YEXNR-12025; No. of pages: 13; 4C: Experimental Neurology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr

F

a

1 0

a r t i c l e

11 12 13 14 15

Article history: Received 6 February 2015 Received in revised form 13 April 2015 Accepted 16 April 2015 Available online xxxx

16 17 18 19 20 21

Keywords: Cellular prion protein Hypoxia Lactate dehydrogenase Monocarboxylate transporter 1 Neuroprotection

O

6 7 8 9

Pfuetzner Science and Health Institute, 55116 Mainz, Germany Department of Neurology, Georg-August University, 37075 Goettingen, Germany Department of Clinical Chemistry, Georg-August University, 37075 Goettingen, Germany d Department of Neuropathology, Georg-August University, 37075 Goettingen, Germany

R O

b c

i n f o

a b s t r a c t

P

5

Sanja Ramljak a,b,1, Matthias Schmitz b,⁎,1, Saima Zafar b, Arne Wrede d, Sara Schumann b, Abdul R. Asif c, Julie Carimalo b,2, Thorsten R. Doeppner b,3, Walter J. Schulz-Schaeffer d, Jens Weise b,4, Inga Zerr b

Although a physiological function of the cellular prion protein (PrPc) is still not fully clarified a PrPc-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion knockout mice (Prnp0/0) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrPc. Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp0/0 knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp0/0 primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp0/0 mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrPc show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrPc was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrPc-overexpression under normoxic conditions. Our data suggest LDH as a direct PrPc interactor with possible physiological relevance under low oxygen conditions. © 2015 Published by Elsevier Inc.

D

4Q2

E

3

Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions

T

2Q1

Regular Article

R

E

C

1

38

R

42 40 39

N C O

41

1. Introduction

44 45

The physiological function of the cellular prion protein (PrPc) remains unclear, although numerous studies point to its neuroprotective role (Walz et al., 1999; McLennan et al., 2004; Weise et al., 2004). PrPc is a multifunctional protein involved in counteraction of oxidative stress (Brown et al., 1997; Wong et al., 2001), modulation of cell death

48

U

Q3 43

46 47

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

⁎ Corresponding author at: Department of Neurology, Georg-August University, Goettingen, Robert-Koch-Str. 40, 37 075 Goettingen, Germany. E-mail address: [email protected] (M. Schmitz). 1 Contributed equally to this work. 2 Dr. J. Carimalo's present address: C'Nano, Laboratory for Photonics and Nanostructures, Route de Nozay, 91460 Marcoussis, France. 3 Dr. Thorsten R. Doeppner's present address: Department of Neurology, University of Duisburg-Essen, 45147 Essen, Germany. 4 PD Dr. J. Weise present's address: Department of Neurology, HELIOS VogtlandKlinikum Plauen, 08529 Plauen, Germany.

(Kuwahara et al., 1999; Bounhar et al., 2001) and activation of several signal transduction pathways known to promote neuronal survival (Mouillet-Richard et al., 2000; Zanata et al., 2002). Interestingly, ablation of the cellular prion protein gene (Prnp) is not crucial for viability (Bueler et al., 1992; Kuwahara et al., 1999). However, under circumstances of increased physiological demands, such as brain seizures or ischemia the presence of PrPc becomes decisive (Walz et al., 1999; Weise et al., 2004). Hence, cerebral PrPc is up-regulated early in response to focal cerebral ischemia and prion protein knockout (Prnp0/0) mice display significantly greater infarct volumes as compared to wild-type (WT) mice following both permanent and transient ischemia (McLennan et al., 2004; Mitteregger et al., 2007). Even though it has been suggested that PrPc exerts neuroprotective effects via phosphatidylinositol 3-kinase (PI3K)/Akt (Weise et al., 2006) and mitogenactivated protein kinase/extracellular signal-regulated kinase (MAPK/ ERK) pathway (Shyu et al., 2005b; Spudich et al., 2005), molecular mechanisms underlying PrPc mediated neuroprotection after ischemic brain injury require further characterization.

http://dx.doi.org/10.1016/j.expneurol.2015.04.025 0014-4886/© 2015 Published by Elsevier Inc.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

2. Material and methods

100

2.1. Preparation and maintenance of primary cortical neurons and hypoxia–re-oxygenation treatment

109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

R

R

O

107 108

C

105 106

N

103 104

Pregnant WT and Prnp0/0 mice were anaesthetized on day 14 of gestation using 2-bromo-2-chloro-1,1,1-trifluoroethane (Sigma-Aldrich, Taufkirchen, Germany) and then sacrificed by cervical dislocation. The genetic background of WT and Prnp0/0 mice is described in detail in the section Transgenic mice. Briefly, brains from day 14 embryos were removed and stripped of meninges. Afterwards, cortex tissue was isolated and mechanically dissociated by several pipetting passages after a 10-min treatment with trypsin/EDTA (Biochrom, Berlin, Germany) at 37 °C. Cortical cells were then centrifuged, counted and finally plated on poly-D-lysine (10 μM)-coated glass cover-slips in culture wells at a density of 1 × 106 cells/well (9.6 cm2). Cell cultures were first grown in DMEM (Dulbecco's Modified Eagle's Medium, Sigma-Aldrich), supplemented with 10% FCS (Biochrom), 20 mM KCl, N-2 (1:100, Gibco/ Invitrogen, Karlsruhe, Germany), B-27 (1:50, Gibco/Invitrogen) supplemented with antioxidants, and 0.1% P/S (penicillin–streptomycin) (Gibco/Invitrogen). Four days after preparation, preexisting medium was replaced by fresh prepared DMEM, containing 10% FCS, B-27, N-2 and the antimitotics uridine (U) and 5-Fuoro-2′-deoxyuridine (FdU) (Sigma-Aldrich) in order to reduce astrocyte proliferation. Hypoxia/re-oxygenation experiments were conducted after 7 days in vitro. Cells were placed in a hypoxic chamber (Labotect, incubator C42, Goettingen, Germany) at 37 °C, 5% CO2, 95% humidity and 1% O2 for various incubation periods (30, 60 and 90 min) followed by 12 h of re-oxygenation under standard conditions (37 °C, 5% CO2 and 95% humidity). For Western blot analysis, cells were washed in phosphatebuffered saline (PBS), scraped on ice in a cell lysis buffer, containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100 (Roth, Karlsruhe,

U

102

E

99

101

2.3. Induction of transient focal cerebral ischemia

147

Animals were anaesthetised with 1%–1.5% isofluran (30% O2, remainder N2O). Rectal temperature was maintained at 36.5–37 °C employing a feedback controlled heating system. In order to assess cerebral blood flow, laser-Doppler flow (LDF) was recorded during all experiments using a 0.5 mm fiberoptic probe (Perimed, Stockholm, Sweden) attached to the skull overlying the core region of the middle cerebral artery (MCA) territory (2 mm posterior, 6 mm lateral from bregma). Focal cerebral ischemia was induced by transient occlusion (60 min) of the MCA using the intraluminal filament technique (Weise et al., 2004). Following a midline neck incision the left common and external carotid artery were isolated and ligated. After placing a microvascular clip (Aesculap, Tuttlingen, Germany) on the internal carotid artery, an 8–0 silicon resin (Xantopren, Deuker, Germany) coated nylon monofilament (Ethilon; diameter 180 to 200 μm; Ethicon, Germany) was introduced through an incision into the distal part of the common carotid artery and, after clip removal, advanced 9 mm distal from the carotid bifurcation for MCA occlusion. The monofilament was withdrawn after 60 min of ischemia to allow reperfusion of the MCA. LDF recording continued for 15 min to monitor appropriate reperfusion.

148

2.4. Visualization of infarcted tissue in WT as compared to Prnp0/0 mice

168

Twenty-four hours after induction of transient ischemia WT and Prnp0/0 mice were euthanized by an overdose of isofluran and their brains removed. Five coronal equidistant slices were cut (thickness: 2 mm) and immersed in 2% 2,3,5-triphenyl-tetrazolium chloride solution for 15 min in order to visualize the infarcted tissue.

169

2.5. Two-dimensional gel electrophoresis (2-DE)

174

For 2-DE mouse brain homogenates were concentrated and desalinated, using a Microcon™YM-3 centrifugal filter (Millipore, Eschborn, Germany), according to supplier's recommendations. Protein samples were diluted with rehydration buffer (7 M urea, 2 M thiourea, 15 mM dithiothreitol (DTT), 4% CHAPS, 2% ampholytes) for first-dimension isoelectric focusing (IEF). IEF on a 7 cm immobilized pH gradient (IPG) strip (pH 3–10, linear) was performed by applying 40 μg of proteins per strip. Focusing of the proteins was initiated at 200 V for 2 h, followed by ramping at 500 V for 2 h, and final focusing at 4000 V for 5 h for a total of 20,000 Vh. After IEF separation, proteins immobilized on the IPG strip were reduced in the buffer containing 6 M urea, 2% sodium dodecyl sulphate (SDS), 30% glycerol, 2% DTT, and 0.375 M Tris–HCl (pH 8.8)

175

F

78 79

132 133

O

76 77

All experimental procedures were performed according to the National Institutes of Health guidelines for the care and use of laboratory animals and approved by local authorities. Adult, male WT and Prnp0/ 0 mice weighing 22–27 g were used in the study. Both WT and Prnp0/0 mice were of mixed (129/Sv × C57BL/6) genetic background. Prnp0/0 mice were generated as described earlier (Bueler et al., 1992). After mice have been sacrificed, brains were taken either as a whole or were dissociated on ice into four brain regions: hippocampus, cortex, cerebellum and olfactory bulb. Subsequently, whole brains/brain regions were cut, complemented with lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 (Roth) and cocktail of protease inhibitors (Roche) and homogenized. Brain lysate samples were rotated for 15 min and centrifuged at 4 °C at 13,000 ×g. Supernatants were transferred in separate tubes and stored at −80 °C.

R O

74 75

131

P

73

2.2. Transgenic mice

D

71 72

Germany), protease and phosphatase inhibitor cocktail (Roche, Mann- 129 heim, Germany). 130

T

69 70

Several earlier reports demonstrated beneficial effects of LDH product, lactate, on post-hypoxic/post-ischemic neuronal tissue (Schurr et al., 1988, 1997, 2001). The astrocyte–neuron lactate shuttle hypothesis postulates that lactate is an essential component of metabolic crosstalk between astrocytes and neurons enabling neuronal recovery under circumstances of high energy demand such as hypoxia/ischemia (Pellerin and Magistretti, 1994). Furthermore, administration of lactate directly after middle cerebral artery occlusion leads to a significant decrease in lesion size and an improvement in neurologic outcome in mice (Berthet et al., 2009). A previous study demonstrated PrPc involvement in the regulation of glutamate-dependent lactate transport of cultured astrocytes (Kleene et al., 2007). We reported a marked PrPc-induced up-regulation of the LDH isoform A (LDH-A) after introduction of PRNP gene into Prnp0/0 cells (Ramljak et al., 2008). Meanwhile, interactome analyses identified the LDH-A isoform not only as a PrPc interaction partner but also as an interactor of Doppel and Shadoo, two mammalian PrPc paralogues (Watts et al., 2009), indicating a potential physiologically relevant association of both proteins. A functional relation between PrPc and LDH could be particularly intriguing in view of the beneficial effects cerebral lactate might exert on neuronal recovery of WT as compared to Prnp0/0 mice after hypoxia/ischemia. In the present study, we verified LDH expression levels in primary cortical neurons derived from WT and Prnp0/0 mice under both normoxic and hypoxic conditions. In addition, we scrutinized LDH expression in WT and Prnp0/0 mice subjected to transient focal cerebral ischemia. Finally, we employed a HEK293 cell line as a cell model and examined regulation of LDH protein levels/activity in PrPcoverexpressing vs. control HEK293 cells (expressing endogenous levels of PrPc) under normoxic and hypoxic conditions. Furthermore, we investigated a possibility of direct interaction between LDH and PrPc by means of immunoprecipitation and co-localization experiments.

C

67 68

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

E

2

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

134 135

136 137 138 139 140 141 142 143 144 145 146

149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167

170 171 172 173

176 177 178 179 180 181 182 183 184 185 186

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

A HEK293 cell line was used due to its shared properties with neuro- 194 nal cells (Shaw et al., 2002) and due to high transfection efficiencies 195

A LDH-B LDH-A

42

betaactin

control nx. 30 min hx. 60 min hx. 90 min hx. WT

400

O

control nx. 30 min hx. 60 min hx. 90 min hx. Prnp0/0

F

37 36

*

R O

*

350 300

P

250 200

D

150

E

100 50

T

0 Prnp0/0 WT

C

Prnp0/0 WT

control nx.

30 min hx.

E

B

Prnp0/0 WT

Prnp0/0 WT

60 min hx.

90 min hx.

Prnp0/0

R

WT

R

nx.

N C O

191

Absolute LDH (A+B) densitometric values

189 190

2.6. Transient overexpression of PrPc in human embryonic kidney 192 (HEK)293 cells 193

for 25 min and were alkylated in the same buffer supplemented with 2.5% iodoacetamide instead of DTT for a further 25 min. Equilibrated strips were placed on top of vertical 12% polyacrylamide gels and electrophoresis was started. Further 2-DE Western blot analysis was performed as described in the Western blot analysis section.

WT

Prnp0/0

90 min hx.

U

187 188

3

100 µm Fig. 1. PrPc-mediated up-regulation of LDH protein expression and concomitant preservation of tubulin cytoskeleton in primary cortical neurons of WT vs. Prnp0/0 mice after hypoxic insult (A, B). (A) The two bands observed on the Western blot represent LDH-A isoenzyme (lower band) and LDH-B isoenzyme (upper band). Primary neurons were exposed either to normal oxygen supply (control nx.) or to reduced oxygen supply for various incubation periods (30; 60; 90 min hx.). LDH expression profile was assessed 12 h post-exposure. Densitometric analysis showed no significant differences between WT (black bars) and Prnp0/0 (grey bars) neurons under normoxic conditions. Thirty minutes after hypoxia LDH was slightly but not significantly up-regulated in WT as compared to Prnp0/0 neurons. The trend of higher LDH expression in WT as compared to Prnp0/0 neurons became significantly pronounced after 60 and 90 min of exposure to limited oxygen supply. An equal protein load (20 μg) is shown by beta-actin expression. The Western blot is representative of three independent experiments. Densitometric analyses were performed from three different Western blots. All densitometric measurements were presented as absolute values (×103). Each bar represents a mean value of total LDH content with corresponding standard deviations. Level of significance: *, p b 0.05. (B) Wild-type (panel WT nx.) and Prnp0/0 (panel Prnp0/0 nx.) cortical neurons show normally structured tubulin skeleton under normoxic conditions as displayed by class III beta-tubulin isotype staining. Twelve hours after re-oxygenation (exposure length 90 min) wild-type neurons (panel WT 90 min hx.) exhibit prominently less damage as assessed by a degree of tubulin structure preservation in comparison to Prnp0/0 neurons (panel Prnp0/0 90 min hx.). The scale bar is 100 μm.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

222

HEK293 cells were grown in DMEM (Sigma-Aldrich) supplemented with 10% FCS (Biochrom), 1% P/S (Gibco/Invitrogen) under standard culture conditions. Cells were passaged twice a week and the medium was exchanged at least every four days. Hypoxia/re-oxygenation experiments were conducted 24–48 h after passaging. Cells were placed in a hypoxic chamber (Labotect, incubator C42) at 37 °C, 5% CO2, 95% humidity and 1% O2 for 90 min followed by 6 h of re-oxygenation under standard conditions. Re-oxygenation time of 12 h was also tested. However, the former condition was employed for further experiments because 12 h of re-oxygenation resulted in complete cell detachment due to experimental procedure exposing cells first to transfection and afterwards to hypoxic stress. Cell lysates were prepared as described in section Preparation and maintenance of primary cortical neurons and hypoxia–re-oxygenation treatment.

223

2.8. Western blot analysis

224 225

Whole cell lysates originating from HEK293 cell line and primary cortical neurons were prepared as described in Preparation and maintenance of primary cortical neurons and hypoxia–re-oxygenation treatment.

226

T

PrPc Age in months

36 Cortex 42

0/0

0/0

3

3

C

220 221

0/0

0/0

+/+

+/+

+/+

+/+

20

20

3

3

20

20

E

218 219

R

216 217

R

215

LDH

beta-actin

O

213 214

C

211 212

36 Hippocampus

N

205 206

U

203 204

F

209 210

202

O

2.7. Maintenance of HEK293 cells under normoxic and hypoxic conditions

200 201

R O

208

198 199

WT and Prnp0/0 mice were sacrificed by an overdose of isofluran at 6 and 24 h (n = 3 per group and time point) following transient cerebral ischemia. In addition, sham-operated WT and Prnp0/0 mice (n = 3 per group) were used as non-ischemic controls. Brains were removed and shock-frozen. After separation of the left (ischemic in operated animals) and right hemisphere, the left hemispheres of individual mice were complemented with lysis buffer, as described in Preparation and maintenance of primary cortical neurons and hypoxia–re-oxygenation treatment, homogenized, centrifuged and the supernatants were used for Western blot analysis. Equal amounts of protein were denatured in 4 × sample buffer Roti®Load (Roth) for 5 min at 95 °C and subsequently separated on 12% SDS-PAGE gels. Following electrophoretic separation, proteins were transferred to polyvinylidene difluoride membranes (AppliChem, Darmstadt, Germany) which were immersed in blocking solution (5% non-fat dry milk, 0.1% Tween-20 in PBS) for 1 h at room temperature (RT). An overnight incubation with primary polyclonal goat anti-LDH that reacts with all LDH isoforms (1:500; ab2101, Abcam, Cambridge, UK), or mouse monoclonal anti-LDH-A (1:1000; MAB2736, Abnova, Heidelberg, Germany), mouse anti-PrP c 12F10 (1:300; AO3221)/SAF32 (1:500; AO3202, SPI-Bio, Massy, France), rabbit polyclonal anti-monocarboxylate transporter 1 (MCT1) (1:200; sc-50324, Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mouse anti-ß-actin (1:5000; ab6276, Abcam) antibodies was performed at 4 °C. Afterwards membranes were extensively rinsed in PBS with Tween-20 and incubated with the corresponding horseradish peroxidase (HRP)-conjugated, donkey anti-goat (705-035-003), donkey anti-mouse (715-035-150) and goat anti-rabbit (111-035-144) secondary antibodies (1:5000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 1 h at RT. Immunoreactivity was detected after immersion of the membranes into enhanced chemiluminescence (ECL) solution and exposition to ECL-Hyperfilm (Amersham Biosciences, Buckinghamshire, UK). Densitometric analyses were carried out using the LabImaging software (Kapelan Bio-Imaging GmbH, Leipzig, Germany). For each

E

207

normally obtained using this cell line. HEK293 cells were transiently transfected with the pCMS-PRNP-EGFP vector, expressing human PRNP. As a control an empty pCMS-EGFP vector was used. For the transfection, 1–1.5 × 106 cells were seeded in a six well plate for 24 h. Plasmid DNA (1.5–2 μg) and 4–6 μL Lipofectamine (Invitrogen, Groningen, Netherlands) were dissolved in 250 μL OptiMEM (Gibco/Invitrogen) for 5 min. Stable DNA complexes were formed after 20–30 min and transferred into the transfection medium, OptiMEM containing 2% foetal calf serum (FCS) (Biochrom) in which the cells were incubated for further 6–8 h or overnight. Thereafter, the transfection medium was replaced by fresh culture medium. The transfection efficiency ranged between 60–80% as monitored by EGFP fluorescence (data not shown).

P

196 197

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

D

4

42

36 Olfactory bulb

LDH beta-actin

LDH

42

beta-actin

36

LDH

Cerebellum 42

beta-actin

Fig. 2. Western blot analysis of LDH expression in four different brain regions of 3 and 20 months old WT and Prnp0/0 mice. Homogenates prepared from cortex, hippocampus, olfactory bulb and cerebellum were examined for LDH expression by Western blotting. LDH expression did not show distinct changes between the four different brain regions nor did it show age dependence. An equal protein load (20 μg) is shown by beta-actin expression.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

262 263

condition analyzed, three Western blots were prepared from three different protein extractions.

264

2.9. Immunoprecipitation

265

PrPc-overexpressing and control HEK293 cells incubated under normoxic and hypoxic (90 min hypoxia) conditions were scraped on

ice in PBS containing a cocktail of protease inhibitors and sonicated. Subsequently, insoluble cell debris was removed by ultracentrifugation with TL100 ultracentrifuge (Beckman Instruments, S.A., Gagny, France) for 15 min at 4 °C. For immunoprecipitation, Dynabeads protein G (Invitrogen) were used following the manufacturer's instructions. Briefly, 10 μg of monoclonal LDH-A antibody was incubated together with 500 μg of total protein extract, originating either from PrPc-overexpressing or

A LDH-B LDH-A

42

betaactin

**

800

24 h rox.

R O

control nx. 6 h rox. WT

*

P

700 600

D

500 400

E

300 200

T

100 0

Prnp0/0 WT 24 h rox.

Prnp0/0 WT 6 h rox.

B

N C O

R

5.6

R

E

Prnp0/0 WT control nx.

C

Absolute LDH (A+B) densitometric values

24 h rox.

O

F

37 36

control nx. 6 h rox. Prnp0/0

LDH-B

7.9 Isolectric point (pH- units)

pI ~5.8

LDH-A

pI ~7.8

37

U

266

5

36

Fig. 3. Regulation and identification of LDH-A and LDH-B protein isoforms in WT and Prnp0/0 mice under physiological conditions and after an experimentally induced transient focal cerebral ischemia (A, B). (A) Two bands observed on the Western blot represent LDH-A isoenzyme (lower band) and LDH-B isoenzyme (upper band) as confirmed by 2-DE Western blotting (see below). No major differences were found in cerebral LDH expression between WT and Prnp0/0 mice under physiological conditions (upper panel, control nx. and control hx.). Six and 24 h after induction of ischemia a marked up-regulation of LDH (A + B) is observed in WT mice as compared to Prnp0/0 mice (upper panel, 6 h rox.; 24 h rox.). Aliquots of the brain homogenates contained an equivalent protein amount (40 μg), as shown by beta-actin expression (lower panel). This figure is representative of three Western blots. Densitometric analysis of LDH Western blots shows a slight up-regulation of total LDH (A + B) under physiological conditions and a significant up-regulation 6 h and 24 h after induction of ischemia in WT mice (black bars) as compared to Prnp0/0 mice (grey bars). All densitometric measurements were presented as absolute values (×103). Each bar represents a mean value of total LDH content with corresponding standard deviations. Densitometric analyses were performed from three different Western blots. Level of significance: *, p b 0.05; **, p b 0.01. (B) Linear IPG strips (pH 3–10) were loaded with 40 μg of the WT mouse brain homogenate. Following IEF, proteins were separated by SDS-PAGE and subsequently immunoblotted with LDH antibody. The inset illustrates a magnified part of 2-DE Western blot with LDH-A isoform located at ~36 kDa and pI ~ 7.8 and LDH-B isoform located at ~37 kDa and pI ~ 5.8, supported by the data obtained from SwissProt database.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

267 268 269 270 271 272 273

282 283

296

The difference between an intra- and an extracellular LDH activity in HEK293 cells under normoxic/hypoxic conditions was measured using a lactate dehydrogenase assay kit (BioVision, Milpitas, CA) according to manufacturer's instructions. In short, each cell lysate sample used to examine an intracellular LDH activity was diluted in an assay buffer to a final volume of 50 μL. The protein concentration of cell lysates was set to 0.5 μg/μL. For measuring an extracellular LDH activity cell culture medium was used. The basal LDH activity of cell culture medium (blank) was subtracted from each of the measurements in which an extracellular LDH activity was examined. Afterwards, the reaction mix consisting of 48 μL assay buffer and 2 μL substrate mix was added either to the 50 μL of cell lysate or the cell culture medium. Optical density was measured at 450 nm prior to and after the incubation period of 30 min at 37 °C. All experiments were performed three times from three different preparations.

297

2.11. Immunocytochemical staining

298

To examine a possible co-localization of LDH and MCT1 with PrPc in HEK293 cells under normoxic/hypoxic conditions we employed polyclonal goat anti-LDH (1:200; Abcam), rabbit polyclonal anti-MCT1 (1:1000; Santa Cruz Biotechnology Inc.) and anti-PrPc mouse monoclonal 3F4 (1:500; gift from Robert Koch Institute, Berlin, Germany) primary antibodies. Following fixation in acetone for 5 min on ice and permeabilisation with 0.5% Triton X-100 (Roth), cells were blocked with 0.2% I-block (Applied Biosystems, Foster City, CA). Secondary antibodies used were Cy3-labelled anti-rabbit (1:200; 111-165-003, Dianova, Hamburg, Germany), HRP-coupled anti-goat (1:100; 305-035-003, Dianova) and Cy2-labelled anti-mouse (1:200; 115-225-003, Dianova). Incubation with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, OR) for 10 min was performed to visualize nuclei. Slides were mounted using fluorescence mounting medium (Dako, Glostrup, Denmark). Staining of HEK293 cells was examined at 1000-fold magnification under BX51 microscope (Olympus, Tokyo, Japan) using a fluorescence unit. Images were acquired and processed using CellF-software (Olympus). An assessment of PrPc and LDH co-localization was performed by calculating fluorescence channel correlations using Pearson's linear correlation coefficient (rp). Three slides with a minimum of 25 cells were examined per experimental condition. To visualize and compare neuronal damage in primary cortical cultures of WT and Prnp0/0 mice subjected to hypoxia we employed Alexa Fluor 488-labelled monoclonal neuronal class III beta-tubulin antibody (cat. no. 302–302, Synaptic Systems, Göttingen, Germany) diluted 1:500. Primary cortical cells under normoxic conditions were used as corresponding controls. Primary cortical cells were fixed in PBS containing 4% paraformaldehyde for 20 min, washed 3 times with PBS and permeabilized by incubation in PBS containing 0.2% Triton X-100 for 10 min. Permeabilization was followed by 1 h of blocking in PBS containing 2% bovine serum albumin (BSA). Subsequently, the specimens were incubated with Alexa Fluor 488-labelled antibody diluted in PBS containing 1% BSA for 2 h at RT. All working steps were carried out in a dark humid chamber and were stopped by washing three times with PBS. Cells were mounted in Mowiol 4–88 (Roth) prior to examination of the staining. Staining of primary cortical cultures was examined using Olympus BX51

303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334

C

301 302

WT

E

299 300

Prnp0/0

R

294 295

R

292 293

We examined primary cortical neurons derived from WT and Prnp0/0 mice under both normoxic and hypoxic conditions. The primary neurons were either provided with normal oxygen supply or were exposed to hypoxic conditions for different incubation periods. Western blot analysis

O

290 291

340 341 342 343

3.1. Post-hypoxic LDH expression is enhanced in WT- vs. Prnp0/0-derived pri- 345 mary cortical neurons 346

C

288 289

344

N

286 287

3. Results

U

284 285

338 339

F

2.10. LDH activity

Densitometric values of Western blot bands as well as LDH activity were statistically evaluated using unpaired two-sided Student's t-test. Means and standard deviations were calculated from at least three independent set of experiments. Differences in protein expression and enzyme activity with p value b 0.05 were considered to be statistically significant.

T

281

337

O

280

2.12. Statistics

R O

278 279

microscope (Olympus) at 400-fold magnification employing a fluores- 335 cence unit. Images were processed with CellF (Olympus) software. 336

P

276 277

control HEK293 cells under normoxic conditions or subjected to 90 min of hypoxia and 6 h of re-oxygenation, under rotation for 1 h at 4 °C. Afterwards, 50 μL of 50% slurry Dynabeads were added into vials and incubated while rotating overnight at 4 °C, followed by three washing steps before the target antigen could be eluted. Immunoprecipitated proteins were investigated by Western blotting using SAF32 antibody. The respective antibody dilutions are indicated in Western blot analysis section.

D

274 275

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

E

6

Fig. 4. 2,3,5-Triphenyl-tetrazolium chloride staining of ischemic infarcts in Prnp0/0 after transient (60-minute) ischemia (Weise et al., 2006). Representative samples are given. Infarct areas are identified by a lack of 2,3,5-triphenyl-tetrazolium chloride staining in left hemispheres. Note increased infarct volumes in Prnp0/0 compared with WT mice. Arrows point at areas of infarction in the basal ganglia (Prnp0/0 and WT mice) and cortical areas (found in Prnp0/0 mice only) after transient ischemia.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

347 348 349 350

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

A

control nx.

90 min hx. PrP

90 min hx.

c+++

control nx. PrP

c+

36

O

F

LDH-A

42

R O

beta-actin

*

*

E

900 800

T

700

C

600 500

E

400

R

300

0

R

200 100

D

29

Absolute LDH-A densitometric values

PrPc

P

35

control nx. 90 min hx. c+++ PrP

N C O

353

conditions 12 h following re-oxygenation. Densitometric analysis 354 revealed no significant differences in LDH expression between WT and 355 Prnp0/0 primary neurons under normoxic conditions (control nx.). Thirty 356

showed two LDH bands in cell lysates of primary cortical neurons which were identified as LDH-A and LDH-B. Fig. 1A displays a representative Western blot of LDH expression profiles under control and hypoxic

control nx.

90 min hx. PrP

c+

Fig. 5. Western blot and densitometric analysis of LDH-A expression in control and PrPc-overexpressing HEK293 cells under normoxic and hypoxic conditions (A). Interaction of LDH-A and PrPc in HEK293 cells as assessed by immunoprecipitation under normoxic and hypoxic conditions (B). Co-localization of LDH and PrPc in HEK293 cells under normoxic and hypoxic conditions as assessed by fluorescence microscopy (C). Increased LDH-activity in PrPc-overexpressing vs. control HEK293 cells under hypoxic conditions (D). (A) Following 90 min of hypoxia PrPc expression was significantly higher (p = 0.039) in hypoxic PrPc-overexpressing HEK293 cells (designated as 90 min hx. PrPc+++) at 6 h post-exposure as compared to PrPc-overexpressing cells under normoxic conditions (designated as control nx. PrPc+++). Similarly, the LDH-A level was noticeably and significantly higher (p = 0.029) in PrPc-overexpressing cells subjected to hypoxia than in the control cells, expressing endogenous levels of PrPc, subjected to the same hypoxic conditions (designated 90 min hx. PrPc+). The LDH-A expression level between the control cells, with normal oxygen consumption (designated as control nx. PrPc+), and the control cells exposed to reduced oxygen supply remained unchanged. An equal protein load (20 μg) is shown by beta-actin expression. The Western blot is representative of three independent experiments. Densitometric analysis displays a significant increase in LDH-A expression in PrPc-overexpressing cells (grey bars) exposed to 90 min of hypoxia vs. PrPc-overexpressing cells exposed to normoxia. Likewise, a significant difference in LDH-A levels was observed between PrPc-overexpressing and control cells (black bars) exposed to the same hypoxic conditions. All densitometric measurements were presented as absolute values (×103). Each bar represents a mean value of LDH-A content with corresponding standard deviations. Densitometric analyses were performed from three different Western blots. Level of significance: *, p b 0.05. (B) Immunoprecipitation (IP) of PrPc in HEK293 cells under normoxic and hypoxic (90 min of hypoxia) conditions by monoclonal LDH-A antibody. Immunoprecipitation with LDH-A antibody, followed by Western blotting using SAF32, revealed a clear di-glycosylated PrPc protein band in PrPc-overexpressing cells under both normoxic (designated as control nx. PrPc+++) and hypoxic (designated as 90 min hx. PrPc+++) conditions. No di-glycosylated protein bands were visible under normoxic and hypoxic conditions in the control cells (designated as control nx. PrPc+ and 90 min hx. PrPc+). (C) Vertical panels show PrPc (green fluorescence), LDH (red fluorescence) and the respective overlays (yellow fluorescence). Cell nuclei are stained blue. Non-transfected control cells under normoxic conditions (horizontal panels designated as PrPc+ nx.) show no visible co-localization of both proteins. On the contrary, a partial co-localization was already detected in non-transfected control cells under hypoxic conditions (horizontal panels designated as PrPc+ 90 min hx.). Furthermore, a marked co-localization presented as punctuate fluorescence signal was seen in PrPc-overexpressing HEK293 cells under normoxic (horizontal panels designated as PrPc+++ nx.) and hypoxic (horizontal panels designated as PrPc+++ 90 min hx.) conditions. The scale bar is 20 μm. Pearson's linear correlation coefficient (rp) shows the numerical values of co-localization for both proteins which is significant under hypoxic conditions (Table 1). (D) Relative LDH activity expressed in arbitrary units (a.u.) is significantly higher in PrPc-overexpressing cells under hypoxic conditions (designated as 90 min hx. PrPc+++) as compared to both PrPc-overexpressing cells under normoxic (designated as control nx. PrPc+++) (p = 0.017) and control cells under hypoxic conditions (designated as 90 min hx. PrPc+) (p = 0.007). Levels of significance: *, p b 0.05; **, p b 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

U

351 352

7

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

8

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

B input

IP

control nx. 90 min hx. 90 min hx. control nx. PrPc+ PrPc+++ PrPc+++ PrPc+

35

29

C

PrPc

Merge

F O

PrP

LDH

c+

R O

nx.

PrPc+

P

90 min hx.

E

D

PrPc+++

T

nx.

C

PrPc+++

R

E

90 min hx.

R

D

160

*

Relative LDH activity (a.u.)

U

N

C

O

**

140 120 100 80 60 40 20 0

control nx.

90 min hx. PrP

c+++

control nx.

90 min hx. PrP

c+

Fig. 5 (continued).

357 358 359

minutes of hypoxia (30 min hx.) resulted in a trend towards higher LDH (A + B) expression in WT as compared to Prnp0/0 neurons. However, a significant (p = 0.024) 1.8-fold increase in LDH (A + B) expression was

observed in WT vs. Prnp0/0 neurons 60 min after induction of hypoxia 360 (60 min hx.). A significant (p = 0.036) 1.7-fold LDH up-regulation in WT 361 as compared to Prnp0/0 neurons was still persistent after 90 min of 362

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

3.2. Hypoxic conditions induced more severe disruption of tubulin skeleton in Prnp0/0 vs. WT primary cortical neurons

384

Prior to proceeding with ischemic experiments, we verified a possibility of altered total LDH expression in different brain regions of WT and Prnp0/0 mice under normoxic conditions. In addition, we examined a possible effect of age on LDH expression in both experimental groups. Four different brain regions, i.e., cortex, hippocampus, olfactory bulb and cerebellum were examined for LDH expression profiles in WT and Prnp0/0 mice under normoxic conditions using Western blot analysis. No differences in LDH expression were observed between WT and Prnp0/0 mice in the tested brain regions (Fig. 2). Likewise, no differences were noticed between 3 and 20 months old WT and Prnp0/0 mice (Fig. 2).

385 386 387 388 389 390 391 392 393 394 395 396 397 398 399

C

378 379

E

376 377

3.4. LDH expression levels do not differ in brain homogenates of WT and Prnp0/0 mice under non-ischemic conditions but are markedly higher in WT vs. Prnp0/0 mice under ischemic conditions

R

374 375

t1:1 t1:2

Table 1 Co-localization degree between PrPc and LDH expressed numerically.

404 405 406 407 408 409 410 411 412 413

N C O

402 403

U

400 401

R

414

In analogy to primary cortical neurons two LDH bands with an apparent molecular weight of 36 and 37 kDa were observed on Western blots of one dimensional-resolved mouse brain homogenates, too (Fig. 3A). By Western blotting and 2-DE analysis the bands could be identified as the LDH-A (36 kDa) and the LDH-B (37 kDa) isoforms (Fig. 3B). The obtained results were in accordance with SwissProt database. Densitometric analyses of Western blots prepared from control and ischemic mouse brain homogenates showed that WT mice exhibited slightly higher though not significantly different LDH (A + B) expression as compared to Prnp0/0 mice under physiological conditions (control nx.) (Fig. 3A). However, 6 and 24 h after re-oxygenation (6 h rox. ; 24 h rox.) LDH expression was significantly higher (p = 0.008 and p = 0.043, respectively) in WT as compared to Prnp0/0 mice at both time points (Fig. 3A). Twenty-four hours after ischemia–reperfusion, total LDH up-regulation was even more prominent in WT mice than in their Prnp0/0 counterparts (2.1-fold) as compared to 6 h after reperfusion (1.8-fold). Both groups exhibited similar increasing trends in LDH

Due to the fact that we repeatedly failed to detect two LDH (A + B) bands in HEK293 cells, by using antibodies listed in section Western blot analysis, we confined to monitor only LDH-A expression under both normoxic and hypoxic conditions. The failure to detect two LDH bands in HEK293 cells could be explained by utilization of different batches of the polyclonal LDH antibody. In order to show that an increase in LDH-A expression following hypoxia could be related to the expression level of PrPc, we maintained control and PrPc-overexpressing HEK293 cells under normoxic conditions or subjected them to 90 min of hypoxia. HEK293 cells were exposed to restricted oxygen supply for 90 min and the LDH-A expression profile was verified 6 h following re-oxygenation (Fig. 5A). Densitometric analyses of Western blots demonstrated marked 1.8-fold (p = 0.039) LDH-A up-regulation after 90 min of hypoxia in PrPcoverexpressing cells (90 min hx. PrPc+++) as compared to PrPcoverexpressing cells provided with normal oxygen supply (control nx. PrPc+++) (Fig. 5A). A significant 2-fold (p = 0.029) increase in LDH-A expression was seen in PrPc-overexpressing cells as compared to the control cells (endogenous PrPc level) exposed to the same hypoxic conditions (90 min hx. PrPc+). Interestingly, no apparent change in LDH-A expression was seen in control cells between normoxic (control nx. PrPc+) and hypoxic conditions (Fig. 5A). Thus, this data clearly show a correlation between LDH-A and PrPc expression levels.

F

3.3. LDH protein expression in different brain regions under normoxic conditions is independent of PrPc and age

372 373

O

382 383

370 371

415 416 417 418 419 420 421

3.5. LDH-A levels are increased in PrPc-overexpressing HEK293 cells as com- 422 pared to HEK293 cells expressing endogenous levels of PrPc after induc- 423 tion of hypoxic stress 424

T

380 381

WT and Prnp0/0 mice-derived primary cortical neurons were either kept under normal cell culture conditions which allowed for normal oxygen consumption or were exposed to oxygen-reduced conditions either for 60 or 90 min. The tubulin labelling was performed 12 h following re-oxygenation. Fig. 1B displays abundant tubulin immunoreactivity under normoxic conditions in both WT (Fig. 1B, panel WT nx.) and Prnp0/0 (Fig. 1B, panel Prnp0/0 nx.) primary neurons. After 60 min of hypoxia no major changes of tubulin cytoskeleton structure were observed between the two groups (data not shown). However, 90 min after exposure to reduced oxygen supply tubulin staining revealed extensive disruption of tubulin cytoskeleton in Prnp0/0 neurons (Fig. 1B, panel Prnp0/0 90 min hx.) while tubulin microstructure was partially maintained in WT primary neurons (Fig. 1B, panel WT 90 min hx.) suggesting reduced extent of neuronal damage.

368 369

R O

367

P

366

expression 6 h post-ischemia as compared to their own corresponding normoxic controls. Yet, 24 h post-ischemia LDH expression is maintained increased in the brains of WT mice while concomitantly decreasing to the normoxic control levels in the brains of Prnp0/0 mice. Visualization of infarct tissue 24 h after induction of ischemia displayed significantly greater lesions in brains of Prnp0/0 as compared to WT mice (Fig. 4, Weise et al., 2006).

D

365

hypoxia (90 min hx.) (Fig. 1A). Both groups, WT and Prnp0/0, exhibited a similar trend in LDH expression 60 and 90 min after re-oxygenation as compared to their corresponding normoxic controls.

E

363 364

9

425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446

3.6. Interaction of LDH-A and PrPc in HEK293 cells as assessed by immuno- 447 precipitation and co-localization experiments 448 To additionally reinforce the finding that an observed increase in LDHA expression following hypoxia in HEK293 cells is causally linked to PrPc expression levels we performed immunoprecipitation experiments under non-hypoxic and hypoxic conditions in PrPc-overexpressing and control cells. Immunoprecipitation using monoclonal LDH-A antibody as a bait, followed by Western blotting using PrPc antibody (SAF32), revealed a clear protein band at molecular weight of 35 kDa which corresponds to a di-glycosylated form of PrPc of the total cell lysate extract (Fig. 5B, designated as input) under both normoxic and hypoxic conditions in PrPc overexpressing HEK293 cells (Fig. 5B, designated as control nx. PrPc+++ and 90 min hx. PrPc+++, respectively) suggesting an interaction of both proteins. However, no clear immunoprecipitation of a diglycosylated PrPc form was evident in HEK293 cells expressing endogenous levels of PRNP under given conditions (Fig. 5B, designated as 90 min hx. PrPc+ and control nx. PrPc+). Therefore, we further assessed a co-localization between LDH and PrPc in PrPc-overexpressing and control cells under normoxic and hypoxic conditions using fluorescence microscopy (Fig. 5C). Examination of the slides employing a fluorescence unit revealed a partial co-localization (yellow fluorescence) of PrPc (green fluorescence) and LDH (red fluorescence) in non-transfected control

t1:3

Hypoxia

PrPc

rp

Co-localization coefficient (red) M1

Co-localization coefficient (green) M2

Percentage of co-localization

t1:4 t1:5

+ −

+ +

0.841 0.545

1 0.999

1 1

3.20 1.31

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

449 450 451 452 453 454 455 456 457 458 459

460 461 462 463 464 465 466 467 468

10

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

A control nx. PrPc+

control nx. PrPc+++

90 min hx. PrPc+

90 min hx. PrPc+++

50 MCT1

37 37

PrPc

F

25

O

50

beta-actin

R O

700

*

600

P

500

D

400 300

E

200

0

control nx.

B

control nx.

90 min hx. PrP

MCT1

c+

Merge

R

PrP

c+

R

PrPc

90 min hx.

c+++

E

PrP

T

100

C

Absolute MCT1 densitometric values

37

O

nx.

C

PrPc+

U

N

90 min hx.

PrPc+++ nx.

PrPc+++ 90 min hx.

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515

524 525

O

3.7. Increased LDH activity in PrP -overexpressing HEK293 cells as compared to control cells under hypoxic conditions The difference between an intra- and an extracellular LDH activity was monitored in PrPc-overexpressing and control HEK293 cells under normoxic and hypoxic conditions. The relative LDH activity is significantly (p = 0.017) higher in PrPc-overexpressing cells subjected to hypoxia (90 min hx. PrPc+++) as compared to PrPc-overexpressing cells under normoxia (control nx. PrPc+++). Moreover, the difference in relative LDH activity is even more significant (p = 0.007) when comparing PrPc-overexpressing to control cells incubated under hypoxic conditions (90 min hx. PrPc+) (Fig. 5D). Interestingly, no differences in relative LDH activity were observed when comparing control cells under both experimental conditions.

Since MCT1 is known as a key regulator of lactate exchange and an increase in its protein expression facilitates lactate uptake under hypoxic conditions in tumour cells (Boidot et al., 2012), we examined its expression under normoxia/hypoxia in control and PrPc-overexpressing HEK293 cells. Western blot shows MCT1, PrPc and beta-actin control load expression. Each condition is represented with two lanes, each lane showing a separate experiment. Remarkably, the expression level of MCT1 was significantly (p = 0.025) higher in PrPc-overexpressing (Fig. 6A upper Western blot panel and graphic depiction, designated as control nx. PrPc+++) as compared to non-transfected control HEK293 cells (Fig. 6A upper Western blot panel and graphic depiction, designated as control nx. PrPc+). No significant differences were observed between the both under hypoxic conditions (Fig. 6A upper Western blot panel and graphic depiction, designated as 90 min hx. PrPc+++ and 90 min hx. PrPc +). Expression levels of MCT1 appear to reflect, at least partially, PrPc expression levels (Fig. 6A upper and middle Western blot panels). Beta-actin expression exhibits an equal protein load (Fig. 6A lower Western blot panel).

The fact that PrPc confers cell survival under stress conditions including hypoxia/ischemia is meanwhile widely accepted (Walz et al., 1999; McLennan et al., 2004; Weise et al., 2004). In particular, the expression level of PrPc seems to be relevant for providing metabolic adaptation to conditions of limited oxygen availability. A previous study has shown that an up-regulation of PrPc upon ischemic injury might depend on phosphorylation of hypoxia-activated transcription factor by ERK1/2 (Shyu et al., 2005b). We previously demonstrated increased postischemic phospho-Akt expression followed by decrease in caspase-3 activation in WT/Prnp0/0 mice comparison (Weise et al., 2006). However, the exact molecular mechanism responsible for attenuation of ischemic injury in WT vs. Prnp0/0 mice (McLennan et al., 2004; Weise et al., 2004; 2006) remains to be elucidated. The following facts led us to undertake the present study: I. a limited oxygen supply enhances LDH-A activation (Semenza et al., 1996), II. the LDH-A and the LDH-B isoforms have been identified as PrPc interaction partners (Rutishauser et al., 2009; Watts et al., 2009; Zafar et al., 2011) and III. the product of LDH, lactate, appears to be neuroprotective under hypoxic/ischemic conditions (Schurr et al., 1988, 1997, 2001; Berthet et al., 2009). Our study demonstrates for the first time PrPc enhancement of LDH protein expression and activity as well as immunoprecipitation and co-localization of both proteins under low oxygen conditions. The regulation of intracellular LDH expression via PrPc under hypoxic/ischemic conditions could be shown in vitro using primary cortical neurons and HEK293 cells, as well as in vivo in the brain homogenates obtained from WT and Prnp0/0 mice. Lactate dehydrogenase expression is obviously augmented by the presence of PrPc in WT neurons/mice vs. Prnp0/0 neurons/mice. The LDH up-regulation seems to be somewhat faster (trend of up-regulation is obvious already at 30 min following hypoxia in WT neurons) and a longer-lasting process (still up-regulated 24 h following 60-min of cerebral ischemia in WT mice) in WT vs. Prnp0/0 experimental models. The more rapid and enduring LDH up-regulation in WT as compared to Prnp0/0 neurons/mice might be crucial for PrPcinduced neuroprotection against hypoxic injury considering the socalled astrocyte–neuron lactate shuttle model (Pellerin and Magistretti, 1994). This model implies that under circumstances of reduced energy supply such as hypoxia/ischemia astrocytes generate high levels of LDH product lactate, which is transported via monocarboxylate transporters (MCTs) found in both astrocytic and neuronal membranes into the extracellular space. Once lactate reaches the mitochondria of neighbouring neurons it is oxidised into pyruvate. The presence of mitochondrial LDH is certain, and lactate was demonstrated to be a good substrate for the

F

c

3.8. Increased MCT1 expression level in PrPc-overexpressing vs. control HEK293 cells under normoxic conditions

523

R O

485 486

4. Discussion

P

484

516 517

D

483

Merging of PrPc (green fluorescence) and MCT1 (red fluorescence) signals employing BX51 microscope with a fluorescence unit did not demonstrate any co-localization (Fig. 6C panels PrPc + nx.; panels PrPc+ 90 min hx.; panels PrPc+++ nx. and panels PrPc+++ 90 min hx.). Notably, following PrPc overexpression under normoxic conditions (Fig. 6C panels PrPc+++ nx.) MCT1 is deposited more centrally, in the vicinity of the nucleus giving a more compact signal.

E

482

T

480 481

C

478 479

E

476 477

R

475

R

473 474

N C O

471 472

cells after 90 min of hypoxia (Fig. 5C, panels PrPc+ 90 min hx.) detected as punctuate fluorescence signals. On the contrary, no co-localization was visible in control cells under normoxic conditions (Fig. 5C, panels PrPc+ nx.). A marked co-localization of both proteins was detected in PrPcoverexpressing cells under non-hypoxic (Fig. 5C, panels PrPc+++ nx.) and hypoxic conditions (Fig. 5C, panels PrPc+++ 90 min hx.). A numerical assessment of co-localization performed using Pearson's linear correlation coefficient (−1 ≤ rp ≤ 1) confirmed a partial co-localization of LDH and PrPc in PrPc-overexpressing cells under normoxic (rp = 0.545) (Table 1) and a marked co-localization of both proteins under hypoxic conditions (rp = 0.841) (Table 1). Co-localization coefficients M1 and M2 ranged between 0 and 1, showing partially co-localized pixels of interest within each channel (Table 1). Both immunoprecipitation and colocalization experiments show an interaction of the two proteins.

U

469 470

11

Fig. 6. Western blot and densitometric analysis of MCT1 expression in control and PrPc-overexpressing HEK293 cells under normoxic and hypoxic conditions (A). Co-localization of MCT1 and PrPc in HEK293 cells under normoxic and hypoxic conditions as assessed by fluorescence microscopy (B). (A) MCT1 expression (upper panel) was markedly higher in PrPc-overexpressing HEK293 cells (designated as control nx. PrPc+++) as compared to control cells (designated as control nx. PrPc+) under normoxic conditions. No differences in MCT1 expression were observed between control (designated as 90 min hx. PrPc+) and PrPc-overexpressing cells after 90-min of hypoxia (designated as 90 min hx. PrPc+++). Noteworthy, MCT1 expression levels seems to partially reflect the PrPc expression levels (middle panel). An equal protein load (40 μg) is shown by beta-actin expression (lower panel). Please note that the first two lanes of the Western blot picture were swapped due to clarity reasons (marker was originally loaded into second lane of the gel). The Western blot is a representative of four independent experiments. The Western blot shows two lanes per condition, each lane represents a separate experiment. Densitometric analysis shows significantly (p = 0.025) higher MCT1 expression in PrPc-overexpressing (grey bars) vs. control HEK293 cells (black bars) under normoxic conditions. No significant differences were observed under hypoxic conditions. All densitometric measurements were presented as absolute values (×103). Each bar represents a mean value of MCT1 content with corresponding standard deviations. Densitometric analyses were performed from three different Western blots. Level of significance: *, p b 0.05. (B) Vertical panels show PrPc (green fluorescence), MCT1 (red fluorescence) and the respective overlays. Cell nuclei are stained blue. No site overlapping was detected between the proteins under either condition. Horizontal panels designated as PrPc+ nx. show non-transfected control cells under normoxic whereas panels designated as PrPc+ 90 min hx. show control cells under hypoxic conditions. Likewise, co-localization between the proteins was neither visible in PrPc-overexpressing cells under normoxic (horizontal panels designated as PrPc+++ nx.) nor under hypoxic (horizontal panels designated as PrPc+++ 90 min hx.) conditions. Please note a compact deposition of MCT1 protein in the vicinity of nuclei following PrPc overexpression only under normoxic conditions (horizontal panels designated as PrPc+++ nx.). The scale bar is 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

518 519 520 521 522

526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631

668

O

F

Conflict of interest

R O

587 588

P

585 586

632 633

D

583 584

demonstrated that nearly 40% of sCJD-specifically regulated proteins are implicated in glycolysis/glucose metabolism (Gawinecka et al., 2010). In addition, increased lactate levels were also measured in human Huntington's disease (HD) striata (Harms et al., 1997) and the LDH inhibitor, oxamate, elicited HD neurodegeneration when delivered intra-striatially to healthy mice (Fox et al., 2007). A new cutting-edge study showed that exposure of neurons to highly toxic misfolded prion protein results in neuronal death via depletion of intracellular NAD+ (nicotinamide adenine dinucleotide) stores followed by decreased ATP production which could be reversed in vitro and in vivo by NAD+ replenishment (Zhou et al., 2015). Interestingly, administration of NAD+ decreases ischemic brain damage in a mouse model of brain ischemia (Zheng et al., 2012) and NAD+ is produced during conversion of pyruvate to lactate by LDH under low oxygen conditions. Taking our data together with investigations of other authors, it is possible to envision that PrPc associated up-regulation of intracellular LDH (A + B), observed under hypoxic/ischemic conditions in WT as compared to Prnp0/0 mice/neurons might lead to an enhanced lactate production (LDH activity is significantly higher in PrPc-overexpressing vs. control HEK293 cells under hypoxic conditions). We observed an increased intracellular LDH (A + B) expression in both primary neurons and whole brains of WT vs. Prnp0/0 mice. Thus, increased levels of lactate produced by the WT astrocytes might be transported via MCT1, shown to be up-regulated by PrPc overexpression but not by hypoxia, to neurons resulting in an increased energy generation in WT vs. Prnp0/0 neurons. Moreover, as already indicated Prnp0/0 neurons might have an impaired capacity for lactate uptake due to a significant downregulation of basigin (Stella et al., 2012). Such a succession of events could ultimately explain the reduced lesions of WT mice as compared to Prnp0/0 mice under ischemic conditions. To further verify the above presumption additional studies employing selective inhibitors of LDH/ lactate transport are mandatory. The presented results demonstrating a direct interaction of PrPc with LDH under low oxygen conditions may prove important not only in the field of prion diseases, but neurodegeneration in general and will certainly open up new avenues for further studies on this issue.

T

581 582

C

579 580

E

577 578

R

575 576

R

573 574

O

572

C

570 571

N

568 569

mitochondrial tricarboxylic acid cycle (Brandt et al., 1987; Brooks et al., 1999). Although the regulation of cytosolic LDH protein expression by PrPc under hypoxic/ischemic conditions is indeed intriguing, immunoprecipitation of PrPc by LDH under hypoxic conditions and co-localization of both proteins in HEK293 cells additionally corroborate this assumption. Immunocytochemical staining performed in this study revealed that PrPc and LDH co-localize not only in the vicinity of the cell membrane but, rather throughout the cell, especially under hypoxic conditions. Remarkably, a recent study monitored PrPc accumulation in the nucleus of neuronal cells subjected to genotoxic stress (Bravard et al., 2014). Cytosolic PrPc has been also described in subpopulation of neurons and in pancreatic β-cells (Mironov et al., 2003; Strom et al., 2007). In line with our observations, earlier report demonstrated that PrPc interacts with another glycolytic enzyme, aldolase C (Strom et al., 2006). Besides, PrPc appears to support the Warburg effect, characterized by a preference of aerobic glycolysis, in colorectal cancer cells through regulation of glucose transporter 1 (Glut1) expression via Fyn-HIF (hypoxia-inducible factor)-2α pathway. Kinetic analysis demonstrated that depletion of PrPc significantly inhibited glycolysis and glucose uptake in cancer cells referred to above (Li et al., 2011). Others reported that knock-down of HIF-1α induced down-regulation of PrPc mRNA and protein expression in neurons under hypoxic conditions and rendered them susceptible to neurotoxic prion peptide (106–126)induced cell death. Nonetheless, although hypoxia increased HIF1α protein expression in PrPc knockout hippocampal neurons these neurons succumbed the cell death and could be rescued only after the introduction of Prnp gene into the knock-out neurons (Jeong et al., 2012). Therefore, the mechanisms of PrPc regulation by HIF-1α and/or HIF-2α still need to be clarified. We observed a marked up-regulation of MCT1 following overexpression of PrPc in HEK293 cells under normoxic but not under hypoxic conditions. As MCT1 does not possess hypoxia-responsive elements (HRE) in its promotor region, unchanged MCT1 expression levels under hypoxic conditions were anticipated. We opted to monitor MCT1 expression as being one of the most ubiquitously expressed MCTs (Halestrap and Wilson, 2012) and the only one involved (alone or in combination with other MCTs) in all decisive processes in neuronal tissues such as lactate uptake from blood via endothelium into the interstitial fluid, lactate efflux from astrocytes and lactate uptake by neurons. An up-regulation of MCT1 in tumour cells was shown to be associated with survival advantage due to lactate uptake (Boidot et al., 2012). Hence, one can surmise that increased expression levels of MCT1 in PrPc-overexpressing as compared to control cells might provide PrPcoverexpressing cells with survival advantage when encountering hypoxic stress. Beyond this, glucose starvation appears to enhance both PrPc and MCT1 expression in vitro (Shyu et al., 2005a; De Saedeleer et al., 2014). On the contrary, hyperglycemia suppresses PrPc expression in islet beta cells in vivo (Strom et al., 2007). Noteworthy, a late study demonstrated a significant decline in GLUT 3 levels in brain tissues of experimentally infected scrapie animals and cell lines suggesting an even broader role for PrPc in glucose homeostasis (Yan et al., 2013). Kleene et al. provided relevant in vitro data on the interplay between PrPc, glutamate receptor 2, α2/β2-adenosine triphosphatase (ATPase), basigin and MCT1 in regulating lactate transport of astrocytes. The authors postulated that this interplay might be functional in the metabolic cross-talk between astrocytes and neurons, especially under stress conditions. Importantly, basigin, which directly stimulates lactate uptake, via its interaction with MCT1, was significantly down-regulated in cerebellar granule neurons of Prnp0/0 as compared to WT mice (Stella et al., 2012). This observation leaves the possibility that lactate uptake in Prnp0/0 neurons might be impaired as compared to WT neurons. Interestingly, lactate levels appear to be increased in brains of sCJD (sporadic Creutzfeldt-Jakob disease) patients (Fujita et al., 2011). Moreover, total LDH levels and LDH-A activities in CSF (cerebrospinal fluid) of CJD patients were shown to be significantly higher than in other dementias (Schmidt et al., 2004) and proteomic characterization of CSF

U

566 567

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

E

12

The authors declare no competing financial interests.

634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667

669

Acknowledgments

670

This work was supported by a grant from the European Commission: Protecting the food chain from prions: shaping European priorities through basic and applied research (PRIORITY, N°222887) Project number: FP7-KBBE-2007-2A. The funding source had no involvement in the study design nor in the analysis, interpretation of data or writing of the manuscript.

671

References

677

Berthet, C., Lei, H., Thevenet, J., Gruetter, R., Magistretti, P.J., Hirt, L., 2009. Neuroprotective role of lactate after cerebral ischemia. J. Cereb. Blood Flow Metab. 240, 1780–1789. Boidot, R., Végran, F., Meulle, A., Le Breton, A., Dessy, C., Sonveaux, P., Lizard-Nacol, S., Feron, O., 2012. Regulation of monocarboxylate transporter MCT1 expression by p53 mediates inward and outward lactate fluxes in tumors. Cancer Res. 72, 939–948. Bounhar, Y., Zhang, Y., Goodyer, C.G., LeBlanc, A.C., 2001. Prion protein protects human neurons against Bax-mediated apoptosis. J. Biol. Chem. 276, 39145–39149. Brandt, R.B., Laux, J.E., Spainhour, S.E., Kline, E.S., 1987. Lactate dehydrogenase in rat mitochondria. Arch. Biochem. Biophys. 259, 412–422. Bravard, A., Auvré, F., Fantini, D., Bernardino-Sgherri, J., Sissoëff, L., Daynac, M., Xu, Z., Etienne, O., Dehen, C., Comoy, E., et al., 2014. The prion protein is critical for DNA repair and cell survival after genotoxic stress. Nucleic Acids Res. http://dx.doi.org/ 10.1093/nar/gku1342. Brooks, G.A., Dubouchaud, H., Brown, M., Sicurello, J.P., Butz, C.E., 1999. Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle. Proc. Natl. Acad. Sci. U. S. A. 96, 1129–1134.

678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

672 673 674 675 676

S. Ramljak et al. / Experimental Neurology xxx (2015) xxx–xxx

829

N C O

R

R

E

C

D

P

R O

O

F

Schurr, A., Payne, R.S., Miller, J.J., Tseng, M.T., Rigor, B.M., 2001. Blockade of lactate transport exacerbates delayed neuronal damage in a rat model of cerebral ischemia. Brain Res. 895, 268–272. Semenza, G.L., Jiang, B.H., Leung, S.W., Passantino, R., Concordet, J.P., Maire, P., Giallongo, A., 1996. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J. Biol. Chem. 271, 32529–32537. Shaw, G., Morse, S., Ararat, M., Graham, F.L., 2002. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 16, 869–871. Shyu, W.C., Chen, C.P., Saeki, K., Kubosaki, A., Matusmoto, Y., Onodera, T., Ding, D.C., Chiang, M.F., Lee, Y.J., Lin, S.Z., et al., 2005a. Hypoglycemia enhances the expression of prion protein and heat-shock protein 70 in a mouse neuroblastoma cell line. J. Neurosci. Res. 80, 887–894. Shyu, W.C., Lin, S.Z., Chiang, M.F., Ding, D.C., Li, K.W., Chen, S.F., Yang, H.I., Li, H., 2005b. Overexpression of PrPc by adenovirus-mediated gene targeting reduces ischemic injury in stroke rat model. J. Neurosci. 25, 8967–8977. Spudich, A., Frigg, R., Kilic, E., Kilic, U., Oesch, B., Raeber, A., Bassetti, C.L., Hermann, D.L., 2005. Aggravation of ischemic brain injury by prion protein deficiency: role of ERK1/-2 and STAT-1. Neurobiol. Dis. 20, 442–449. Stella, R., Cifani, P., Peggion, C., Hansson, K., Lazzari, C., Bendz, M., Levander, F., Sorgato, M.C., Bertoli, A., James, P., 2012. Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons. J. Proteome Res. 11, 523–536. Strom, A., Diecke, S., Hunsmann, G., Stuke, A.W., 2006. Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry. Proteomics 6, 26–34. Strom, A., Wang, G.S., Reimer, R., Finegood, D.T., Scott, F.W., 2007. Pronounced cytosolic aggregation of cellular prion protein in pancreatic beta-cells in response to hyperglycemia. Lab. Invest. 87, 139–149. Walz, R., Amaral, O.B., Rockenbach, I.C., Roesler, R., Izquierdo, I., Cavalheiro, E.A., Martins, V.R., Brentani, R.R., 1999. Increased sensitivity to seizures in mice lacking cellular prion protein. Epilepsia 40, 1679–1682. Watts, J.C., Huo, H., Bai, Y., Ehsani, S., Jeon, A.H., Shi, T., Daude, N., Lau, A., Young, R., Xu, L., et al., 2009. Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperons. PLoS Pathog. 5. http://dx.doi.org/10.1371/journal.ppat.1000608. Weise, J., Crome, O., Sandau, R., Schulz-Schaeffer, W., Bahr, M., Zerr, I., 2004. Upregulation of cellular prion protein (PrPc) after focal cerebral ischemia and influence of lesion severity. Neurosci. Lett. 372, 146–150. Weise, J., Sandau, R., Schwarting, S., Crome, O., Wrede, A., Schulz-Schaeffer, W., Zerr, I., Bahr, M., 2006. Deletion of cellular prion protein results in reduced Akt activation, enhanced post-ischemic caspase-3 activation, and exacerbation of ischemic brain injury. Stroke 37, 1296–1300. Wong, B.S., Liu, T., Li, R., Pan, T., Petersen, R.B., Smith, M.A., Gambetti, P., Perry, G., Manson, J.C., Brown, D.R., et al., 2001. Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein. J. Neurochem. 76, 565–572. Yan, Y.E., Zhang, J., Wang, K., Xu, Y., Ren, K., Zhang, B.Y., Shi, M., Chen, C., Shi, Q., Tian, C., et al., 2013. Significant reduction of the GLUT3 level, but not GLUT1 level, was observed in the brain tissues of several scrapie experimental animals and scrapie-infected cell lines. Mol. Neurobiol. http://dx.doi.org/10.1007/s12035-013-8574-8. Zafar, S., von Ahsen, N., Oellerich, M., Zerr, I., Schulz-Schaeffer, W.J., Armstrong, V.W., Asif, A.R., 2011. Proteomics approach to identify the interacting partners of cellular prion protein and characterization of Rab7a interaction in neuronal cells. J. Proteome Res. 10, 3123–3135. Zanata, S.M., Lopes, M.H., Mercadante, A.F., Hajj, G.N., Chiarini, L.B., Nomizo, R., Freitas, A.R., Cabral, A.L., Lee, K.S., Juliano, M.A., et al., 2002. Stress-inducible protein 1 is a cell surface ligand for cellular prion that triggers neuroprotection. EMBO J. 21, 3307–3316. Zheng, C., Han, J., Xia, W., Shi, S., Liu, J., Ying, W., 2012. NAD+ administration decreases ischemic brain damage partially by blocking autophagy in a mouse model of brain ischemia. Neurosci. Lett. 512, 67–71. Zhou, M., Ottenberg, G., Sferrazza, G.F., Hubbs, C., Fallahi, M., Rumbaugh, G., Brantley, A.F., Lasmézas, C.I., 2015. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment. Brain http://dx.doi.org/10.1093/brain/awv002.

E

T

Brown, D.R., Schulz-Schaeffer, W.J., Schmidt, B., Kretzschmar, H., 1997. Prion proteindeficient cells show altered response to oxidative stress due to decreased SOD-1 activity. Exp. Neurol. 146, 104–112. Bueler, H.R., Fischer, M., Lang, Y., Bluethmann, H., Lipp, H.P., DeArmond, S.J., Prusiner, S.B., Aguet, M., Weissmann, C., 1992. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577–582. De Saedeleer, C.J., Porporato, P.E., Copetti, T., Pérez-Escuredo, J., Payen, V.L., Brisson, L., Feron, O., Sonveaux, P., 2014. Glucose deprivation increases monocarboxylate transporter 1 (MCT1) expression and MCT1-dependent tumor cell migration. Oncogene 33, 4060–4068. Fox, J.H., Kama, J.A., Lieberman, G., Chopra, R., Dorsey, K., Chopra, V., Volitakis, I., Cherny, R.A., Bush, A.I., Hersch, S., 2007. Mechanisms of copper ion mediated Huntington's disease progression. PLoS ONE http://dx.doi.org/10.1371/journal.pone.0000334. Fujita, K., Harada, M., Yuasa, T., Sasaki, M., Izumi, Y., Kaji, R., 2011. Temporal evolution of sporadic Creutzfeldt-Jakob disease monitored by 3-Tesla MR spectroscopy. J. Neurol. 258, 1368–1370. Gawinecka, J., Dieks, J., Asif, A.R., Carimalo, J., Heinemann, U., Streich, J.H., Dihazi, H., Schulz-Schaeffer, W., Zerr, I., 2010. Codon 129 polymorphism specific cerebrospinal fluid proteome pattern in sporadic Creutzfeldt-Jakob disease and the implication of glycolytic enzymes in prion-induced pathology. J. Proteome Res. 9, 5646–5657. Halestrap, A.P., Wilson, M.C., 2012. The monocarboxylate transporter family-role and regulation. IUBMB Life 64, 109–119. Harms, L., Meierkord, H., Timm, G., Pfeiffer, L., Ludolph, A.C., 1997. Decreased N-acetylaspartate/choline ratio and increased lactate in the frontal lobe of patients with Huntington's disease: a proton magnetic resonance spectroscopy study. J. Neurol. Neurosurg. Psychiatry 62, 27–30. Jeong, J.K., Seo, J.S., Moon, M.H., Lee, Y.J., Seol, J.W., Park, S.Y., 2012. Hypoxia-inducible factor-1α regulates prion protein expression to protect against neuron cell damage. Neurobiol. Aging 33, 1006.e1-10. http://dx.doi.org/10.1016/j.neurobiolaging.2011. 09.037. Kleene, R., Loers, G., Langer, J., Frobert, Y., Buck, F., Schachner, M., 2007. Prion protein regulates glutamate-dependent lactate transport of astrocytes. J. Neurosci. 27, 12331–12340. Kuwahara, C., Takeuchi, A.M., Nishimura, T., Haraguchi, K., Kubosaki, A., Matsumoto, Y., Saeki, K., Yokoyama, T., Itohara, S., Onodera, T., 1999. Prions prevent neuronal cell-line death. Nature 400, 225–226. Li, Q.Q., Sun, Y.P., Ruan, C.P., Xu, X.Y., Ge, J.H., He, J., Xu, Z.D., Wang, Q., Gao, W.C., 2011. Cellular prion protein promotes glucose uptake through Fyn-HIF-2α-Glut1 pathway to support colorectal cancer cell survival. Cancer Sci. 102, 400–406. McLennan, N.F., Brennan, P.M., McNeill, A., Davies, I., Fotheringham, A., Rennison, K.A., Ritchie, D., Brannan, F., Head, M.W., Ironside, J.W., et al., 2004. Prion protein accumulation and neuroprotection in hypoxic brain damage. Am. J. Pathol. 165, 227–235. Mironov Jr., A., Latawiec, D., Wille, H., Bouzamondo-Bernstein, E., Legname, G., Williamson, R.A., Burton, D., DeArmond, S.J., Prusiner, S.B., Peters, P.J., 2003. Cytosolic prion protein in neurons. J. Neurosci. 23, 7183–7193. Mitteregger, G., Vosko, M., Krebs, B., Xiang, W., Kohlmannsperger, V., Nölting, S., Hamann, G.F., Kretzschmar, H.A., 2007. The role of the octarepeat region in neuroprotective function of the cellular prion protein. Brain Pathol. 17, 174–183. Mouillet-Richard, S., Ermonval, M., Chebassier, C., Laplanche, J.L., Lehmann, S., Launay, J.M., Kellermann, O., 2000. Signal transduction through prion protein. Science 289, 1925–1928. Pellerin, L., Magistretti, P.J., 1994. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc. Natl. Acad. Sci. U. S. A. 91, 10625–10629. Ramljak, S., Asif, A.R., Armstrong, V.W., Wrede, A., Groschup, M.H., Buschmann, A., SchulzSchaeffer, W., Bodemer, W., Zerr, I., 2008. Physiological role of the cellular prion protein (PrPc): protein profiling study in two cell culture systems. J. Proteome Res. 7, 2681–2695. Rutishauser, D., Mertz, K., Moos, R., Brunner, E., Ruelicke, T., Calella, A.M., Aguzzi, A., 2009. The comprehensive native interactome of a fully functional tagged prion protein. PLoS ONE http://dx.doi.org/10.1371/journal.pone.0004446. Schmidt, H., Otto, M., Niedmann, P., Cepek, L., Schroter, A., Kretzschmar, H.A., Poser, S., 2004. CSF lactate dehydrogenase activity in patients with Creutzfeldt-Jakob disease exceeds that in other dementias. Dement. Geriatr. Cogn. Disord. 17, 204–206. Schurr, A., Dong, W.Q., Reid, K.H., West, C.A., Rigor, B.M., 1988. Lactic acidosis and recovery of neuronal function following cerebral hypoxia in vitro. Brain Res. 438, 311–314. Schurr, A., Payne, R.S., Miller, J.J., Rigor, B.M., 1997. Brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon re-oxygenation: an in vitro study. Brain Res. 744, 105–111.

U

694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762

13

Please cite this article as: Ramljak, S., et al., Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions, Exp. Neurol. (2015), http://dx.doi.org/10.1016/j.expneurol.2015.04.025

763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828