g.153

In subsequent studies in cattle and horses, it was

reported that the concentration varied in the peripheral compared to the central vitreous, with

20

an increase towards the core.154

In 1994, Lundquist et al. reported on the vitreous glucose

concentration in living human diabetics and provided some control data. They reported a

fasting concentration of 3.5 ±1.8 mM /L.150

Casson et al. noted similar results, but with a

lower variance.

In a somewhat obscure publication, Weiss noted that the vitreous glucose concentration

declined in rabbits during periods of retinal ischaemia. The inference was that the vitreous

was serving as an energy substrate reservoir.155

In the rat, streptozocin-induced diabetes and

intraperitoneal injection of glucose significantly increases the concentration in the vitreous

but only minimally in the retina.62

This corresponds to Reddy and Kinsey’s findings and the

limited human data.9 Furthermore, Casson et al. recently reported that topical application of

glucose significantly elevates the vitreous glucose concentration in pseudophakic but not

phakic patients.151

1.7.3 Glucose neuroprotection of the retina

In the retina, providing glucose to retinal neurons affords a robust neuroprotective effect in

the presence of ischemia or hypoxia.62

In retinal cultures, Wood et al. demonstrated a dose-

dependent protective effect of glucose against mitochondrial inhibition of retinal neurons.156,

157 In vivo, Casson et al. then showed that elevating vitreal glucose levels, either by short-

term diabetes, or a systemic bolus of glucose, or intraocular glucose delivery produced a

remarkable degree of protection against acute high-intraocular pressure-induced ischaemic

retinal injury or experimental glaucoma.3, 62, 149

Others have shown that chronically

hypoglycaemic transgenic mice develop retinal degeneration158

and that regular

administration of high dietary glucose can rescue retinal structure and function in this

model.159

Interestingly, these results are consistent with a previous study in 1993 showing

that a traditional Chinese herb Honghua (rich in high glucose) protected the retina from

experimental ischaimia.160

In contrast, hypoglycaemia exacerbates experimental ischaemic

21

retinal injury.148

This finding is consistent with the well-described clinical situation of

increased ischaemic retinal features in diabetic patients with rapidly reducing blood glucose

levels. Casson et al. also showed that intraocular lactate delivery was not protective62

and that

inhibition of lactate transport did not exacerbate ischaemic retinal injury in vivo. All together,

these findings indicate that glucose and not lactate is responsible for the protective effect. In a

first-to-man, double blind randomized trial, Casson et al. recently showed ocular glucose

delivery temporarily recovered contrast sensitivity and visual acuity in patients with severe

primary open-angle glaucoma.151

Although a rapid pathway from basic bioenergetic research to clinical translation has been

developed, the current limitation of this potentially transformative research programme is that

the mechanism by which high concentration of glucose protects retina from ischemia remains

poorly understood.161

1.7.4 Possible mechanisms by which glucose protects the retina

1.7.4.1 Role of glycolysis-derived ATP underlying glucose neuroprotection in the retina

The significant role of glycolysis with regard to retinal energy metabolism in the vertebrate

animal was first shown by Noell in 1951.73

In 2003, Winkler and his research team showed

that the retina can increase glycolytic ATP production in the presence of reduced oxygen

tension: the Pasteur effect.162

When O2 is unavailable, more than 50% of the glucose is

metabolized via anaerobic glycolysis and then converted to lactate.19, 65

When mitochondrial

function is inhibited, 25mM glucose maintain 70% of normal ATP levels in rat retinal

cultures.157

Moreover, unlike the CNS, the mammalian retina tends to generate energy from

glycolysis even in the presence of normal oxygen tension: the Warburg effect.25

1.7.4.2 Does the pentose phosphate pathway play a role?

Apart from the important role that glucose plays in cellular energy production, glucose is also

the primary substrate for the PPP. Parallel to glycolysis, the PPP is a biochemical pathway

22

involving oxidation of glucose. The PPP acts as a key pathway for the manufacture of the

cytosolic cellular reducing equivalent, NADP(H),163, 164

which assists neurons to combat

oxidative stress.164, 29

There is evidence that the reducing power generated from the PPP can

completely prevent ROS-induced neuronal apoptosis.163, 165

NADPH is also involved in the

recycling of photopigments in photoreceptors through the PPP.166, 167

And its precursor,

nicotinamide has been shown to rescue neurons from acute ischemia injury in a rat model.168

In rat retinal cultures, inhibition of the PPP by the administration of the 6-phosphogluconate

dehydrogenase inhibitor, 6-AN, reduced the protective effect of glucose against rotenone-

induced retinal cell toxicity.157

However, previous research indicated that ATP production

from glycolysis rather than PPP-derived NADPH is the most important neuroprotective

mechanism by which glucose protects the intact retina from ischaemia. Winkler et al.169

showed that the portion of total glucose metabolised via the PPP does not increase

significantly in the isolated retina when glucose was elevated from 5mM to 30mM. The

protective effect of the PPP may be dependent on the experimental model, in particular the

amount of ROS-induced injury involved.170

23

1.8. Conclusion

It is clear that an understanding of retinal energy metabolism is far from complete. Local

oxygen tension, blood supply, and glucose delivery are key factors contributing to retinal

energy metabolism. Glucose predominantly provides ATP via glycolysis even in the presence

of normal oxygen tension (Warburg effect). Under ischemic conditions, the retina upregulates

glycolysis and maintains ATP generation, displaying the Pasteur effect. Glycolytic

intermediates may be required for the biosynthetic demands of photoreceptor turnover. Also,

mitochondrial function and its derived energy source are also important for the retinal

neurotransmission and phototransduction. Glucose entry into the PPP is important to maintain

cellular reducing power. Finally, the finding that ocular glucose delivery recovers visual

psychophysical parameters in individuals with severe glaucoma provides information about

the pathogenesis and evidence for the existence of “sick” neurones. Although delivery of

glucose may not be a viable long-term therapeutic approach, a bioenergetic strategy in the

treatment of retinal diseases where energy failure is part of the pathogenesis warrants further

study in the laboratory with potential clinical translation.

24

25

2. Mechanisms of neuroprotection by glucose in rat retinal cell cultures

subjected to respiratory inhibition

26

A Han, G., Wood, J.P.M., Chidlow, G., Mammone, T. & Casson, R.J. (2013) Mechanisms of neuroprotection by glucose in rat retinal cell cultures subjected to respiratory inhibition. Investigative Ophthalmology and Visual Science, v. 54(12), pp. 7567-7577

NOTE:

This publication is included on pages 26-39 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://doi.org/10.1167/iovs.13-12200

40

41

3. Rotenone induced calpain-µ activation in neurons and induction of ER

stress and GSK3β in glial cells

The previous paper proved that the rotenone model represents a valid in vitro system to

experimentally mimic retinal cell mitochondrial dysfunction, as may occur in glaucoma or

LHON. In brief, it has been proven that at the concentration employed, rotenone is associated

with the generation of ROS and the depletion of ATP in rat retinal cell cultures. These

outcomes do not provide information on which cell death and signalling pathways are

specific to each cell type, however. In addition, it was determined that both apoptotic and

non-apoptotic modes of neuronal demise occur in this system. It was therefore thought to be

of interest and relevance to the present thesis to investigate mechanisms of induction of cell

death by rotenone in these cultures in more detail.

ER stress is clearly involved in a number of optic nerve pathologies, reflecting perturbations

in cellular homeostasis and alterations in calcium dynamics.171

In a chronic rodent glaucoma

model, ER stress was observed in the early phases of injury and was associated with RGCs

undergoing apoptosis.172, 173

Furthermore, it was shown that induction of the ER stress

marker, CHOP, ultimately heralds cell death in susceptible cells174

It is also known that ER

stress is highly reliant on cellular ATP levels since its primary purpose is to correct

incidences of protein misfolding and this is an active, energy-dependent process.175

It is

therefore interesting to ask whether in our in vitro model of retinal mitochondrial

dysfunction, there is a role for ER stress in the mediation of cell death, and whether this

process has a different role to play in glia and neurons, depending on how these cells use

glucose to derive their ATP. In addition, the role of the associated intracellular mediators

calpain-µ and GSK3β, was also investigated in vitro.

A Han, G., Casson, R.J., Chidlow, G. & Wood, J.P.M. (2014) The mitochondrial complex I inhibitor rotenone induces endoplasmic reticulum stress and activation of GSK-3B in cultured rat retinal cells. Investigative Ophthalmology and Visual Science, v. 55(9), pp. 5616-5628

NOTE:

This publication is included on pages 42-63 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://doi.org/10.1167/iovs.14-14371

64

65

4. Emerging aspects of glucose metabolism in the retina: The expression of

pyruvate kinase in rodent retina

This thesis has examined aspects of glucose energy metabolism in the retina and the

possibility of clinically exploiting the retina’s unusual metabolism in disease states where

energy failure is part of the problem. Over 90 years ago, Warburg noted that the glycolytic

metabolism of the isolated retina was similar to that of some cancers.76

This aspect of retinal

physiology has received relatively little attention. In the third section of the thesis, we

describe some background to the concept that the retina has a cancer-like metabolism and

present novel data indicating the presence of glycolytic enzymes generally associated with

cancerous and proliferating tissue, specifically pyruvate kinase M2.

Expression of Pyruvate Kinase (PKM) in the rodent retina

Guoge Han1, 2

, John PM Wood1, 2

, Glyn Chidlow1, 2

, Robert J Casson, 1,2

*

1Ophthalmic Research Laboratories, South Australian Institute of Ophthalmology,

Hanson Institute Centre for Neurological Diseases, Frome Rd, Adelaide, SA 5000,

Australia

2Department of Ophthalmology, University of Adelaide, Frome Rd, Adelaide, SA

5000, Australia

*Address correspondence to: casson.robert@gmail.com

Address: Ophthalmic Research Laboratories, Hanson Centre for Neurological

Diseases, Frome Rd, Adelaide, SA 5000, Australia

66

Abstract

PURPOSE. Pyruvate kinase, which is a rate-limiting enzyme in glycolysis, has

emerged as a key regulator of cancer metabolism. The relatively inactive isozyme

PKM2 is an important mediator of the Warburg effect and the “metabolic budget

system” in tumours. To our knowledge, the localization of pyruvate kinase isozymes

in the retina has never been reported. To better understand retinal metabolism,

particularly its similarity to cancer, we studied the distribution pattern of PKM1 and

PKM2 in the rodent retina and compared this to the distribution in brain.

METHODS. Protein expression of PKM1 and PKM2 in retinal, brain, and cancerous

tissues was determined using Western blotting and the spatial localization in the retina

and brain was determined using immunohistochemistry. Optimization of

commercially available antibodies to PKM1 and PKM2 was performed.

RESULTS. PKM2 was expressed in both mouse and rat retina tissue, and was

immunolocalized to the photoreceptors, particularly the inner segments. This finding

was supported by the results from rat retinal cultures. In addition, PKM1 was present

in the ganglion cell layer and inner nuclear layer in the mouse retina. Currently

available PKM1 antibodies are not specific to PKM1 in the rat.

CONCLUSION. The present data provide the basis for future studies that can

explore the roles that pyruvate kinase may play in retinal metabolism at the

mechanistic level.

Abbreviations: RRC, Rat retinal cells; ATP, adenosine triphosphate; PKM pyruvate

kinase; OXPHOS, oxidative phosphorylation; PEP, phosphoenolpyruvate.

67

Introduction

Cellular adenosine triphosphate (ATP) is produced by two related processes:

oxidative phosphorylation (OXPHOS) and cytoplasmic glycolysis. The final step of

the glycolytic pathway requires pyruvate kinase (PK), to catalyze the conversion of

phosphoenolpyruvate (PEP) to pyruvate, which then enters the Krebs cycle.1

The mammalian retina, unlike other parts of the central nervous system, has a

propensity for “aerobic glycolysis” whereby pyruvate is converted to lactate via

glycolysis, despite the presence of sufficient oxygen.2, 3

This metabolic pattern is

similar to many tumors and has been termed the Warburg effect.4 In recent years,

cancer researchers have recognized the importance of the enzyme pyruvate kinase

(PK) as a key mediator of the Warburg effect in cancer.5

PK exists in mammalian cells as four isoforms, which are coded by PKLR and PKM

genes. The expression of R and L forms of PK are controlled by an upstream

promoter of PKLR either containing CAAT or TATA in red blood cells and liver

tissue respectively.6 The M1 and M2 isoforms of PK are alternatively spliced forms of

the PKM gene, distinguished by the presence of exclusively exon 9 or exon 10 in each

forms.7 In fact, it has been estimated that PKM2 is the predominate isoform of PK in

tumor tissue: this hypothesis was supported by a series of immunohistochemistry

studies.8-10

Subsequently, Christofk et al.11

also reported that the switch from

expression of PKM1 to PKM2 in cancer cells is responsible for the Warburg effect.

Although this notion was overturned by the finding that PKM2 was expressed in

normal tissue and tumors, the up-regulation of this isoform has been proved to

68

associated with aerobic glycolysis.12

Furthermore, PKM2 exists in two isoforms: an active tetrameric form and a relatively

inactive dimeric form. When in the inactive state, glycolytic intermediates upstream

of PEP accumulate and are utilized for biosynthesis. When biosynthetic building

blocks are excessive and energy levels fall, PKM2 switches to its active tetrameric

form re-establishing glycolytic flow. This feedback mechanism providing the cell

with an opportunity to regulate biosynthesis and energy production is known as the

“metabolic budget system” and is an important part of cancer metabolism (Fig. 1). It

is closely linked with the Warburg effect. To our knowledge, its presence in a

non-proliferating tissue has never been reported, but the presence of PKM2 would be

strong circumstantial evidence for its existence. In addition, to the best of our

knowledge, pyruvate kinase and its possible regulation of the Warburg effect has not

been fully understood in the rodent retina. Therefore, the present study sought to

investigate the expression of the two PKM isoforms and their localization patterns in

vivo and in vitro rat retina, and compare the findings in the retina with those in the

brain.

69

Material and Methods

Tissue preparation

C57BL/6 mice and Sprague-Dawley (SD) rats were obtained from University of

Adelaide and the experimental procedures were conducted in accordance with the

Animal Ethics Committees of SA Pathology and conformed to the Australian Code of

Practice for the Care and Use of Animals for Scientific Purposes, 7th edition, 2004 as

well as the ARVO Statement for the Use of Animals in Ophthalmic and Vision

Research. Briefly, rats were killed under deep anesthesia by trans-cardiac perfusion

with 0.9%NaCl to exsanguination. The eyes were then enucleated and immediately

immersion-fixed in fresh 10% (w/v) neutral buffered formalin overnight or in

Davidson’s solution (2 parts 37% formaldehyde, 3 parts 100% ethanol, 1 part glacial

acetic acid and 3 parts water) for 24 h followed by 70% ethanol processing. In some

cases, mice and rat liver, brain cortex and muscle samples were also prepared (for

Western immunoblot or immunohistochemistry). Tissue sections of tumor tissue,

which served as positive controls in this study, were obtained from the Surgical

Diagnostic facility of SA Pathology (Adelaide, Australia).

Immunohistochemistry

Immunohistochemistry and fluorescence labeling were performed as previously

described.13

The eyes were hand-processed briefly according to the following steps:

70% ethanol (30 min), 3×100% ethanol (30 min), 2×xylene (30 min), 50%

xylene/50% wax (30 min) at 62°C, 2 × waxes (30 min) at 62°C. After cutting by using

a rotary microtome (4µm section), the retina section were deparaffinised and was

70

blocked with H2O2. Subsequently, antigen retrieval was performed by microwaving

sections in 1 mM EDTA (pH 8.0) and non-specific binding was blocked with PBS

comprising 3% normal horse serum (PBS-HS). After that, sections were incubated at

room temperature overnight in primary antibody (diluted in PBS-HS), followed by

incubations with biotinylated secondary antibody for 30min(Vector, Burlingame, CA)

and streptavidin-peroxidase conjugate (Pierce, Rockford, IL) for 1h. Primary

antibodies used in immunohistochemsitry are described in Table 1. Color

development was performed by using NovaRed substrate kit (Vector, Burlingame, CA)

for less than 3 min. Sections were counterstained with hematoxylin, dehydrated,

cleared in histolene and mounted on glass slides.

For double labelling fluorescent immunohistochemistry cases, one antigen was

stained by a 3-step procedure (primary antibody, biotinylated secondary antibody,

streptavidin-conjugated AlexaFluor 594 (Invitrogen 1:500 for 1h), whilst the second

antigen was labelled by a 2-step procedure (primary antibody, secondary antibody

conjugated to AlexaFluor 488 (Invitrogen 1:250 for 1h), according previously

described methods.14

Finally, the sections were then mounted using anti-fade

mounting medium and observed under a confocal fluorescence microscope (Olympus,

Mount Waverly, Australia).

Rat retinal cell cultures

Rat retinal cell cultures comprising both neurons and glia were prepared using a

trypsin-mechanical digest procedure as previously described.15, 16

Briefly, retinas were

enucleated from 1-2 day old rat pups obtained from Adelaide University and

71

incubated in physiological buffer (Solution 1; 120mM NaCl, 5.4mM KCl, 24mM

NaHCO3, 0.1mM NaH2PO4, 3 g/L BSA, 20mM Glucose, and 0.15mM MgSO4, 28µM

phenol red) containing 0.1mg/ml trypsin (Sigma Aldrich, Castle Hill, NSW,

Australia), at 37°C for 8 minutes. After the reaction was stopped, cells were

resuspended in minimal essential medium (MEM) containing 10% fetal bovine serum

(FBS), 10 mg/ml gentamicin sulfate, 200µM glutamine and 25mM glucose and

applied to 13mm diameter borosilicate glass coverlips (immunocytochemistry), 6-well

plates (Western blot) or 12-well plates (ATP assay and ROS determination), all of

which had previously been coated with 10µg/ml poly-D-lysine, for 2h. Cells were

maintained in saturating humidity with 5% CO2 at 37°C and were used 7 days after

culture.

Cell culture media and fetal bovine serum (FBS) were purchased from Invitrogen

(Mulgrave, Victoria, Australia). Unless stated, all other chemical reagents were from

Sigma-Aldrich Chemical Company (Castle Hill, New South Wales, Australia).

Antibodies used for Western blotting and immunocytochemsitry are described in

Table 1. Culture plates and plasticware were from Sarstedt Pty (Adelaide, Australia).

Some rat retinal cultures were maintained for up to 28 days with medium being

changed every 2 days. Comparing with 7 day mixed rat retinal cell,these cultures were

identified to comprise predominantly Müller glial cells as shown previously.15

Their

use in the present study was to identify the expression of PKM in retinal Müller cell.

Immunocytochemistry

Cells were fixed with neutral-buffered formalin for 15 minutes, washed in

72

phosphate-buffered saline (PBS; 137 mM NaCl, 5.4 mM KCl, 1.28 mM NaH2PO4, 7

mM Na2HPO4; pH 7.4) and then permeabilised with PBS containing 0.1% Triton

X-100 (PBS-T). Preparations then underwent blocking in normal horse serum (NHS;

3.3 % v/v in PBS; PBS-HS) and were subsequently labeled with a range of neuron-

and glia-specific antibodies, diluted in PBS-HS, at 4°C over-night. Labelling was

visualized by consecutive incubations with appropriate biotinylated second antibodies

(Vector Laboratories, Abacus ALS, Brisbane, Australia; 1:250 in PBS-HS; 30 minutes)

and streptavidin-AlexaFluor 488 or streptavidin-AlexaFluor 594 (Invitrogen,

Mulgrave, Victoria, Australia; 1:500 in PBS-HS; 1 hour); nuclei were counterstained

using 4’,6-diamidino-2-phenylindole (DAPI; 500ng/ml in PBS; 5 minutes). Finally,

cells on coverslips were mounted using anti-fade mounting medium (DAKO, Botany,

New South Wales, Australia) and examined under a confocal fluorescence microscope

(Olympus, Mount Waverly, Australia).

Western blotting

Cells were initially harvested from plates by scraping into PBS; cell samples or tissue

were then sonicated in homogenisation buffer (20 mM Tris-HCl, pH 7.4; containing 2

mM EDTA, 0.5 mM EGTA, 1 mM dithiothreitol, 50 µg/mL leupeptin, 50 µg/mL

pepstatin A, 50 µg/ mL aprotinin and 0.1 mM phenylmethylsulfonyl fluoride). An

equal volume of sample buffer (62.5 mM Tris-HCl, pH 7.4, containing 4% w/v SDS,

10% v/v glycerol, 10% v/v -mercaptoethanol and 0.002% w/v bromophenol blue)

was added and samples were heated to 70°C for 3 minutes. Electrophoresis was

performed as reported previously 17

using 10% containing 0.1% (w/v) SDS. Proteins

73

were transferred to PVDF membrane and blots subsequently labelled with antibodies

diluted as described in Table 1. Membrane labeling was carried out with a two-step

procedure using appropriate biotinylated secondary antibodies (Vector Laboratories;

1:500; 30 minutes) followed by a streptavidin-peroxidase conjugate (Pierce, Rockford,

Il, USA) reaction as described previously18

. -actin was assessed in cell extracts as a

positive control and quantification was performed using Adobe Photoshop (version

CS2).

74

Results

PKM1

Specificity of the PKM1 antibody

To evaluate the specificity of the PKM1 antibodies, three distinct commercially

available antibodies of PKM1 (CST, Novus, and Proteintech, respectively) were

obtained. All antibody information is summarised in Table 1.

Consistent with previous studies, PKM1 was expressed in the brain, and skeletal

muscle.19

Both the CST and Proteintech antibody detected a clear single band at a

MW of 60kDa in murine tissue, whereas none of the antibodies produced a

satisfactory signal to background ratio in rat tissue, over a range of dilutions (Fig.2).

Alpha-tublin was selected as the house-keeping control protein and IgG1 antibody

was selected as a negative control for this experiment (data not shown).

Immunohistochemistry experiments were performed to analyze the specificity and the

quality of the antibodies. Mouse muscle and cortex brain tissue were also selected as a

positive control (Fig.3). The PKM1 antibodies from Proteintech and CST identified

the cortical neurons in mouse brain tissue and was visualized ubiquitously in the

mouse muscle tissue, with a satisfactory signal to background ratio. In contrast, in rat

tissue samples, the CST antibody failed to detect any proteins at the correct molecular

mass. The antibody from Novus produced a weak or non-specific signal in terms of

the immunolabelling pattern in the rat tissue. Evaluation of antibodies on different

tissue samples is summarized in Table 2.

75

PKM1 expression in the rodent retina

The anti-PKM1 antibodies from Proteintech and CST were able to distinguish protein

at the predicted band around MW of 60kDa in the mouse retinal tissue (Fig.2). When

incubated with rat tissue extracts, these antibodies yielded weaker patterns of

immunoblotting. Further investigation by immunohistochemistry showed

immunoreactivity within the ganglion cell layer and inner nuclear layer in mouse

retina (Fig.3). In contrast, in rat retinal sections, no specific PKM1 immunolabelling

could be observed. Similarly, in the 7-day rat retinal cell cultures, comprising both

neurons and glial cells, PKM1 revealed no positive staining in any sample.

PKM2

Specificity of the PKM2 antibody

In order to verify the specificity of the PKM2 antibodies, two commercially available

antibodies of PKM2 were obtained: from CST and another one from Novus

Western Blotting analysis has demonstrated that the M2 splice form of PK is

exclusively expressed in human cancer cell lines derived from different tissue (A549,

H1299, 293T, HeLa, MCF10a).11

As a consequence, HeLa cell lines served as a

positive control in the present immunoblotting study. Both the two antibodies from

CST detected a single band at a MW of 60kDa in mouse retina (Fig.2). Moreover, in

mouse brain cortex and liver samples a protein with a mass of 60 kD was detected,

but this band was weakly distinguished in muscle protein specimens. In agreement

with these findings, when analyzing the rat retina samples, a protein with a mass of 60

kDa was also detected. Of particular interest, the ratio of PKM1/PKM2 determined by

76

immunoblotting in brain tissue was considerably higher than that in the retinal tissue.

In mouse and rat cortical brain section, PKM2 was expressed in the astrocytes and

blood vessels, but not neurons (Fig.3).

PKM2 expression in the rat retina

The localization of PKM2 in mouse and rat eyes was investigated by

immunohistochemistry. In C57BL/6 mice and adult SD rats, PKM2 immunostaining

was observed in the photoreceptor inner segments layer with both of the antibodies.

The double labeling of PKM2 and RET-P1 (rhodopsin) also showed that the

photoreceptor cells were strongly PKM2 labeled (Fig.4).

Consistent with the results in vivo, immunocytochemical analysis demonstrated

PKM2 was again expressed in RET-P1 (rhodopsin) positive neurons from 7-day rat

retinal cultures (Fig.5). Both of antibodies resulted in cytoplasmic staining, consistent

with the known subcellular localization of PKM2. In addition, both 7-day and 28-day

rat retinal cultures showed weak PKM2 signal throughout the cells with rabbit cell

signal antibody.

77

Discussion

Energy compromise is definitely or probably part of the pathogenesis of a number of

common eye diseases, including retinal vascular disease, diabetic retinopathy,

glaucoma15

and dry age-related macular degeneration (ARMD).20

Therefore, a better

understanding of the retinal energy metabolism is of great importance. Previous

researchers have shown that, although the retina is an out-pouching of the brain, its

energy metabolism is characterized by “aerobic glycolysis”, more akin to that of a

cancer.21

This phenomenon has been named “the Warburg effect”. However, the

mechanism in the retina remains unknown.

Previous research on the Warburg effect, has shown that the M2 splice variant of PK

is involved in this mechanism due to its important role in regulating glycolysis.22, 23

However, to best of our knowledge, even the basic question that how the pyruvate

kinase distributed and located in the normal and cancer tissue, seems still

controversial. This situation, firstly, resulted from the inappropriately validation of the

specificity of the PKM2 antibody, which could lead to misleading in the publication.

Secondly, this also strongly depends on the specific method employed by the

researchers, as well as the tissue preparation. In the present study, we firstly

undertook a series of experiments which tested and optimized the commercially

available mouse and rat antibodies for PKM1 and PKM2. Important results of the

evaluation of the specificity of the each antibody are summarized as followed: (1)

Western blot analysis showing both that some of the commercially available

antibodies bind specifically to a correctly sized antigen and such a band is also

78

observed in positive control tissue; IgG was used as a negative control in this

immunoblotting system. (2) Antibodies that demonstrated clear and distinct bands at

the expected molecular weights on immunoblotting also produced satisfactory

immunostaining (3) Reassuring lack of signal in negative control tissues (4)

Immunocytochemistry results supported the findings in vivo. (5) PKM1 antibodies

employed in this study had more affinity with mouse than rat tissue, despite the

commercial specification claiming application in the rat. On checking the amino acid

sequence of the PKM1 protein in rats and mice, we noted that the mouse demonstrates

considerably more homology with the human protein than does the rat. There are

differences between the rat and mouse PKM1 protein that presumably account for the

poor affinity of the currently available PKM1 rat antibodies.

Some cells use different isoforms of PK to regulate their metabolic pathways,

according to its need.24

PKM1 is constitutively active; it catalyzes the reaction of PEP

into pyruvate and then enters TCA cycle for generating energy, with a higher

consumption of oxygen and lower production of lactate.25

Conversely, when PKM2 is

in its relatively less active (monomer/dimeric) form, it drives the conversion into

glycolysis, contributed to the Warburg effect.1, 25

In addition, in proliferating cells,

due to the low activity of the enzyme, all glycolytic intermediates preceding PK can

accumulate and become available as precursors for biomass synthesis (such as:

nucleotides, amino acids and lipids).1 This could provide proliferating tissue with a

powerful metabolic strategy known as the “metabolic budget system”.26

Therefore, it

has been proposed that the PKM gene coded protein is a key mediator of the Warburg

79

effect in cancer tissue.5, 27, 28

However, this has not been investigated in a

non-proliferating tissue, such as the retina.

In this study, we investigated PKM2 expression in mouse and rat retina. Consistent

with previous studies on the distribution of other glycolytic enzymes (eg. G3PD, LDH)

in the retina,13

PKM2 was also located in rhodopsin positive cells in cultures, and in

the photoreceptor layer (highly concentrated in rod inner segments). Each mammalian

rod outer segment (ROS) consists of a stack of ~1500 distinct discs enclosed by the

plasma membrane. Approximately 60% of the dry weight of the disc membrane is

protein, and opsin comprises 90% of the protein content. Hence, rhodopsin, which is a

G protein-coupled receptor, forms a large structural component of the rod disc

membrane. Young showed that rhodopsin was constantly renewed as the disc

membranes moved in a scleral direction along the ROS towards the retinal pigment

epithelium. Interestingly, the rhodopsin turnover parallels the degree of aerobic

glycolysis found in different species.29

Furthermore, the relatively low rate of

photoreceptor turnover in lower vertebrates is temperature dependent, increasing at

higher temperatures,29

reflecting the temperature-dependent Warburg effect.30

Hence,

we proposed that the mammalian retina shares similar biosynthesis requirements to

neoplastic tissue due to the prodigious turnover of the opsin protein in the disc

membranes of the outer segments.31

And rhodopsin turnover drives aerobic glycolysis

mediated by PKM2.

Double labeling the 7-day retinal and 28-day Müller cell cultures with the rabbit

PKM2 antibody (Cell Signaling) also resulted in weak signals, but the staining

80

seemed to be throughout the cells,, which corresponded with reports that PKM2 is

present in both in the cytoplasm and the nucleus. Retinal Müller cells, which have the

potential capacity of re-entering a mitotic cell cycle and proliferating,14

display

marked aerobic glycolysis and lactate production. In cancer cells, it has been reported

that monomeric PKM2 can translocate into the nucleus, where it upregulates the

expression of c-Myc and cyclin D1, thereby promoting the Warburg effect and cell

cycle progression.22

However, only a few Müller cells re-enter the mitotic cycle in

certain pathological conditions.32

The possible explanation for the ubiquitous

expression of PKM2 within untreated Müller cells could be: (1) the preparation called

“28-day cultures”, in which the majority of the cells are Müller cells, was obtained

from neonatal SD rat pups, which have differentiation and proliferation potential, i. e.

they are not representative of adult Müller cells in vivo;33

(2) the frequent medium

change could be a potential insult to the Müller cells; (3) PKM2 requires other

promoter or enzyme in regulating the Warburg effect in these cells.23, 32

Regarding PKM1, of the three commercially available antibodies tested in this study,

two provided satisfactory results: specific bands were at the expected protein mass

detected in Western blotting and a strong signal was present in control tissue (mouse

skeletal muscle). Subsequently we tested this antibody on rat brain sections and

retinal tissue. This antibody revealed strong immunolabelling with cortical neurons in

the mouse and rat brain sections, which is consistent with the preferred metabolic

pattern in these cells. This antibody appears to localize to cells that prefer oxidative

phosphorylation. In the retina sections, PKM1 was localized in the ganglion cell layer,

81

inner plexiform layer and inner nuclear layer: the inner rodent retina displayed an

immunostaining patern similar to the brain, whilst the outer retina displayed a staining

pattern more similar to a proliferating tissue. Given the specific distribution of the M1

form of PK within the retina, we hypothesized that the M1 positive cells may

preferentially metabolize glucose by oxidative phosphorylation rather than rely on

aerobic glycolysis.

In conclusion, we have demonstrated the spatial localization of PKM isozymes in the

rodent retina, and have optimized the immunostaining. We have shown that the

currently available PKM1 antibodies for use on the rat are not satisfactory, probably

due to the lack of homology between the rat and the mouse PKM1 protein. The PKM2

immunoreactivity was displayed in the rod photoreceptors in vivo and in rhodopsin

positive cells in vitro, while PKM1 is expressed in the ganglion cell layer, inner

plexiform layer and inner nuclear layer in the mouse retina. This information provides

a basis for investigating the mechanism by which PKM2 contributes to the Warburg

effect in mammalian retina.

82

Acknowledgements

This work was supported by grants from the NHMRC (565202, 626964). Guoge Han

receives financial support from Council Scholarship of China (CSC: 2010627027).

We also thank Ms.Teresa Mammone and Mr. Mark Daymon for their kindness and

skilled technical assistance. The authors declare no other potential conflicts of

interest.

83

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pyruvate kinase is important for cancer metabolism and tumour growth. Nature

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12.Bluemlein K, Gruning NM, Feichtinger RG, et al. No evidence for a shift in

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15.Flammer J, Mozaffarieh M. What is the present pathogenetic concept of

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16.Wood JP, Chidlow G, Graham M, Osborne NN. Energy substrate requirements of

rat retinal pigmented epithelial cells in culture: relative importance of glucose, amino

acids, and monocarboxylates. Invest Ophthalmol Vis Sci 2004;45(4):1272-80.

17.Wood JP, Mammone T, Chidlow G, et al. Mitochondrial inhibition in rat retinal

cell cultures as a model of metabolic compromise: mechanisms of injury and

neuroprotection. Invest Ophthalmol Vis Sci 2012;53(8):4897-909.

18.McAlpine CS, Bowes AJ, Khan MI, et al. Endoplasmic reticulum stress and

glycogen synthase kinase-3beta activation in apolipoprotein E-deficient mouse

models of accelerated atherosclerosis. Arteriosclerosis, thrombosis, and vascular

biology 2012;32(1):82-91.

19.Yang P, Li Z, Fu R, et al. Pyruvate kinase M2 facilitates colon cancer cell

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20.Feigl B. Age-related maculopathy - linking aetiology and pathophysiological

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21.Holman MC, Chidlow G, Wood JP, Casson RJ. The effect of hyperglycemia on

hypoperfusion-induced injury. Invest Ophthalmol Vis Sci 2010;51(4):2197-207.

22.Yang W, Lu Z. Nuclear PKM2 regulates the Warburg effect. Cell Cycle

2013;12(19):3154-8.

23.Yang W, Xia Y, Ji H, et al. Nuclear PKM2 regulates beta-catenin transactivation

upon EGFR activation. Nature 2011;480(7375):118-22.

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24.Sun Q, Chen X, Ma J, et al. Mammalian target of rapamycin up-regulation of

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25.Anastasiou D, Yu Y, Israelsen WJ, et al. Pyruvate kinase M2 activators promote

tetramer formation and suppress tumorigenesis. Nat Chem Biol 2012;8(10):839-47.

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in Muller glial proliferation in mouse retina. Glia 2012;60(10):1579-89.

33.Germer A, Kuhnel K, Grosche J, et al. Development of the neonatal rabbit retina in

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86

Figure 1: Metabolic Budget System & the Warburg Effect in the Retina

Fig. 1 Metabolic budget system and the Warburg effect. PKM2 exists as an active

tetrameric form and an inactive dimeric form. It’s transition is controlled by the

glycolytic intermediate, fructose 1,6 biphosphate. It is a co-activator of HIF-1α. We

propose that glucose-derived amino acids are required for rhodopsin synthesis, and

that the Warburg effect is HIF-dependent. PPP = pentose phosphate pathway; PEP =

phosphoenolpyruvate; PKM2 = pyruvate kinase M2; LDHA = lactate dehydrogenase

A; PDH = pyruvate dehydrogenase; PDK1 = pyruvate dehydrogenase kinase 1; PSPT

= phosphoserine phosphatase

87

Figure 2: Evaluation of PKM1 and PKM2 antibodies in C57BL/6 mice and

Sprague-Dawley (SD) rats by western blotting.

88

PKM1 and PKM2 immunoblotting using various commercially available antibodies

on cortex, liver, skeletal muscle and retina samples in (A) C57BL/6 mice and (B)

Sprague Dawley rats. Labelling for α-tublin is shown as control group. Two

antibodies from CST detected a single expected band at MW of 60kDa in mouse and

rat retina. In rat retina preparations, no specific PKM1 immunolabelling could be

observed.

89

Figure 3: Representative images of PKM1 and PKM2 immunolabelling in C57bl/6.

mice

Formalin-fixed, paraffin-embedded sections of mouse cortical brain and retina,

immunostained with PKM1(#7067) and PKM2 (#4053) antibodies. In the retina,

PKM2 was expressed in the photoreceptors, particularly the photoreceptor inner

segments. PKM1 was present in the ganglion cell layer and inner nuclear layer in the

mouse retina(C-D). In the cortical brain, PKM2 was expressed in the astrocytes and

blood vessels, but not neurons, whereas PKM1 was located in the cortical neurons

(A-B). Scale bar = 30 mm

90

Figure 4: Immunohistochemical localization of PKM2 in the SD rat. Double labelling

immunofluorescence of vimentin (A–D) and rhodopsin (E–H) with PKM2 using CST

(#3198). In rat retina. PKM2 was evident in the photoreceptors, particularly the inner

segments (arrow). Scale bar = 25μm.

91

Figure 4. Immunocytochemical localization of PKM2 (#3198) in rat retinal cultures.

Double labelling immunofluorescence of rhodopsin (A–D) and vimentin (E–H) with

PKM2 CST (#3198). PKM2 was particularly evident in rhodopsin-positive cells.

Scale bar = 25μm.

92

Table 1 Antibodies used in this research

ICC, immunocytochemistry; IHC, immunohistochemistry;WB, Western blotting; CST,Cell Signaling Technology.

Antibody Name(source) Host Cat No. Dilution(IHC) Dilution(WB) Dilution(ICC)

PKM1

CST Rabbit 7067 1:1200 1:1000 1:1000

Proteintech Rabbit 15821-1-AP 1:3600 1:1000 1:500

Novus Rabbit NBP2-14833 1:10,000 1:1000 1:500

PKM2

CST Rabbit 4053 1:2400 1:1000 1:1000

CST

Novus

Rabbit

Rabbit

3198

NBP2-19852

1:3600

1:3600

1:1000

1:1000

1:1000

--

α-tublin Biogen

Mouse

4583

--

1:10000

--

β-actin Sigma Mouse 030M4788 -- 1:10,000 --

93

Table 2. Antibodies evaluation in this research. Evaluation of PKM1 and PKM2 antibodies in different preparation(mice, rat and rat retinal cells)

were summarized in Table 2. (+):satisfactory result.(-): not working properly. (±) needed to be further identified. ICC, immunocytochemistry;

IHC, immunohistochemistry;WB, Western blotting.

C57BL/6 mice retina SD rat retina Rat retinal cells

Antibody Name(source) WB IHC WB IHC ICC

PKM1

CST ± + - - ±

Proteintech ± + - - -

Novus - - - - -

PKM2

CST + + + + +

CST

Novus

+

-

+

-

+

-

+

-

+

-

94

95

96

97

5. Glucose-Induced Temporary Visual Recovery in Primary Open-Angle

Glaucoma

The current body of work focusses mainly on neuroprotection for glaucoma. Currently, the

only effective treatment therapy for Primary Open-Angle Glaucoma (POAG) is to lower the

intraocular pressure. Although this strategy is successful in some individuals with POAG,

some continue to progress despite optimal IOP management. This suggests that IOP

reduction alone is an insufficient clinical strategy in some individuals. Hence, alternative

neuroprotective strategies are highly clinically desirable.

Previous studies from our group have demonstrated that an increased vitreous glucose

concentration protects the retina from acute and chronic retinal ischemic injury and

experimental glaucoma. We aimed subsequently to proceed to a human trial using topical

glucose as a neurorecovery agent in glaucoma patients.

NOTE:

This publication is included on pages 98-106 in the print copy of the thesis held in the University of Adelaide Library.

A Casson, R.J., Han, G., Ebneter, A., Chidlow, G., Glihotra, J., Newland, H. & Wood, J.P.M. (2014) Glucose-induced temporary visual recovery in primary open-angle glaucoma: A double-blind, randomized study. Ophthalmology, v. 121(6), pp. 1203-1211

ne

6/j

It is also available onli

http://doi.org/10.101

to authorised users at:

.ophtha.2013.12.011

107

6. Conclusions and future research directions

6.1 General conclusions and relationship of the thesis to the literature

This thesis provides valuable additional information to what is known about retinal

metabolism and the role of glucose in this process. In brief, the work presented here

demonstrates the central viability-sustaining roles that glucose plays in the retina via

glycolytic ATP generation, pentose phosphate pathway-derived reducing equivalent

production and prevention of ER stress and the unfolded protein response. Furthermore, a

“proof-of-principle” means to translate the concept of bioenergetic-based neuroprotection to

the clinic was demonstrated.

Previous studies have shown that providing glucose to the retina in vivo affords a robust

protective effect to neurons in an acute model of retinal ischemia.3 The first paper derived

from this thesis addressed the question of the mechanisms of glucose protection to the retina

against energy depletion and oxidative stress.157

Some of the key mechanistic findings were

also validated in adult rat retinal explants, as shown in the relevant supplemental figures. Of

significance, it was demonstrated that ATP generated from glycolysis takes a pivotal role in

retinal energy production. Furthermore, glucose also enters into the pentose phosphate

pathway to counteract the effects of excess ROS generation derived from inefficient oxidative

phosphorylation in situations of metabolic compromise.

Mitochondrial dysfunction has been implicated in central nervous system diseases, such as

Parkinson disease176

and certain optic neuropathies, including glaucoma.177

Rotenone is a

plant-derived ketonic pesticide, which acts as a mitochondrial complex I (NADH-quinone

reductase) inhibitor and which has been used to experimentally induce cellular metabolic

dysfunction.157, 178-180

The mechanisms by which neurons and glia die in rat retinal cultures in

response to rotenone, however, had not previously been elucidated. To this end, the second

108

study of this thesis provided important data which demonstrated that toxicity of rotenone to

neurons and glia in retinal cultures was mediated through distinct mechanisms. Pathology

was implemented via a variety of mechanisms including ATP depletion, ROS elevation, ER

stress ,activation of GSK-3 or calpain-μ, ROS elevation and ATP delpetion. Since ER

stress-induced cell apoptosis is energy-dependent whereas necrosis is ATP independent, it is

likely that the two different death pathways in neurons and glia are related to their different

levels of available ATP. Since neurons produce more of their energy from mitochondrial

oxidative metabolism, as compared to the predominantly glycolytic glia, then it was

hypothesised that it would be likely that the former cells would be more energetically

affected by rotenone. This was indeed the case. The presented data therefore provide a clear

link between the degree of reduction in ATP caused by different forms of metabolic

dysfunction and the cell death pathway taken by the affected cell. In addition, the dosage of

rotenone also determines the necrotic or apoptotic cell death. For instance, in chapter

three,1μM rotenone induced more non-apoptotic than apoptotic cell death. In contrast, lower

level rotenone exposure (5-100nM ) result in apoptotic death in PC12 cells.181

Therefore, we

presume that longer treatment with lower concentration of rotenone may lead to higher

portion of cell apoptosis in the cultures preparation.

Although the retina is part of the brain, its energy metabolism pattern, particularly the outer

retina, has greater similarities with cancerous tissue: both tissues preferentially produce the

majority of their energy via aerobic glycolysis (the Warburg effect).3 The appendix paper

suggested that the mammalian retina shares similar biosynthesis requirements to neoplastic

tissue due to the prodigious turnover of the rhodopsin in the disc membranes of the outer

segments.76, 182

Furthermore, the third paper showed the expression and localization of the

key glycolytic mediator enzyme, pyruvate kinase, in the retina in vitro and in vivo and

attempted to relate the differential expression of the two isoenzymes, PKM1 and PKM2 to

109

specific cell functioning. The presented results may help to explain the overall preferential

use of glycolysis for energy production in the retina, even in the presence of oxygen.

The thesis culminated in the clinical study. In this chapter, we conducted a prospective,

randomized, double-masked pilot study testing the hypothesis that topical glucose acts as a

neurorecoverant and improves contrast sensitivity and visual acuity in glaucoma patients.

Initially, it was found that 50% topical glucose had no adverse effects and could significantly

elevate the vitreous glucose concentration in pseudophakic patients by approximately 30%.

This result indicated that the crystalline lens acts as a physical or metabolic barrier in phakic

eyes. In the second phase of the study, we showed that 50% topical glucose temporarily

elevates the average contrast sensitivity at 12 clycles/degree. This clinical trial explored a

new neuroprotective strategy for a variety of eye diseases where energy insufficiency is part

of the pathogenesis. The thesis advanced our bioenergetic neuroprotection research

programme, and addressed the question: how does glucose protect the retina/optic nerve

against ischaemia and glaucomatous injury? It also provides a better understanding of energy

metabolism in the retina and motivates further bioenergetics-based research.

6.2 Limitations of the studies presented in this thesis

6.2.1 In vitro study

The rat retinal cells used in this study are dissociated from 1-2 days old pups. As it was

described in previous studies, this preparation comprises neurons such as horizontal, bipolar

and amacrine cells and Müller glia cells, astrocytes and microglia.183

Therefore, this culture

has been suggested as an ideal in vitro model of retinal cell function and is used widely to

disseminate mechanisms of retinal energy- or trophic factor-deprivation or as a surrogate

model for LHON.5, 134, 156, 157, 184, 185

However, neonatal retina which is used to prepare the

present type of cultures comprises non-terminally differentiated cells and differs from mature

110

retina in many ways, which likely include energy production and usage. Furthermore,

immature ganglion cells do not readily survive the culture procedure and so these neurons,

which are of great interest in studies of glaucoma and LHON, are only present in low

quantities. In addition, the nature of the Tau and PGP9.5 positive neurons has not been

identified so far and we do not have information about cell specific responses. This requires

further study. Therefore, although results obtained from these cultures add useful information

to that known about retinal metabolism, they still have to be placed in context with results

from other experimental systems, such as retinal explants.

For these reasons, some of the key data was also validated in an adult rat retinal explant

model (see supplementary data section of the first paper). This model has some advantages

over the use of dissociated retinal cultures. First, it allows the use of adult retinal cells, which

are obviously more relevant to the study of glaucoma, which is generally considered to be an

ageing disease. Secondly, as the cells are maintained in their in vivo interrelationships then

results are more reflective of the true state of the retina. It must be borne in mind, however,

that results obtained from these explants, while based on a more relevant model than the

cultures, also likely vary from the in vivo condition and therefore also only act as a guide to

what may be the case in situ. Here, it is pertinent to realise that this tissue preparation lacks

its physiological anatomical structure, particularly with reference to having a vascular

supply.186

The generation of an in vivo model which could be used to study mechanistic

aspects of retinal metabolism would be a major advance and is a future aim of our lab.

6.2.2 Validation of the antibodies

In metabolic terms, the retina acts in an unusual manner. Even in the presence of oxygen, the

mammalian retina will preferentially produce the majority of its ATP from glycolysis. As

mentioned previously, this is termed aerobic glycolysis.76

As also stated, this means that the

111

metabolism of the retina bears more than a passing resemblance to tumour cells, which also

preferentially undertake aerobic glycolysis. It has been speculated that the reason for this is

that glycolysis produces metabolic intermediates which can be used to produce different

amino acids and both tissue types need to provide substrates for biomass production: the

retina for turnover of photoreceptor outer segment discs and tumour cells for proliferation.

Recent data have postulated that the switch to aerobic glycolysis occurs because of the

expression of a specific form of a normal glycolytic enzyme, pyruvate kinase. The M2 splice

variant (PKM2; M2-PK) of this enzyme was suggested to be the key to this mechanistic

metabolic switch by Christofk and colleagues. However, this role is somewhat

controversial.187

In order to investigate the potential function of PKM2 in the retina, with the

view to relating this role to the tissue preference for aerobic glycolysis, expression and

localisation of different isoforms of this enzyme were investigated. Care was needed to be

taken with antibody selection and testing because inappropriate validation of the specificity

of any PKM1/2 antibodies, could lead to incorrect findings and conclusions.

In the third paper, in order to accurately address the expression and localisation of pyruvate

kinase isoenzymes in the retina, a number of PKM1 and PKM2 antibodies and appropriate

control tissues were employed. Important results concerning the specificity evaluation of each

antibody are summarized as followed: (1) Western blot analysis showed that some of the

commercially available PKM2 antibodies bind specifically to a correctly sized antigen and

also that such a band can be observed in their correct positive tissue. (2)

Immunohistochemical analyses showed that the PKM2 antibodies used for Western blotting

gives cell-type specific labelling with satisfactory signal-to-noise ratios in their respective

positive control sections. In comparison, the PKM1 antibody only provided satisfactory

blotting results in mouse and human, but not rat tissue.

112

At the last step in glycolysis, phophoenolpyrvate is converted into pyruvate with the

concurrent generation of two molecules of ATP. This reaction is catalyzed by pyruvate kinase

38. In cancer, the dimeric form of PKM2 has a low affinity for its substrate and therefore it

predominates over the tetrameric form.188, 189

This leads to inefficient PEP conversion and as

a consequence glycolytic intermediates upstream of PEP accumulate and are available for

biosynthesis in cell proliferation. Unfortunately, the current commercial available antibodies

of PKM2 have not been identified to recognize the dimeric form of this enzyme by western

blotting. Thus, it is hard to test to what extent the dimeric form of PKM2 contributes to the

existence of predominant aerobic glycolysis in the retina.

6.2.3 Clinical testing study

In chapter five, based on the finding from cultures and animals, we tested the ability of

intensive topical glucose to temporarily recover aspects of vision in human paitents. We did

not employ any electrophysiology testing and it is not certain that the effects were due to

neurorecovery at the level of the retina. Although we had no evidence for it, conceivably, an

unexplained optical effect could have produced the results. In addition, we currently have no

explanation as to why some patients responded and others did not. Whether glucose delivery

in some form to the eye is a realistic method of long-term neurprotection seems doubtful.

Adverse effects, especially diabetic retinopathy would be a concern. Nevertheless, the

possibility of glucose delivery, perhaps in acute conditions, could conceivably be a valid

treatment strategy in the future.

113

6.3 Other possible speculations on the neuroprotective mechanism of

glucose

6.3.1 Age and the protective effect of glucose

Age is another important risk factor for the progression of POAG; this is thought to relate to

effects on mitochondrial function and ATP generation.190-192

This is supported by data

showing that the possibility of suffering from glaucoma increases by seven times after fifty

five years old.190, 193

From an epidemiological perspective, this result is not only restricted to

glaucoma but also applies to other age-related central nervous diseases such as Parkinson’s

disease.190

Emerging genetic data has suggested that somatic mtDNA mutations which

accumulate with age rather than are inherited could contribute to glaucomatous

retinopathy.191, 194

And this mitochondrial DNA mutations, as well as subsequent reduced

metabolic reserve (such as ATP synthesis and production) accelerate the oxidative stress. In

addition, mtDNA are highly susceptible to oxygen deprivation195

, and it has been suggested

that glucose protects cells by decreasing mitochondrial membrane potential and against the

oxidative damage.196

It is interesting to hypothesize, therefore, that high glucose could

potentially protect mtDNA and mitochondrial function from the age-related deterioration.

Aging is also associated with the build-up of oxidative stress products such as ROS, which

result from insufficient oxidative phosphorylation as by-products. ROS are a group of

reactive free radicals containing an unpaired valence electron.89

It has been suggested that in

the brain, neuronal mtDNA is particularly susceptible to oxidative stress-induced damage and

this ultimately leads to mutations.197

Qi et al. hypothesized, for example, that antioxidant

defence could rescue the G11778A mutation in mitochondrial DNA in patients with Leber’s

hereditary optic neuropathy patients.198

Since we have demonstrated that short term

hyperglycaemia can protect the retina from ischemic injury by counteracting excess ROS

114

production, it seems reasonable to propose that glucose can also protect the retina from age-

related deterioration.

6.3.2 Mechanical stress and the protective effect of glucose

Mechanical stress such as that produced from increased IOP is associated with the loss of

RGCs and degeneration of the optic nerve in glaucoma.199, 200

This stress could initially

impair mitochondrial function and axonal transport at the level of the lamina cribrosa.201

It

has been reported that hydrostatic pressure triggers mitochondrial fission and induces

intracellular energy depletion.202, 203

In addition, IOP-associated axonal transport disruption

could decrease cellular ATP levels in the optic nerve head.204

In human eyes, this mechanical

stress can cause a remodelling of the optic nerve head region by activating astrocytes and

other glial cells which usually provide beneficial factors to local neurons.205

In addition, these

stresses may have metabolic effects. Impairment of glia cells in the optic nerve head may

perturb lactate coupling and, consequently, mitochondrial energy generation in neurons.206

Elevated IOP can also influence cellular redox status and modulates oxidative stress via

activation of glia cells.207

Hernandez et al. has showed that the possible protective

mechanism by which glia protects neurons is via the production of glutathione and the supply

of precursors for GSH generation.208

GSH, via its antioxidant property, can rescue injured

RGCs from oxidative stress.209

In addition, astrocytes can also deactivate superoxide by

increasing expression of superoxide dismutase.210

All these findings provide further proof for

the role of mitochondrial dysfunction in mechanical stress-related glaucomatous optic nerve

degeneration.

6.3.3 PKM2 role in glucose metabolism in cancer and retina

It has been reported that the switch from expression of physiological PKM1 to PKM2 in

cancer cells is responsible for the Warburg effect.31

However, this conclusion is supported by

115

studies describing the knock-down PKM2 gene expression and/or transfection of cells with

the PKM1 gene. The same authors also published Western blotting data for PKM1/PKM2

from many types of tumour cell which agreed with these data.31

This conclusion was latterly

described to be based on the misinterpretation of Western blots from PKM1-expressing

mouse muscle tissue. However, Bluemlein et al.187

opposed this suggestion by using

proteomics to definitively show that PKM2 was present in both tumours and normal control

tissue (not displaying the Warburg effect), undermining the notion that the switch from

PKM1 to PKM2 was responsible for the Warburg effect. Subsequently, Luo et al.32

summarized the above results into a coherent explanation by suggesting that the Warburg

effect in HeLa cells is modulated via PKM2 serving as a co-activator for HIF-1α. They also

proved that prolyl hydroxylation of PKM2 accelerates the interaction between PKM2 and

HIF-1α, thereby increasing HIF-1α binding to the hypoxia response elements of other

glycolysis-associated target genes in order to convert glucose to lactate. They proposed that

the above interactions are not expressed together under normal conditions in most tissues, but

were promoted in cancers due to the relatively high expression of hypoxia-associated HIF

stabilization factors. In addition, Hughes et al. and his group have recently suggested that

human retina under physiological conditions also expresses baseline HIF-1α.211

Other factors

such as oncogene c-Myc, is also involved in PKM2 mediated actions in some cancer tissue.

In summary, all the emerging evidence indicates that PKM2 plays a pivotal role in promoting

and/or maintaining aerobic glycolysis and therefore the Warburg effect in cancer.212, 213

The Warburg effect has also been described in the retina.4, 6, 25, 76

Winkler et al. in 2000

reported that retinal cells such as Müller cells predominantly use glycolysis by converting

glucose to lactate even in the presence of physiological levels of oxygen.21

This metabolic

pattern is similar to that which exists in tumours and other proliferative tissues. Casson et al.

recently postulated that the high turnover rate of photoreceptor outer segments may

116

contribute to the retina promoting a Warburg effect, ie, preferring to generate most of its

energy via glycolysis in the presence of oxygen.76

This suggestion is favoured by the

conclusion that the rate of glycolysis parallels the turn-over rate of rhodopsin, which is

constantly renewed in rod outer segments.214

In future studies, therefore, we propose to

investigate this phenomenon by injecting 14

C-labelled glucose into the eye and determining to

what extent glucose directly contributes to rhodopsin renewal in the retina. As well as the

identification of PKM2 in photoreceptors these data will provide novel information

concerning preferential retinal aerobic glycolysis.

117

6.4 Future directions on bioenergetics therapies

Retinal ganglion cells are very susceptible to mitochondrial impairment and a variety of

neurodegenerative diseases display cellular energy defecits. Bioenergetic therapies, aimed at

rescuing mitochondria from ischemia injury, are becoming a promising potential

neuroprotective strategy. Providing enough glucose to an oxygen- and glucose-deprived

tissue can decrease OXPHOS defects, alter mitochondrial membrane potential and diminish

reactive oxygen species production. Further research which could define whether the

heterogeneous nature of glaucoma relates to the different degree of energy depletion in

different situations or different patients may provide a new perspective for re-tackling

traditional IOP management therapies.

Long term glucose treatment and delivery is not safe and practical due to the potential

complication of hyperglycaemia. Therefore, our group employed topical glucose eye drops to

study the beneficial effect of supplying high levels of glucose to glaucoma patients. In this

randomized pilot clinical trial, topical glucose temporarily improved visual acuity and

contrast sensitivity in patients. This rapid and temporary increase of psychophysical visual

parameters reflected improved cellular and mitochondrial function. In the next stage, we plan

to confirm this conclusion by employing retinal electrophysiology to directly determine the

neurorecoverant effect of topical glucose administration on RGC function. In addition, in this

study we did not find an elevation of the vitreous glucose concentration in phakic patients.

Therefore, we still need to seek other possible administration routes/solutions in a bid to

increase glucose delivery to the retina via the vitreous. In recent animal experiments

(unpublished data), subconjunctival injections of glucose were shown to significantly

increase vitreous glucose concentrations in rats with intact lenses. Future clinical trials will

focus on similar efficient approaches to deliver glucose in different ophthalmic diseases

118

where mitochondrial dysfunction may be part of the pathogenesis. Although the application

of topical glucose could improve visual acuity temporally, the pharmacological use of the

glucose is restricted due to rapid diffusion and non-targeted treatment. We would therefore

also like to consider developing a slow releasing glucose or ATP system which could be

encapsulated by liposomes or other suitable biomaterials.

Apart from glucose, several other agents have also been found to have protective effects

against a variety of neurodegenerative disease. For instance, nicotinamide serves as a

substrate for complex І in the mitochondrial electron transport chain, and acts as an NADP

precursor in scavenging ROS and inhibiting cell apoptosis.215, 216

Oral supplementation of

nicotinamide decreases lesions caused by excitotoxic brain injury.217

It has also been reported

by Osborne et al that nicotinamide can rescue RGCs from acute ischaemic injury.168

In

addition, emerging evidence has also indicated that creatine, which is a nitrogenous guanidine

agent, might provide a protective effect to neural or other high energy demanding tissues.218-

220 Oral and subcutaneous creatine have both displayed potential protection to neurons in

animal models of acute ischemic injury.183, 217, 219, 221

Therefore, due to its safe nature and

good bioavailability, creatine is another promising candidate for bioenergetic therapy of

neurons subjected to diseases associated with metabolic dysfunction.

118

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8. Appendix

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134

A Casson, R.J., Chidlow, G., Han, G. & Wood, J.P.M. (2013) An explanation for the Warburg effect in the adult mammalian retina. Clinical and Experimental Ophthalmology, v. 41(5), pp. 517

NOTE:

This publication is included on page 134 in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://doi.org/10.1111/ceo.12050