Quantitative Proteomic Analysis of the Orbital Frontal Cortex in Rats

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Quantitative proteomic analysis of the orbital frontal cortex in rats following extended exposure to caffeine reveals extensive changes to protein expression: implications for neurological disease Jane L. Franklin, Mehdi Mirzaei, Travis A. Wearne, Judi Homewood, Ann K. Goodchild, Paul A. Haynes, and Jennifer L. Cornish J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01043 • Publication Date (Web): 04 Mar 2016 Downloaded from http://pubs.acs.org on March 6, 2016

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Journal of Proteome Research

Quantitative proteomic analysis of the orbital frontal cortex in rats following extended exposure to caffeine reveals extensive changes to protein expression: implications for neurological disease Jane L. Franklin 1, Mehdi Mirzaei 2, Travis A. Wearne 1, Judi Homewood 3, Ann K. Goodchild 4, Paul A. Haynes 2, Jennifer L. Cornish* 1 1

Department of Psychology, Macquarie University, North Ryde, NSW 2109, Australia

2

Department of Chemistry and Biomolecular Sciences, Macquarie University, North Ryde, NSW 2109, Australia 3

Faculty of Human Sciences, Macquarie University, North Ryde, NSW 2109, Australia

4

Department of Biomedical Sciences, Macquarie University, North Ryde, NSW 2109, Australia

*CORRESPONDING AUTHOR Associate Professor Jennifer L. Cornish, Department of Psychology, Macquarie University, North Ryde, NSW 2109, Australia. Email: [email protected] Telephone (+612) 9850 1185 Fax: (+612) 9850 7759 Keywords: caffeine / cytoskeletal regulation / nanoflow LC-MS/MS/ orbitofrontal cortex / proteomics/ synaptic plasticity / neurological disease

ACS Paragon Plus Environment


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Abstract Caffeine is a plant-derived psychostimulant and a common additive found in a wide range of foods and pharmaceuticals. The orbitofrontal cortex (OFC) is rapidly activated by flavours, integrates gustatory and olfactory information, and plays a critical role in decision-making, with dysfunction contributing to psychopathologies and neurodegenerative conditions. This study investigated whether long-term consumption of caffeine causes changes to behavior and protein expression in the OFC. Male adult Sprague Dawley rats (n=8 per group) were treated for 26 days with either water or a 0.6 g/L caffeine solution. Locomotor behaviour was measured on the first and last day of treatment, then again after 9 days treatment free following exposure to a mild stressor. When tested drug free, caffeine-treated animals were hyperactive compared to controls. Two hours following final behavioural testing, brains were rapidly removed and prepared for proteomic analysis of the OFC. Label free shotgun proteomics found 157 proteins differentially expressed in the caffeine-drinking rats compared to control. Major proteomic effects were seen for cell-to-cell communication, cytoskeletal regulation and mitochondrial function. Similar changes have been observed in neurological

disorders

including

Alzheimer’s

disease,

schizophrenia.


Parkinson’s

disease

and

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Abbreviations ACN, Acetonitrile; AD, Alzheimer's disease; ADD2, Adducin 2 (beta); ADHD, attention deficit hyperactivity disorder; ADORA1, A1, Adenosine A1 receptor; ADORA2A, A2A, Adenosine A2A receptor; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ANP32A, Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A, Aβ, Amyloid-beta; APP, Amyloid Beta (A4) Precursor Protein; CADPS2, Ca++-dependent secretion activator 2; CAF 0.6g/l caffeine solution; CALB2, Calbindin; CAMK2B, Calcium/calmodulin-dependent protein kinase II alpha; CANX, Calnexin; CDK5, Cyclindependent kinase 5; control, Tap water; DLG4, Discs, large homolog 4 (Drosophila); DSTN, Destrin (actin depolymerizing factor), DTT, Dithiothreitol; EDTA, Ethylenediaminetetraacetic acid; FDR, False discovery rate; HTT, Huntingtin; i.p., Intraperitoneal; IPA, Ingenuity Pathway Analysis; ITPR1, Inositol 1,4,5-trisphosphate receptor, type 1; KIF5A, Kinesin family member 5A; LDT, Last day of treatment day 26; MAPT, Microtubule-associated protein tau; MFN2, mitofusin 2; MTOR, Mechanistic target of rapamycin (serine/threonine kinase); NF1, Neurofibromin 1, NH4HCO3, Ammonium bicarbonate NMDA, N-methyl-D-aspartate; NPTN, Neuroplastin; NPTX1, Neuronal pentraxin I solute carrier family; NSAF, Normalized spectral abundance factor; OFC, Orbitofrontal cortex; PH, Procedural habituation day, PD, Parkinson’s disease; PFC, Prefrontal cortex; PP2A, phosphatase 2A; PSEN1, Presenilin-1, RAB3A, RAB3A member RAS oncogene family; RAB6A, RAB6A, member RAS oncogene family; SC, Saline challenge day; SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SDS, Sodium dodecyl sulfate; SEM, Standard Error of the Mean; SH3GL2, SH3-domain GRB2-like 2; SLC1A2, Glial high affinity glutamate transporter, member 2; STMN1, Stathmin 1; SYN2, Synapsin II; TD1, First Day of treatment day 1; TD2, Type 2 diabetes; THY1, Thy-1 cell surface antigen; TUBB, Tubulin, beta class I; TUBB2A, Tubulin, beta 2A class IIa TUBB3, Tubulin, beta 3 class III; USP5, Ubiquitin specific peptidase 5 (isopeptidase T);



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1

INTRODUCTION

Caffeine is a bitter tasting plant-derived psychostimulant and a common additive in a wide range of foods and pharmaceuticals 1. It is arguably the world’s most commonly consumed psychoactive drug as most adults eat or drink moderate amounts of caffeinated products daily

2, 3

, yet surprisingly little is known about the mechanisms of its many biological

effects. In general, low to moderate caffeine consumption appears to be protective for liver function, type 2 diabetes (TD2), Alzheimer’s (AD) and Parkinson’s disease (PD), improves attention in animal models of attention deficit disorder (ADHD) and decision making in sleep deprived humans, and broadly has been associated with a slightly reduced cancer risk 2, 4-8

.

The psychoactive effects of caffeine derive from its ability to easily cross the blood brain barrier and are primarily the consequence of its actions as an antagonist at adenosine A1 (ADORA1) and A2A (ADORA2) receptors. Adenosine promotes and maintains sleep, regulates arousal, and plays a critical role in coupling cerebral blood flow to energy demand 9

. Caffeine administration increases arousal, stimulates locomotor activity and has either no

rewarding effect, appetitive or aversive properties depending on the dose and amount of exposure 10-13. In the prefrontal cortex (PFC), the orbitofrontal cortex (OFC) is rapidly activated by taste, mediating the hedonic response to sweet and bitter tastes as well as integrating gustatory and olfactory information. Damage to the OFC can alter these processes and furthermore affect habituation to aversive stimuli

14-17

. Data links changes in OFC functioning to a similar

range of psychological disorders and neurodegenerative conditions to those affected by caffeine exposure including ADHD, AD and PD, as well as to schizophrenia 18-22. Similarly, both administration of caffeine and OFC functioning influence decision-making 23, 24 25.


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The aim of this study was to evaluate the effect on locomotor activity in rats of chronic lowlevel caffeine consumption, approximately equivalent to the caffeine in 1 to 2 cups of coffee per day for an adult human consumer, and to employ quantitative shotgun proteomics to establish if caffeine consumption is associated with changes to protein expression in the OFC. The information obtained from pathway analysis was used to specifically examine the downstream molecular functions associated with prolonged antagonism of adenosine receptors by chronic caffeine administration.



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2 2.1

MATERIALS AND METHODS Animal handling and diet.

Sixteen adult male Sprague-Dawley rats (250-325 g; Animal Resources Centre Canning Vale, WA, Australia) were homed in groups of four in standard laboratory housing conditions, with temperature maintained at 21 ± 1 oC, lights on at 08:00 and lights off at 20:00 hr. All aspects of this study were approved by Macquarie University Animal Ethics Committee and followed the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (8th edition National Health and Medical Research Council 2013). Upon arrival, rats underwent a 3-day acclimation period, and were then handled for 3 days prior to treatment. They had ad libitum access to standard rat chow [(13/kJ/g), fat (≈ 6% energy), sugar (≈ 4.1% energy) and carbohydrate (≈ 37.2% energy)] throughout. Rats were divided into two groups, each containing 2 cages of 4 animals that were randomly assigned to receive either ad libitum tap water (control) or 0.6 g/L caffeine in tap water (CAF), for the duration of the 26-day treatment (n=8 in each treatment group). Body weight, chow and fluid consumption were measured every 3 days. 2.2

Behavioural experiments

Locomotor activity during chronic treatment was measured on the first Day (T1) and last day (LDT) of treatment in 16 identical Med Associates chambers (St Albans, VT, United States) each fitted with 4 pairs of infrared detectors. The animals were acclimated to the locomotor chambers for 12 hours with water only and food access prior to the first day of treatment. Standard lab chow and either tap water or CAF solution was provided during treatment test sessions. Locomotor activity was measured in the dark active period, in at 20:00 and out at 08:00 on the first day of treatment and again over the last 12 hours of the


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dark active period of the 26-day treatment period. Animals, treatment and chow were weighed before and after each testing session. Locomotor activity in response to a mild stressor, saline intraperitoneal injection (i.p.)

26

,

was measured 9 days post-treatment in perspex locomotor boxes of a different design and location to those used during treatment measures to prevent any motor effects of cue reexposure. To further differentiate from treatment locomotor testing, activity was measured in the light active period. Locomotor activity was recorded by automated video tracking software (Motion Mensura, Cooks Hill, NSW Australia).

Animals first underwent a

procedural habituation (PH) day, followed by a saline challenge day. On the PH test, animals were first acclimated to the boxes for 15 minutes, removed and injected with saline (0.9%, 1 mL/kg i.p.) and replaced into the chamber for locomotor behaviour measurements over a further 60 minutes. For the challenge day (CD) this procedure was repeated, animals were injected again with saline as a mild stressor 26 and then behaviour was measured for 60 minutes. Two hours after the saline challenge, rats were anaesthetised with an i.p. injection of 1 mL pentobarbitone sodium 325 mg/mL that was diluted with 1 mL saline (Virbac, Milperra, Australia). Once non-responsive to tail pinch, they were decapitated by guillotine. The brains were rapidly removed, snap frozen in liquid nitrogen then stored at -80 °C. A brain matrix was used to remove 1 mm thick coronal sections at the level of the OFC 4.2 mm rostral to bregma 27. The medial, ventral lateral and dorsolateral orbital frontal cortices were isolated for proteomic analysis. Using a dissecting microscope, structural landmarks visible on both sides of the 1 mm slice were compared to Paxinos brain atlas images to ensure collection of tissue only within the boundary of the OFC

27

. The OFC samples were

homogenized in a buffer (0.32 M sucrose, 2 mM EDTA, with 1% SDS) with a dounce



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homogenizer and then centrifuged at 13,000 rpm at 4oC for 15 minutes. The supernatant was stored at -80 °C until prepared for proteomic analysis. 2.3

Protein extraction, fractionation and digestion.

Aliquots of protein from the supernatant were combined with a 5 x SDS-PAGE sample buffer containing 200 mM DTT. For the proteomic study, three biological replicates were randomly selected from the control and CAF animals that were included in the behavioural analyses. Proteins were separated by electrophoresis on a Bio-Rad 10% Tris-HCl precast gel. Protein concentration in the prepared extracts was estimated using a micro-BCA assay. Volumes were adjusted and 100 µg of each extract was loaded onto each lane of the SDSpage gels. The high degree of reproducibility in raw numbers across the replicates confirms the amount of protein loaded onto the gels was consistent. The gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad) for 1 hour, and then de-stained overnight in Milli-Q water. Each gel lane was divided into 16 equal pieces and further chopped into smaller pieces, which were then transferred into a 96 well plate: 16 wells (fractions) for each biological replicate. To further destain the gel pieces, they were washed 3 times with 150 µL of ACN (50%)/100 mM NH4HCO3 (50%) for 20 minutes, then dehydrated with 100% ACN twice for 10 minutes. The samples were air dried and reduced with 50 µL of 10 mM DTT/ NH4HCO3, (50 mM) at 37oC for 1 hour, before alkylating in the dark with 50 µL of 50 mM iodiacetamide/NH4HCO3 (50 mM) at room temperature for 1 hour. The gel pieces were washed with 150 µL of ACN (50%)/100 mM NH4HCO3 (50%) for 20 minutes, 3 times, then again dehydrated with 100% ACN twice and air-dried for 10 minutes. Finally, samples were digested with 30 µL of trypsin (6.6 ng/µL in 50 mM NH4HCO3) for 30 minutes at 4°C, then incubated overnight at 37°C.


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The peptides that resulted from the trypsin digestion were extracted from each well into a labelled Eppendorf tube. Sixteen tubes were used, one for each fraction, of each rat tested. Each well was washed with 60 µL of ACN (50%) formic acid (2%) then incubated for 30 minutes at 37°C. The peptides were again extracted and added to the peptides previously extracted. This extraction process was repeated three times. Each extract was dried using a vacuum centrifuge and reconstituted to 10 µL with 2% formic acid immediately prior to analysis in the mass spectrometer. 2.4

Nanoflow liquid chromatography-tandem mass spectrometry

The 16 reconstituted fractions of each biological replicate were analyzed using nanoflow LC-MS/MS using an LTQ-XL linear ion trap mass spectrometer (Thermo, San Jose, CA) as previously described

28-30

. In a fused silica capillary with an integrated electrospray tip,

reversed-phase columns were packed in-house to approximately 7 cm (100 µm I.D.) using 100 A, 5 µm Zorbax C18 resin (Agilent Technologies, CA, USA). The tip was prepared using a Sutter Instruments P-2000 laser puller, and had a diameter of approximately 10 to 15µm. An electrospray voltage of 1.8 kV was applied via a liquid junction upstream of the C18 column. Samples were injected onto the column using a surveyor autosampler, which was followed by an initial wash step with a buffer (5% v/v ACN, 0.1% v/v formic acid) for 10 minutes at 1 µL/minutes. Peptides were eluted from the column with 0-50% buffer (95% v/v ACN, 0.1 v/v formic acid) for 58 minutes at 500 nL/minutes. The column elute was directed into the nanospray ionization source of the mass spectrometer. Spectra were scanned over the range of m/z 400-1500, using Xcalibur software (Version 2.06, Thermo) automated peak recognition, dynamic exclusion (repeat count 1, repeat duration 30 seconds, list size 500, exclusion duration 90 seconds, exclusion by mass with 1.5 Dalton tolerance) and MS/MS of the top six most intense precursor ions at 35% normalization collision energy



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were performed 31. The samples were injected in rows of 8 from the 96 well plate. Thus, 16 fractions were injected in two rows of 8 for each animal tested. The control replicates were analyzed before the tissue from the CAF animals. Standards were run before and after data acquisition to ensure optimum system performance. 2.5

Database search for protein/peptide identification.

Raw nano LC-MS/MS data files were converted to mzXML format and searched against the National Centre for Biotechnology Information Rattus Norvegicus database (30296 proteins January 2013), using the global proteome machine software (version 2.1.1) and the X!Tandem algorithm. The 16 fractions of each replicate were processed sequentially with output files for each fraction, then merged, and a nonredundant output file was generated containing protein identifications with log (e) values < -1. Carbamidomethylation of cysteine was considered as a complete modification and partial modifications included the oxidation of methionine and tryptophan. Additional searches against a reversed sequence database were carried out, allowing evaluation of the false discovery rate (FDR). For the X!Tandem searches, the mass tolerance for fragment ions was 0.4 Da, with tolerance for parent ions +3 Da and -0.5 D, and the enzyme specificity was set to trypsin. The proteins identified from the three biological replicates of the treatment conditions were further filtered using two criteria: only proteins with a total spectral count greater than or equal to 6, and also present in all replicates of at least one treatment condition, were considered to be high stringency, reproducibly identified proteins and retained for quantitation 32. 2.6

Data processing and statistical analysis

Statistical analysis of behavioural and physiological data was conducted using SPSS version 21. A General Linear Model was used for all analyses. A Greenhouse Geisser Epsilon


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adjustment was applied if the assumption of sphericity was not met. One CAF and one control rat were excluded from the procedural habituation and saline challenge data, as their activity was more than 2 standard deviations removed from the mean response. These animals were also excluded from the proteomic analysis. We used a label free quantitative approach that utilizes normalized spectral abundance factors (NSAF) to compare the relative abundance of protein in each treatment group. For each reproducibly identified protein the NSAF value was calculated, including addition of a spectral fraction of 0.5 to all counts to compensate for null values when calculating fold changes

28, 33

. To identify any difference in protein abundance the natural log NSAF values

were analyzed using a two sample Student’s t-test. The significance level was set at p .05). Exposure to caffeine did not change the rate of weight gain or the volume of chow consumed across the 26-day period (Figure 1A and B). There were no between treatment group differences measured in the average daily fluid consumed and this volume remained constant across the experiment (Figure 1C). On average the control and CAF rats daily consumed 32.7 and 31.7 ± 3.6 mL fluid, respectively. Caffeine has a bitter taste that can be aversive at higher concentrations 13, 39, however, as the volume consumed did not differ from control this suggests the taste of the solution was either not aversive or that the concentration used was below the taste threshold for the rats. Oral administration of caffeine results in comparable pharmacokinetics in humans and rats as well as equivalent blood plasma curves

10, 40

. Rats, however, metabolize caffeine faster

than humans. The concentration of caffeine was 0.6 g/L, double the g/L used in a previous study by Svenningsson et al 40, and was chosen to model moderate to high levels of caffeine consumption, and account for the different metabolic rate of rats compared to humans. The animals in this study drank less fluid than Svenningsson et al,

40

and as a consequence our

caffeine mg/kg dose [M = 53.4, SEM 1.2] ultimately modeled low-level caffeine consumption, which in a rat is ~ 60mg/kg daily. This is approximately equivalent to the caffeine in 1 to 2 cups of coffee per day for an adult human consumer 40. This study did not analyze precise caffeine levels in the blood, as caffeine levels and caffeine metabolites change as tolerance develops

40

. This study used locomotor response as an indicator that a



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pharmacologically effective dose had been achieved and tolerance to the caffeine had developed. 3.2

Caffeine increased locomotor behaviour in the first 12 hours of treatment with tolerance to this effect evident in the last 12 hours of treatment

Caffeine exposure increased locomotor activity over the first 12 hours of exposure compared to control [F (1,14) = 12.074, p = 0.004.] the stimulatory effect in the CAF rats developed over the first locomotor session, with the difference to control becoming significant by the 4th hour of exposure, peaking in the 5th before reducing to match levels measured in the first hour. A second peak in activity is evident in the 10th hour of testing (Figure 2A). Tolerance to the stimulatory effect of caffeine was evident as there were no between group differences in total locomotor activity measured over the 12 hours dark active period on the last day of treatment (LDT). However, there is a pattern of high and low activity evident in the control animals, whereas the CAF animals after a period of lower activity remain consistently active between the 4th to 10th hours of testing. In the 11th hour of testing there is a sharp significant increase in activity in the CAF animals, 1 hour earlier than the increase in activity observed in controls (Figure 2B). The locomotor stimulant response on T1 and the development of tolerance following extended caffeine exposure are consistent with previous findings

40-42

. Although there was

no difference in total activity in the CAF rats compared to control over the 12 hour dark active LDT testing session, there was evidence that long-term exposure to low dose caffeine can promote low level constant activity and disrupt the normal cycle of peaks and troughs of activity that were observed in the control rats (Figure 2B). If replicated in humans, these


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data have particular implications as changes to activity levels could have consequences for effective learning 43, 44. 3.3

Caffeine treated rats are hyperactive when treatment free

After 9 days treatment free and 24 hours after the procedural habituation day (PH) and on the final saline challenge day, the CAF treated rats were significantly more active than control [F (1, 12) = 9.225, p = 0.01] (Figure 3A and C). This increase in activity measured in the CAF rats was also evident on the PH [F (1, 12) = 23.086, p < 0.0001] (Figure 3A and B). Comparing the difference between total locomotor activity revealed a significant decrease in activity in control rats from the PH to saline challenge (SC) test, suggesting the rats had habituated to the testing conditions. In contrast for the CAF animals there was no difference measured in total active seconds between PH and SC tests, suggesting a resistance to habituation (Figure 3A). In both the PH and SC test sessions, to reduce activity induced by novelty and introduction to the test chamber, all rats were placed into the testing chambers for 15 minutes prior to the saline challenge. When the PH was compared to the SC, the difference in how the CAF and control animals respond to the equipment in the 15 minutes before the i.p. injection and the locomotor response immediately after the injection is particularly revealing. On the PH, the first exposure to the test equipment, the highest activity measured was across the first 15 minutes of habituation to the equipment and before the injection. The control animals’ activity dropped 10 minutes after the injection with activity remaining lower than CAF throughout testing. Activity in the CAF rats also dropped in the first 10 minutes, though not as much as control, however 20 minutes after the injection the CAF rats activity increased before returning to levels similar to those measured at 10 minutes post -injection.



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Locomotor activity in the CAF rats did not fall to match that measured in the control until 40 minutes after the injection (Figure 3B). On the SC in the first 15 minutes, activity in the control rats reduced whereas the CAF rats remained more active. A peak in activity is clearly evident in the 5 minutes post the saline i.p. injection in the control rats, not seen in the controls, which may be attributable to higher overall activity. The control rats show a marked locomotor decrease, with activity of the CAF rats also dropping in the 5 minutes post injection, but not to the extent of the control animals, and they remain more active for the rest of the session. Changes in activity and other withdrawal symptoms in humans and rodents vary dependent on the caffeine dose, peaking at 48 hours

45-48

. However, with exception to withdrawal

headaches, these symptoms generally resolve after 4 to 5 days. Locomotor depression during caffeine withdrawal has been observed in humans and rodents

45, 48-50

. In this study,

the extended 9-day treatment free period and hyperactivity to a stressor, as opposed to locomotor depression, suggest that the behavioural response to the saline challenge in CAF rats is unlikely to be a consequence of withdrawal. Increased activity has been measured in rats with OFC lesions and furthermore, the OFC plays a critical role in habituation, including habituation to mildly aversive stimuli

17, 51, 52

.

The locomotor activity on the PH, and the absence of reduced locomotor behaviour on the SC, suggests the CAF rats may not be habituating to the testing equipment or the saline injection. Beyond this lack of habituation, the CAF rats were more hyperactive on both treatment free test days, which may reflect altered OFC function. Evidence for this was found in the proteomic data, however it is not possible to make a conclusive statement as to whether these changes are indicative of a reduction in OFC activity or whether it would be of sufficient magnitude to explain the behavioural change measured. The OFC has not been


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reported to directly mediate motor control, however, it has dense projections to other nuclei that are well known for regulating locomotor activity, such as the ventral tegmental area and medial prefrontal cortex 53, 54. As the treatment was systemic, changes to protein expression in the other key locomotor nuclei known to drive the psychostimulatory effects of caffeine could also be mediating this treatment free hyperactivity. This, in turn, may drive changes to protein expression in the OFC as a consequence of altered input.

Thus, while our

examination of the OFC cannot conclusively explain what mechanisms underlie the hyperactivity observed, this behavioural evidence suggests CAF exposure can result in persistent neural adaptations. 3.4

Five are implicatedCaffeine treatment changed more than 12% of the proteins in the OFC

Label free quantitative shotgun proteomic analyses reproducibly identified a total of 1246 non-redundant proteins in the OFC. This included 1041 proteins from the control rats and 1107 proteins from the CAF rats. The protein and peptide FDR were 0.004% and 0.0003% respectively. CAF exposure up-regulated 52 proteins and down-regulated 105 with 1089 proteins statistically unchanged relative to control (Tables 1, 2 and S1). It is worth noting that the 157 statistically significant differentially expressed proteins include 23 proteins that were reproducibly present in one condition and not detected in the other. This does not necessarily prove that they are absent from one condition, as it remains possible that such proteins are below the level of detection in one condition and detected in the other. Nonetheless, these proteins may be worthy of particular attention in follow up studies. Shotgun proteomic studies are exploratory in nature and thus cannot make causal statements without additional experimental testing that directly manipulate the proteins of interest. Furthermore, it is not possible to make definitive statements about the biological



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significance of the differentially expressed proteins, regardless of the magnitude of the fold change, until the range of normal variation in expression is known for the protein in question. Thus, while our examination of the differentially expressed proteins has focused on a pathway analysis approach, a limited number of differentially expressed proteins will be discussed in order to explore a biological process or pathway identified as important consequences of CAF treatment. This focus on pathways rather than proteins informed the decision not to include western blotting analysis to validate the data, as it is clearly not practical to validate all the proteins present in a pathway. Western blotting is a valuable technique and an important element in any study that is seeking to identify specific biomarkers in a disease model, however, the aim here was to explore the consequences of long-term caffeine consumption on protein expression in the OFC and to identify the key pathways changed by this treatment. The top molecular and biological functions in order of significance were ‘cellular assembly and organization’, ‘cellular function and maintenance’, ‘cell morphology’ cell-to-cell signalling and interaction’ and ‘cellular development’. Neurological disease was the top disease and disorder identified by IPA with > 36% of the differentially expressed proteins potentially implicated. Ranked number 1 in the Top Networks was ‘neurological disease, nervous system development and function and cell-to-cell signalling and interaction’ with a score of 54. Scores in excess of 2 indicate > 99% confidence that these associations are not generated by random chance alone (Table 3). Ingenuity Pathway Upstream Regulator Analysis can be used to identify molecules that are also able to regulate expression of the proteins changed by CAF. The list of upstream regulators includes chemicals and drugs that are used to treat disease, drugs of abuse, environmental toxins, and proteins that are expressed in neural tissue. Overall, IPA


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identified a total of 226 potential upstream regulators of the differentially expressed proteins following exposure to CAF (Table S2). An upstream regulator can be considered either activated or inhibited if the associated z-score is greater than +/- 2

55

. In a study such as

this, where the effects of exposure to a drug are being explored, for the activation status to be relevant the protein concerned must also be differentially expressed as a consequence of that treatment. None of the upstream regulators identified as either activated or inactivated were also differentially expressed by CAF. Thus in this analysis the upstream regulator list was used to perform comparisons between the effects of caffeine and other important regulators of neural function. The 9th ranked upstream regulator was the adenosine A2A receptor gene ADOR2A (Table 4 and S2; p=2.12E-05, 7 of 157 CAF proteins). Importantly the biological and neurological effects of caffeine that are experienced by human consumers primarily result from the antagonism of adenosine receptors

40, 56

. There are 4 types of adenosine receptors known;

however, the effect that caffeine has on neural function and behaviour mainly results from 10-13

antagonism of adenosine A1 and A2A receptors

. Adenosine A1 receptors are highly

expressed throughout the brain. While low levels of A2A receptors are expressed in the prefrontal cortex (PFC), in the striatum they are expressed at a 20 fold higher density than in the rest of the brain

57-59

. Studies using knockout mice have revealed caffeine-induced

arousal and the aversion to high dose caffeine are mediated by A2A and not A1 receptors. Neither receptor alone or in combination mediates caffeine reward, which furthermore appears not to be dopamine dependent

13, 60

. However, caffeine-induced changes to

locomotor behaviour can be linked to the antagonism of both A1 and A2A receptors as dopamine neurotransmission in the striatum is altered via two mechanisms. Firstly, by antagonising presynaptic A1 receptors caffeine increases dopamine levels via glutamate



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dependent and independent mechanisms, and secondly by antagonising A2A receptors it prevents the inhibitory control of dopamine D2 receptors by adenosine 61, 62. There is evidence that chronic exposure to caffeine can alter A1, but not A2A, receptor density, and that locomotor behavior may be affected when the balance in expression of these receptors is changed

63

. More recently, it has been revealed by selectively knocking

out different populations of A1 and A2A, receptors that caffeine-induced locomotor behavior, and potentially its effects on other behaviors and neuronal health, depend on the interplay between both receptor types and their level of expression in different brain areas

59

.

Notwithstanding that the numbers of A2A receptors expressed in the PFC are far lower than in the striatum, there is evidence to suggest these extrastriatal receptors may play a disproportionate role in mediating psychomotor behaviour. If CAF exposure has resulted in a persistent change in the expression ratio of A1 and A2A in the OFC, this could provide a potential neurochemical explanation for the post-treatment hyperactivity observed if similar effects occurred in the striatum. The behavioural and proteomic changes measured here could represent persistent compensatory changes that are the consequence of an altered expression of these receptors. This may be particularly important as selective targeting of the extrastriatal A2A receptors can result in improved treatment of neurological disorders including psychosis, anxiety, depression and addictive behaviour 59. Ingenuity Upstream Regulator Analysis, used to identify upstream regulators that may be responsible for the changes to protein expression in the dataset analyzed, suggests the majority of the proteins changed by CAF cannot be directly related to antagonism of adenosine receptors. Adenosine A1 was not identified as an upstream regulator of any of the differentially expressed proteins measured in the OFC following exposure to CAF. Adenosine A2A was however identified as an upstream regulator of 7 proteins (Table 4).


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Of these, five are implicated in multiple processes while two have defined functions. Ubiquitin specific peptidase 5 (isopeptidase T) (USP5) was down-regulated 1.5 fold by CAF; reduced expression of this protein can increase DNA sensitivity to damage and increase the risk of cellular death

64, 65

. Our data set included two isoforms of the RAS

oncogene RAB6A; one was detected only in CAF (up-regulated 73.1 fold by CAF) and the other was detected only in control (down regulated 87.5 fold by CAF). Alternative splicing of the Rab6A gene results in two isoforms, RAB6A and RAB6A′, which have different functional roles. Both isoforms are involved in vesicular trafficking in the Golgi, with RAB6A′ implicated specifically in retrograde trafficking

66, 67

. Unfortunately, western

blotting can not be utilized to resolve this issue as there are no suitable antibodies that can differentiate between the two isoforms. An assessment of expression levels of mRNA for each isoform could be achieved using qPCR, however the level of expression of mRNA and protein, even in samples taken from the same animal at the same time point, do not always match 68. The remaining proteins changed by CAF linked by IPA to potential ADORA2A antagonism are destrin (actin depolymerizing factor) (DSTN) up-regulated 2.2 fold, kinesin family member 5A (KIF5A) up-regulated, 23.6 fold, RAB3A, member RAS oncogene family (RAB3A) up-regulated 1.2 fold, tubulin, beta 2A class IIa (TUBB2A) down-regulated 1.3 fold and tubulin, beta 3 class III (TUBB3) down-regulated 1.3 fold. All are involved in a range of cellular functions that include regulation of cytoskeletal structure and protein trafficking, synthesis and degradation, processes that are implicated in the pathophysiology associated with neurological disease 69-71. The top four upstream regulators identified by IPA are all proteins that are critically involved in the pathophysiology associated with neurological disease. These were



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Microtubule-associated protein tau (MAPT), p = 2.75E-29 (31/157 CAF proteins), Amyloid Beta (A4) Precursor Protein (APP), p = 5.49E-28 (43/157 CAF proteins) APP, Presenilin-1 (PSEN1), p= 4.45E-25 (32/157 CAF proteins) and Huntingtin (HTT), p = 7.22E-12 which also regulates 26 of 157 CAF proteins (Table 3,4 and S1).

3.5

Of the 157 proteins changed by caffeine treatment, 58 were mapped by IPA to neurological disease

IPA linked these 58 differentially expressed proteins to 49 possible disorders and diseases. A total of 12 proteins were linked to AD, 13 to Dementia, 18 to Tauopathy, 9 to PD and 14 to Schizophrenia (Table 5). IPA linked many of the proteins differentially expressed to more than one disorder. As an example, none of the proteins associated with AD are uniquely associated with this disease (Figure 4). As these proteins have implications for more than one disorder, discussion will focus on three cellular processes that these neurological diseases and disorders have in common: cytoskeletal organisation; mitochondrial and endoplasmic reticulum function and interaction; and finally, tau hyperphosphorylation and amyloid plaque formation (Figure 5). 3.5.1 Cytoskeletal Organisation Changes to the cytoskeleton and the proteins that interact with it have been linked to a wide range of diseases 72-74. IPA analysis associated 34 of the differentially expressed proteins to cytoskeletal organisation p = 1.78E-09, 32 to microtubule dynamics p = 4.72E-10 and 13 proteins to processes that control the actin cytoskeleton. Microtubules are important for intracellular transport and structure and are formed from α and β-tubulins. CAF exposure down-regulated the expression of two α-tubulins and six β-tubulins. IPA identified five of


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the six tubulin proteins changed by CAF as of specific importance in the aetiology of tauopathy (Figure 4). MAPT, APP and PSEN1, the top 3 upstream regulators of the differentially expressed proteins in the OFC of the CAF treated rats, are also upstream regulators of all the down-regulated tubulin proteins. These upstream regulating proteins are all intimately linked with the pathophysiology of a range of neurological diseases including AD and PD 75-78. Microtubules are not static structures

79

, however

CAF did not change any of the

microtubule associated stabilising proteins or any of the key proteins that regulate the number, or the length, of microtubules. Stathmin 1 (STMN1), which is involved in the disassembly of microtubules, was up-regulated 3 fold by CAF

80

. In the prefrontal cortex,

changes in expression of STMN1 have been associated with disrupted cognitive and emotional control

81

. As the brain ages, STMN1 levels decline, with greater reductions

measured in PD post-mortem neural tissue

82

. Thus, the increased expression of STMN1

may underlie the previous finding that low to medium level CAF consumption is protective against developing PD. Numerous proteins interact with microtubule proteins to allow intracellular trafficking. Acidic (leucine-rich) nuclear phosphoprotein 32 family, member A (ANP32A), which was up-regulated 14.5 fold by CAF, binds with microtubules to facilitate the localisation of golgi apparatus, mitochondria, lysosomes and peroxisomes

83

. Kinesin proteins act as molecular

motors moving cargo, such as vesicles or mRNA to where they are required, along the microtubules

84

. KIF5A up-regulated by CAF, specifically transports dopamine in post-

synaptic cells and is essential to mitochondrial and GABA-A receptor transport. Changes in the expression of kinesin proteins can result in transport defects that have been associated with a range of neurodegenerative diseases 85.



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Actin is another crucial cytoskeletal protein

Page 24 of 52

86-88

. DSTN, up-regulated by CAF, potentially

as a direct consequence of A2A receptor antagonism, is a member of a family of actin depolymerizing proteins that regulate recycling of ADP-actin monomers into ATP-actin. Changes to normal actin dynamics, which are intimately dependent on actin depolymerizing factors such as DSTN, have been associated with the progression of dementia and other neurodegenerative diseases 89. 3.5.2 Mitochondrial and endoplasmic reticular function and interaction The number and distribution of mitochondria is dependent on maintaining a perfect balance of mitochondrial fission and fusion. This balance is disturbed in AD and other neurodegenerative diseases

90

. CAF up-regulated Mitofusin 2 (MFN2) 2.1 fold, this

mitocondrial fusion protein, stimulates clustering of small fragmented mitochondria, thereby reducing the mitochondrial membrane potential. Changes to MFN2 have been linked to mitochondrial dysfunction and can initiate apoptotic processes which are critically implicated in the pathophysiology of neurological disease

91

. MFN2 also maintains the

interface between the endoplasmic reticulum (ER) and mitochondria, either in a homodimer or in a dimer with Mitofusin 192. Disruption to coupling of the ER to mitochondria can affect Ca2+ levels

93

. Inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1), down-regulated

1.5 fold by CAF, is expressed in the ER and functions as a Ca2+ release channel. Transfer of Ca2+ between mitochondria and the ER needs to be maintained to ensure optimal mitochondrial and ER function. Changes to ER coupling and Ca2+ homeostasis can also impact ATP production. The final phase of ATP synthesis in Complex V of the electron transport chain (ETC), in the mitochondria, is dependent on rotations of the gamma subunit. ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 (ATP5C1), was up-regulated 140.3 fold by CAF. This protein changes the conformation of


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the α and β subunits to allow phosphate to bind to the ADP to then form and release as ATP 94

. Reduction in Complex V and other ETC protein expression levels has been associated

with decreased mitochondrial function

95

. Whether these changes represent improved

mitochondrial function or represent a compensatory change would require further investigation. 3.5.3 TAU hyperphosphorylation and amyloid plaques Several of the proteins differentially expressed by CAF are implicated in Tau hyperphosphorylation and the formation of amyloid plaques; key pathologic indicators of AD and other tauopathies. CANX, down-regulated 1.6 fold by CAF, is expressed in the ER where it prevents misfolded proteins from being transported to the Golgi

96

. Down-

regulation of CANX can result in increased levels of misfolded proteins - including amyloid precursor protein - being transferred from the ER to the Golgi 97. Accumulation of tau and disease induced misfolded proteins can induce ER stress. Therefore, prevention of these processes can reduce the neurodegeneration induced by tau 98. Mechanistic target of rapamycin (serine/threonine kinase) (MTOR) was down-regulated 1.8 fold by CAF, has been implicated in numerous diseases including cancer and neurological diseases. Many of these diseases are also associated with aging and there is evidence that inhibiting MTOR can extend life span

99-101

. Dysregulation of MTOR has been linked to

type 2 diabetes (TD2), which has been associated with an increased risk of developing AD. It is interesting that caffeine consumption has also been associated with a reduced risk of developing TD2

99, 102

. In mice that over express tau, reducing mTOR reduces levels of

hyperphosphorylation, which reduces tau pathology and associated behavioural deficits 103

101,

. Thus, this protein could represent a protective molecular mechanism that explains some

of the health benefits associated with low to moderate caffeine consumption.



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ANP32A, in addition to interacting with microtubules, plays a role in a wide range of biological processes including apoptosis, cell differentiation and proliferation, gene expression and cellular signalling

104

. ANP32A is an important mediator of

neurodegenerative diseases including AD, and, as a tumour suppressor, is capable of inhibiting several types of cancer 105. This protein has a powerful inhibitory effect on protein phosphatase 2A (PP2A) (unchanged by CAF), an important regulator of abnormal hyperphosphorylation of tau

83

. The overexpression of PP2A in PC12 cells results in the

abnormal hyperphosphorylation of tau at the same sites as seen in AD and can also result in cell death

106

. Thus, the up-regulated expression of ANP32A by CAF could represent a

mechanism that underpins the lower risk of developing tau related neurological diseases that have been observed in moderate to low level consumers of caffeine. CDK5, up-regulated 10 fold by CAF, plays a major role in the pathophysiology associated with AD and PD107. It is implicated in neuronal cell death and in the disruption to synaptic function that is observed as these diseases progress

108

. CDK5 contributes to the

hyperphosphorylation of tau that can result in the neurofibrillary tangles, a primary marker of AD and other tauopathies 107, 109, 110. Amyloid beta, found in amyloid plaques, is a protein fragment snipped from APP, which is a substrate of CDK5. CDK5 only becomes active when it is associated with its regulatory binding partners, p35 or p25.

111

. Amyloid beta

generation increases the cleavage of p35 to p25, which, in turn, up-regulates CDK5. This can generate a positive feedback loop that has been linked to the pathology associated with AD

111

. Neither of the regulatory binding partners p35 or p25 were detected in this study,

which may be explained by the very short half-life of these proteins. Thus, whether activation of CDK5 has occurred cannot be determined. CDK5 is implicated in a wide range of processes in addition to mediating amyloid beta generation and hyperphosphorylation of


Page 27 of 52

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tau, therefore it is possible that the up-regulated expression of this protein by CAF has quite different consequences for cellular function. Down-regulation of MTOR and up-regulation of ANP32A could represent protective mechanisms against hyperphosphorylation of tau, whereas up-regulation of CDK5 could increase tau pathology, but only if it has been activated by p25. Determining the activation state of CDK5 and whether CAF suppresses hyperphosphorylation of tau as a consequence of altering MTOR and ANP32A expression would be an interesting line of research to pursue, particularly given the epidemiological evidence that caffeine consumers may have a lower risk of developing AD 1, 112.

3.6

Conclusions

This study found that extended CAF consumption in rats produced enhanced locomotor behaviour compared to control when tested treatment free and exposed to a mild stressor. Changes to habituation and hyperactivity measured in the CAF rats are consistent with reduced OFC function, yet will likely incorporate neuroadaptations to locomotor circuitry produced by systemic CAF exposure

17, 52

. Label free quantitative shotgun proteomic

analysis of the orbitofrontal cortex revealed extensive changes to protein expression that can be linked to a wide range of biological functions including pathways implicated in neuropathology. While the CAF effect can be explained for 7 of the proteins measured via antagonism of A2A receptor, the mechanisms that underlie how CAF changed the vast majority of the differentially expressed proteins in the OFC remain unclear. Thus, the observed changes to protein expression could reflect compensatory changes due to altered input to the OFC from other brain areas 113, 114. A proteomic study such as this is not able to



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make definitive statements as to how caffeine might modify the risk for developing any specific neurological disorder. However, it does provide evidence for potential mechanisms to explain epidemiological findings that regular caffeine consumers have a lower risk of developing certain neurological diseases, and also highlights the importance of diet in maintaining neural function.

ACKNOWLEDGEMENTS The authors would like to thank Dana Pascovici for assistance with the Ingenuity Pathway Analysis and statistical analyses and Christine Sutter and Wayne McTegg for animal care. JLF and TAW are recipients of Australian Postgraduate Awards and would like to acknowledge the support of Macquarie University in the form of the Psychology Department Higher Degree Research Grant. The authors acknowledge support from the Australian Research Council in the form of an Industrial Transformation Training Centre grant on which PAH is a chief investigator and MMZ is a member of the project leadership team. PAH also wishes to thank Margo van Staaveren for continued support and encouragement. AKG and JLC acknowledge support from the Hillcrest Foundation.

CONFLICT OF INTEREST The authors declare no competing financial interest.


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88. Bamburg, J. R., Proteins of the ADF/cofilin family: essential regulators of actin dynamics. Annual review of cell and developmental biology 1999, 15, (1), 185-230. 89. Bamburg, J. R.; Wiggan, O. N. P., ADF/cofilin and actin dynamics in disease. Trends in cell biology 2002, 12, (12), 598-605. 90. Wang, X.; Su, B.; Zheng, L.; Perry, G.; Smith, M. A.; Zhu, X., The role of abnormal mitochondrial dynamics in the pathogenesis of Alzheimer’s disease. Journal of Neurochemistry 2009, 109, 153-159. 91. Huang, P.; Yu, T.; Yoon, Y., Mitochondrial clustering induced by overexpression of the mitochondrial fusion protein Mfn2 causes mitochondrial dysfunction and cell death. European Journal of Cell Biology 2007, 86, (6), 289-302. 92. de Brito, O. M.; Scorrano, L., An intimate liaison: spatial organization of the endoplasmic reticulum–mitochondria relationship. The EMBO Journal 2010, 29, (16), 27152723. 93. Jacobson, J.; Duchen, M. R., Interplay between mitochondria and cellular calcium signalling. Mol Cell Biochem 2004, 256, (1-2), 209-218. 94. Logue, S. E.; Cleary, P.; Saveljeva, S.; Samali, A., New directions in ER stressinduced cell death. Apoptosis 2013, 18, (5), 537-546. 95. Navarro, A.; Boveris, A., The mitochondrial energy transduction system and the aging process. Am J Physiol Cell Physiol 2007, 292, (2), C670-86. 96. Ni, M.; Lee, A. S., ER chaperones in mammalian development and human diseases. FEBS Lett 2007, 581, (19), 3641-3651. 97. Taguchi, J.; Fujii, A.; Fujino, Y.; Tsujioka, Y.; Takahashi, M.; Tsuboi, Y.; Wada, I.; Yamada, T., Different expression of calreticulin and immunoglobulin binding protein in Alzheimer’s disease brain. Acta Neuropathol 2000, 100, (2), 153-160. 98. Pereira, C. M. F., Crosstalk between endoplasmic reticulum stress and protein misfolding in neurodegenerative diseases. ISRN Cell Biology 2013, 2013. 99. Dazert, E.; Hall, M. N., mTOR signaling in disease. Current Opinion in Cell Biology 2011, 23, (6), 744-755. 100. Roizen, M., Rapamycin fed late in life extends lifespan in genetically heterogenous mice Harrison DE, Strong R, Sharp ZD, et al (The Jackson Laboratory, Bar Harbor, ME; The Univ of Texas Health Science Ctr at San Antonio; et al) Nature 460: 392-395, 2009. Year Book of Anesthesiology and Pain Management 2010, 2010, 15-16. 101. Caccamo, A.; Magrì, A.; Medina, D. X.; Wisely, E. V.; López-Aranda, M. F.; Silva, A. J.; Oddo, S., mTOR regulates tau phosphorylation and degradation: implications for Alzheimer's disease and other tauopathies. Aging Cell 2013, 12, (3), 370-380.


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102. Steen, E.; Terry, B. M.; Rivera, E. J.; Cannon, J. L.; Neely, T. R.; Tavares, R.; Xu, X. J.; Wands, J. R.; de la Monte, S. M., Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer's disease - is this type 3 diabetes? Journal of Alzheimer's Disease 2005, 7, (1), 63-80. 103. Wang, C.; Yu, J.-T.; Miao, D.; Wu, Z.-C.; Tan, M.-S.; Tan, L., Targeting the mTOR signaling network for Alzheimer's disease therapy. Mol Neurobiol 2014, 49, (1), 120-135. 104. Khan, M. Z.; Vaidya, A.; Meucci, O., CXCL12-mediated regulation of ANP32A/Lanp, a component of the inhibitor of histone acetyl transferase (INHAT) complex, in cortical neurons. Journal of Neuroimmune Pharmacology 2011, 6, (1), 163-170. 105. Haesen, D.; Sents, W.; Ivanova, E.; Lambrecht, C.; Janssens, V., Cellular inhibitors of protein phosphatase PP2A in cancer. Biomed Res 2012, 23, 197-211. 106. Tsujio, I.; Zaidi, T.; Xu, J.; Kotula, L.; Grundke-Iqbal, I.; Iqbal, K., Inhibitors of protein phosphatase-2A from human brain structures, immunocytological localization and activities towards dephosphorylation of the Alzheimer type hyperphosphorylated tau. FEBS Lett 2005, 579, (2), 363-372. 107. Cheung, Z. H.; Ip, N. Y., Cdk5: a multifaceted kinase in neurodegenerative diseases. Trends in Cell Biology 2012, 22, (3), 169-175. 108. Cheung, Z. H.; Fu, A. K. Y.; Ip, N. Y., Synaptic Roles of Cdk5: Implications in Higher Cognitive Functions and Neurodegenerative Diseases. Neuron 2006, 50, (1), 13-18. 109. Dhavan, R.; Tsai, L. H., A decade of CDK5. Nature reviews. Molecular cell biology 2001, 2, (10), 749-59. 110. Lee, K.-Y.; Clark, A. W.; Rosales, J. L.; Chapman, K.; Fung, T.; Johnston, R. N., Elevated neuronal Cdc2-like kinase activity in the Alzheimer disease brain. Neurosci Res 1999, 34, (1), 21-29. 111. Cruz, J. C.; Tsai, L.-H., Cdk5 deregulation in the pathogenesis of Alzheimer's disease. Trends in Molecular Medicine 2004, 10, (9), 452-458. 112. Cano-Marquina, A.; Tarín, J.; Cano, A., The impact of coffee on health. Maturitas 2013, 75, (1), 7-21. 113. Nehlig, A.; Daval, J.-L.; Boyet, S.; Vert, P., Comparative effects of acute and chronic administration of caffeine on local cerebral glucose utilization in the conscious rat. Eur J Pharmacol 1986, 129, (1–2), 93-103. 114. Dager, S. R.; Layton, M. E.; Strauss, W.; Richards, T. L.; Heide, A.; Friedman, S. D.; Artru, A. A.; Hayes, C. E.; Posse, S., Human brain metabolic response to caffeine and the effects of tolerance. American Journal of Psychiatry 1999, 156, (2), 229-237.



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Table 1. Up-regulated proteins identified in the OFC changed in CAF rats relative to control. Fold change direction indicated by +/-, Type 1 = Enzyme, 2 = Growth Factor 3 = Ion Channel, 4 = Kinase, 5 = other, 6 = Peptidase, 7 = Phosphatase, 8 = Transcription Regulator, 9 = Translation Regulator 10 = Transmembrane receptor, 11 = Transporter.

Ensembl ENSRNOP00000019108 ENSRNOP00000008337 ENSRNOP00000015601 ENSRNOP00000020443 ENSRNOP00000017692 ENSRNOP00000061946

Symbol ACAD9 ACO1 AK4 ANP32A ARF4 ATP5C1

ENSRNOP00000020670

ATP5D

ENSRNOP00000029426

ATP5J2

ENSRNOP00000009737 ENSRNOP00000022943 ENSRNOP00000011052 ENSRNOP00000010318 ENSRNOP00000024033 ENSRNOP00000000568 ENSRNOP00000047029 ENSRNOP00000035649 ENSRNOP00000007794 ENSRNOP00000028725 ENSRNOP00000013494 ENSRNOP00000012346 ENSRNOP00000048546 ENSRNOP00000041042 ENSRNOP00000007721 ENSRNOP00000052539 ENSRNOP00000010117 ENSRNOP00000012616 ENSRNOP00000052147 ENSRNOP00000009552 ENSRNOP00000050300 ENSRNOP00000027819 ENSRNOP00000009179 ENSRNOP00000026392 ENSRNOP00000025104 ENSRNOP00000027345 ENSRNOP00000005471 ENSRNOP00000001743 ENSRNOP00000007676 ENSRNOP00000063309 ENSRNOP00000022574 ENSRNOP00000008685 ENSRNOP00000049498 ENSRNOP00000020647 ENSRNOP00000024575 ENSRNOP00000022863

ATP6V1F CALB2 CDK5 CMPK1 COX4I1 CUTA DIRAS1 DPY30 DSTN FKBP2 GMFB GSTA4 HBB HIST2H4 KIF5A MFN2 NDUFA12 NDUFB9 PDE1B Pdhx PDXP PIN1 PPIA RAB3A RAB6A REEP5 RPL23 S100B SKP1 SSR4 STMN1 THY1 TOLLIP TPI1 TPM1 TPPP3

Entrez Gene Name Acyl-CoA dehydrogenase family, member 9 Aconitase 1, soluble Adenylate kinase 4 Acidic (leucine-rich) nuclear phosphoprotein 32 family, memberA ADP-ribosylation factor 4 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit ATP synthase, H+ transporting, mitochondrial Fo complex, subunit F2 ATPase, H+ transporting, lysosomal 14kDa, V1 subunit F Calbindin 2 Cyclin-dependent kinase 5 Cytidine monophosphate (UMP-CMP) kinase 1, cytosolic Cytochrome c oxidase subunit IV isoform 1 CutA divalent cation tolerance homolog (E. coli) DIRAS family, GTP-binding RAS-like 1 Dpy-30 homolog (C. elegans) Destrin (actin depolymerizing factor) FK506 binding protein 2, 13kDa Glia maturation factor, beta Glutathione S-transferase, alpha 4 Hemoglobin, beta Histone cluster 2, H4 Kinesin family member 5A Mitofusin 2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9, 22kDa Phosphodiesterase 1B, calmodulin-dependent Pyruvate dehydrogenase complex, component X Pyridoxal (pyridoxine, vitamin B6) phosphatase Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 Peptidylprolyl isomerase A (cyclophilin A) RAB3A, member RAS oncogene family Ras-related protein Rab6A Receptor accessory protein 5 Ribosomal protein L23 S100 calcium binding protein B S-phase kinase-associated protein 1 Signal sequence receptor, delta Stathmin 1 Thy-1 cell surface antigen Toll interacting protein Triosephosphate isomerase 1 Tropomyosin 1, alpha Tubulin polymerization-promoting protein family member 3


Type 1 1 4 5 1 11

Fold 1.873 1.695 3.049 14.511 1.393 140.325

p.value 0.019 0.006 0.022 0.001 0.045 0.000

11

1.648

0.014

11

2.43

0.020

1 5 4 4 1 5 1 5 5 1 2 1 11 5 11 1 1 1 1 5 7 1 1 1 1 11 5 5 8 5 5 5 5 1 5 5

4.735 44.828 9.964 2.587 2.279 5.287 9.964 4.183 2.216 2.116 1.535 17.543 1.613 2.583 23.606 2.085 2.370 2.070 4.183 1.819 3.513 2.320 1.239 1.217 73.113 3.433 1.609 2.321 3.200 5.287 3.021 1.286 5.596 1.154 55.439 16.027

0.034 0.020 0.001 0.023 0.028 0.030 0.000 0.040 0.042 0.022 0.036 0.000 0.010 0.003 0.000 0.012 0.012 0.049 0.045 0.035 0.026 0.033 0.020 0.035 0.000 0.027 0.013 0.038 0.014 0.030 0.007 0.016 0.042 0.030 0.000 0.008

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Down-regulated proteins identified in the OFC changed in CAF rats relative to control. Fold change direction indicated by +/-, Type 1 = Enzyme, 2 = Growth Factor 3 = Ion Channel, 4 = Kinase, 5 = other, 6 = Peptidase, 7 = Phosphatase, 8 = Transcription Regulator, 9 = Translation Regulator 10 = Transmembrane receptor, 11 = Transporter. Ensembl ENSRNOP00000011166 ENSRNOP00000029144 ENSRNOP00000004673 ENSRNOP00000027773 ENSRNOP00000021491 ENSRNOP00000054218 ENSRNOP00000026738 ENSRNOP00000013914

Symbol ABCB1 ACO2 ACSF2 ACTN4 ADD2 AP1B1 AP2A2 API5

Entrez Gene Name ATP-binding cassette, sub-family B (MDR/TAP), member 1 Aconitase 2, mitochondrial Acyl-CoA synthetase family member 2 Actinin, alpha 4 Adducin 2 (beta) Adaptor-related protein complex 1, beta 1 subunit Adaptor-related protein complex 2, alpha 2 subunit Apoptosis inhibitor 5

ENSRNOP00000006918 ENSRNOP00000009964 ENSRNOP00000003932 ENSRNOP00000060948 ENSRNOP00000002727 ENSRNOP00000033679 ENSRNOP00000004613 ENSRNOP00000026793 ENSRNOP00000059787 ENSRNOP00000046543 ENSRNOP00000040859 ENSRNOP00000025824 ENSRNOP00000052096 ENSRNOP00000062298 ENSRNOP00000027897 ENSRNOP00000001695 ENSRNOP00000026496 ENSRNOP00000010210 ENSRNOP00000025211 ENSRNOP00000057073 ENSRNOP00000014817

ATL1 ATP1A2 ATP1B1 ATP2B4 ATP6V1A BRSK1 CADM3 CADM4 CADPS2 CAMK2B CANX CCT3 CNTNAP1 COL23A1 COL5A3 COL6A2 CORO1A CSE1L DLG4 DNM1 EIF3S6IP

ENSRNOP00000049629 ENSRNOP00000036774 ENSRNOP00000022032 ENSRNOP00000024952 ENSRNOP00000063176 ENSRNOP00000045180 ENSRNOP00000038073

EIF4G1 EVI5L FXYD6 GDI2 GLS H2afv HADHA

ENSRNOP00000014637

HADHB

ENSRNOP00000055371 ENSRNOP00000009556 ENSRNOP00000024548 ENSRNOP00000019862 ENSRNOP00000054924 ENSRNOP00000043221 ENSRNOP00000007247 ENSRNOP00000009288 ENSRNOP00000024895 ENSRNOP00000059177 ENSRNOP00000036336 ENSRNOP00000014167

HOOK3 HSP90AA1 HSPA12A IARS IMMT ITIH3 ITPKA ITPR1 KIAA1967 L1CAM LRRC47 MTOR

Atlastin GTPase 1 ATPase, Na+/K+ transporting, alpha 2 polypeptide ATPase, Na+/K+ transporting, beta 1 polypeptide ATPase, Ca++ transporting, plasma membrane 4 ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A BR serine/threonine kinase 1 Cell adhesion molecule 3 Cell adhesion molecule 4 Ca++-dependent secretion activator 2 Calcium/calmodulin-dependent protein kinase II, beta Calnexin Chaperonin containing TCP1, subunit 3 (gamma) Contactin associated protein 1 Collagen, type XXIII, alpha 1 Collagen, type V, alpha 3 Collagen, type VI, alpha 2 Coronin, actin binding protein, 1A CSE1 chromosome segregation 1-like (yeast) Discs, large homolog 4 (Drosophila) Dynamin 1 Eukaryotic translation initiation factor 3, subunit 6 interacting protein Eukaryotic translation initiation factor 4 gamma, 1 Ecotropic viral integration site 5-like FXYD domain containing ion transport regulator 6 GDP dissociation inhibitor 2 Glutaminase H2A histone family, member V Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), beta subunit Hook homolog 3 (Drosophila) Heat shock protein 90kDa alpha (cytosolic), class A member 1 Heat shock 70kDa protein 12A Isoleucyl-tRNA synthetase Inner membrane protein, mitochondrial Inter-alpha-trypsin inhibitor heavy chain 3 Inositol-trisphosphate 3-kinase A Inositol 1,4,5-trisphosphate receptor, type 1 KIAA1967 L1 cell adhesion molecule Leucine rich repeat containing 47 Mechanistic target of rapamycin (serine/threonine kinase)


Type 11 1 1 5 5 11 11 5

Fold -6.965 -1.655 -6.518 -1.114 -1.864 -1.302 -1.419 -2.761

p.value 0.023 0.001 0.036 0.041 0.011 0.022 0.009 0.012

1 11 11 11 11 4 5 5 5 4 5 5 5 5 5 5 5 11 4 1 5

-1.521 -1.338 -1.456 -1.615 -1.268 -5.540 -1.445 -13.197 -6.518 -1.308 -1.635 -1.650 -2.468 -21.228 -2.390 -7.003 -1.538 -2.374 -1.486 -1.270 -2.183

0.021 0.015 0.045 0.024 0.024 0.041 0.044 0.000 0.047 0.014 0.020 0.019 0.021 0.000 0.034 0.028 0.009 0.002 0.023 0.039 0.040

9 5 3 5 1 5 1

-3.602 -17.213 -5.540 -1.388 -1.471 -79.452 -1.213

0.019 0.000 0.030 0.003 0.027 0.000 0.021

1

-13.197

0.001

5 1 5 1 5 5 4 3 6 5 5 4

-6.272 -1.400 -1.526 -5.176 -1.536 -17.213 -7.003 -1.492 -7.003 -1.674 -2.596 -1.804

0.034 0.003 0.022 0.044 0.049 0.000 0.023 0.044 0.020 0.008 0.042 0.020


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Ensembl ENSRNOP00000016229 ENSRNOP00000057594 ENSRNOP00000022485 ENSRNOP00000012091 ENSRNOP00000005067 ENSRNOP00000006361 ENSRNOP00000008375 ENSRNOP00000036285 ENSRNOP00000026316 ENSRNOP00000037928 ENSRNOP00000029540 ENSRNOP00000008719 ENSRNOP00000048878 ENSRNOP00000043537 ENSRNOP00000048749 ENSRNOP00000052297 ENSRNOP00000019087 ENSRNOP00000026528 ENSRNOP00000061453 ENSRNOP00000007223 ENSRNOP00000016520

Symbol NCDN NFASC NGEF NPTN NPTX1 NSF OGDH PACSIN1 PC PSMD3 PTBP1 PTPRZ1 RAB4A RAB6A RAP1GAP RAP1GDS1 RASGRF2 RPS5 RRAGD SACM1L SAMM50

ENSRNOP00000008999 ENSRNOP00000062103

SH3GL2 SLC12A5

ENSRNOP00000046952

SLC1A2

ENSRNOP00000025196

SLC3A2

ENSRNOP00000063290

SLC4A10

ENSRNOP00000009066 ENSRNOP00000049322 ENSRNOP00000011292 ENSRNOP00000056103 ENSRNOP00000013935 ENSRNOP00000006322 ENSRNOP00000004797 ENSRNOP00000001095 ENSRNOP00000023611 ENSRNOP00000023582 ENSRNOP00000023452 ENSRNOP00000013863 ENSRNOP00000024947 ENSRNOP00000033950 ENSRNOP00000020785 ENSRNOP00000041134 ENSRNOP00000052715 ENSRNOP00000025316

SRGAP3 STXBP5 SYN2 TNR TRIM2 TUBA1A TUBA4A TUBB TUBB2A TUBB2B TUBB3 TUBB4B TUBB6 UBA1 USP5 USP7 WDR47 WDR7

Entrez Gene Name Neurochondrin Neurofascin Neuronal guanine nucleotide exchange factor Neuroplastin Neuronal pentraxin I N-ethylmaleimide-sensitive factor Oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) Protein kinase C and casein kinase substrate in neurons 1 Pyruvate carboxylase Proteasome (prosome, macropain) 26S subunit, non-ATPase, 3 Polypyrimidine tract binding protein 1 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 RAB4A, member RAS oncogene family Ras-related protein Rab6A RAP1 GTPase activating protein RAP1, GTP-GDP dissociation stimulator 1 Ras protein-specific guanine nucleotide-releasing factor 2 Ribosomal protein S5 Ras-related GTP binding D SAC1 suppressor of actin mutations 1-like (yeast) Sorting and assembly machinery component 50 homolog (S. cerevisiae) SH3-domain GRB2-like 2 Solute carrier family 12 (potassium/chloride transporter), member 5 Solute carrier family 1 (glial high affinity glutamate transporter), member 2 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 Solute carrier family4, sodium bicarbonate transporter, member 10 SLIT-ROBO Rho GTPase activating protein 3 Syntaxin binding protein 5 (tomosyn) Synapsin II Tenascin R Tripartite motif containing 2 Tubulin, alpha 1a Tubulin, alpha 4a Tubulin, beta class I Tubulin, beta 2A class IIa Tubulin, beta 2B class IIb Tubulin, beta 3 class III Tubulin, beta 4B class IVb Tubulin, beta 6 class V Ubiquitin-like modifier activating enzyme 1 Ubiquitin specific peptidase 5 (isopeptidase T) Ubiquitin specific peptidase 7 (herpes virus-associated) WD repeat domain 47 WD repeat domain 7


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Type 5 5 5 5 5 11 1 4 1 5 1 7 1 1 5 5 5 5 1 7 5

Fold -1.332 -1.722 -2.742 -1.869 -2.661 -1.393 -1.382 -1.380 -1.561 -7.003 -7.003 -3.053 -75.436 -87.483 -4.729 -1.446 -6.518 -1.526 -25.243 -2.439 -13.197

p.value 0.015 0.012 0.011 0.031 0.022 0.044 0.042 0.042 0.028 0.017 0.024 0.009 0.000 0.000 0.049 0.043 0.032 0.036 0.000 0.003 0.005

1 11

-1.459 -1.844

0.020 0.011

11

-1.645

0.015

11

-1.782

0.022

11

-1.720

0.022

5 5 5 5 1 5 5 5 5 5 5 5 5 1 6 6 5 5

-1.872 -1.637 -1.420 -1.521 -3.328 -1.259 -1.291 -1.397 -1.328 -1.372 -1.334 -1.418 -1.449 -1.604 -1.506 -2.803 -2.157 -1.751

0.037 0.008 0.031 0.029 0.016 0.015 0.015 0.009 0.026 0.017 0.030 0.010 0.036 0.017 0.012 0.024 0.015 0.038

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Ingenuity Pathway Analysis of OFC following extended exposure to caffeine Top Networks Neurological Disease, Nervous System Development and Function, Cell-To-Cell Signaling and Interaction

Score 54 48

Developmental Disorder, Hereditary Disorder, Metabolic Disease Cancer, Hematological Disease, Dermatological Diseases and Conditions

41

Cellular Assembly and Organization, Cellular Function and Maintenance, Hereditary Disorder

33

Cell-To-Cell Signaling and Interaction, Renal and Urological System Development and Function, Tissue Development

28

Top Diseases and Disorders Neurological Disease Cancer Dermatological Diseases and Conditions Hematological Disease Inflammatory Disease

p-value 1.92E-13 - 1.48E-02 1.44E-10 - 1.48E-02 1.44E-10 - 1.48E-02 1.44E-10 - 1.48E-02 1.44E-10 - 1.48E-02

no. 58 54 16 21 13

Molecular and Cellular Functions Cellular Assembly and Organization Cellular Function and Maintenance Cell Morphology Cell-To-Cell Signaling and Interaction Cellular Development

p-value 4.72E-10 - 1.48E-02 4.72E-10 - 1.48E-02 3.79E-08 - 1.48E-02 7.11E-08 - 1.48E-02 1.12E-07 - 1.48E-02

no. 55 47 49 35 26

p-value 7.02E-11 1.39E-06 1.73E-06 2.41E-06 2.41E-06

Ratio 10/68 (0.147) 9/148 (0.061) 10/209 (0.048) 9/165 (0.055) 9/177 (0.051)

p-value of overlap 1.21E-35 3.27E-27 6.92E-26 2.45E-15 9.05E-12

no. 30 42 31 25 11

Top Canonical Pathways Remodeling of Epithelial Adherens Junctions Epithelial Adherens Junction Signaling Breast Cancer Regulation by Stathmin1 Germ Cell-Sertoli Cell Junction Signaling Gap Junction Signaling Top Upstream Regulators MAPT APP PSEN1 HTT NFE2L2* * Predicted Activation State - Activated



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 4. Top upstream regulators and proteins identified by ingenuity pathway analysis that can explain the differentially proteins following chronic exposure to caffeine

Rank Upstream Regulator 1 MAPT

Description Microtubuleassociated protein tau

TOP UPSTREAM REGULATORS Molecule Type Activation zp-value of score overlap Kinase 2.75E-29

2

APP

Amyloid Beta (A4) Precursor Protein

Other

3

PSEN1

Presenilin-1

Peptidase

4

HTT

Huntingtin

Transcription regulator

0.496

7.22E-12

5

BDNF

Brain-derived Neurotrophic Factor

Growth Factor

-0.954

8.39E-08

6

CD 437

RARγ-selective agonist.

Chemical Drug

0.632

1.26E-06

7

ATN1

Atrophin 1

8

MEPH

Transcription Regulator Chemical Toxicant G-protein Coupled Receptor

9

Mono-(2-ethylhexyl) phthalate ADORA2A Adenosine A2 A Receptor

0.518

5.49E-28

4.45E-25

3.75E-06 1.50E-05 -0.378

2.12E-05

Target molecules in dataset ACO2, ANP32A, ARF4, ATP1A2, ATP1B1, ATP5C1, ATP5D, ATP6V1A, CANX, DLG4, DSTN, GDI2, H2afv, HBB, Hist2h4, HSP90AA1, PPIA, S100B, SH3GL2, SLC1A2, STMN1, TPM1, TUBA1A, TUBA4A, TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4B, TUBB6 ACO2, ANP32A, ARF4, ATP1A2, ATP1B1, ATP5C1, ATP5D, ATP6V1A, CANX, CDK5, DLG4, DNM1, DSTN, GDI2, GSTA4, H2AFV, HBB, HIST2H4, HSP90AA1, L1CAM, NCDN , NF1, NPTX1, PACSIN1, PDXP, PIN1, PPIA, S100B, SH3GL2, SLC1A2, STMN1, SYN2, THY1,TPM1, TUBA1A, TUBA4A, TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4B, TUBB6 ACO2, ANP32A, ARF4, ATP1A2, ATP1B1, ATP5C1, ATP5D, ATP6V1A, CANX, DLG4, DSTN, GDI2, H2afv, HBB, Hist2h4, HSP90AA1, L1CAM, PPIA, S100B, SH3GL2, SLC1A2, STMN1, Tpm1, TUBA1A, TUBA4A, TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4B, TUBB6 ANP32A, ATP5J2, CAMK2B, CANX, CDK5, COX4I1, DLG4, DNM1, FXYD6, ITPR1, MTOR, NDUFA12, NGEF, NPTX1, NSF , PC, PDE1B, PSMD3, PTBP1, RAP1GAP, SLC1A2, STMN1, SYN2, TUBA4A, TUBB CALB2, HSP90AA1 ,ITIH3, ITPR1, RAB3A, REEP5, SLC12A5, SLC1A2, SYN2, TUBA1A, TUBB3 API5, ATP5C1, CCT3, HADHA, HSP90AA1, NDUFB9, PIN1, PSMD3, SKP1 ,TUBA1A, ATP1B1, DLG4, NPTX1, PDE1B, PTBP1, STMN1,TUBA4A, TUBB ACO1,ACO2,ATP5D,COX4I1,HADHA, NDUFB9, OGDH, PC DSTN , KIF5A, RAB3A, RAB6A, TUBB2A, TUBB3, USP5


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 5. The top IPA associated network functions linked to the differentially expressed up- and down-regulated proteins in OFC of rats following chronic exposure to caffeine

TOP RELATED NETWORK FUNCTION AND ASSOCIATED PROTEINS p-value Up Regulated p-value Down Regulated Biological Function Neurological Disease 2.68E-04- TOLLIP, RAB3A, THY , KIF5A, 1.53E-09- CADM3, NCDN, NPTN, CADPS2, 4.25E-02 ANP32A, ATP5C1, STMN1, 2.93E-02 SH3GL2, EIF4G1, TUBB, NPTX1, CDK5, PPIA, S100B, PDE1B, SLC12A5, TUBB2B, HADHB, NSF, PIN1, PGRMC1, MFN2, Camk2b, MTOR, FXYD6, CSE1L, BRSK1, DLG4, CNTNAP1, PGRMC1, ABCB1, TUBB3, NGEF, TUBB4B, ATL1, GLS, TUBB2A, TUBA4A, CANX, L1CAM, NFASC, ITPR1, ATP6V1A, TUBA1B, TUBA1A, NF1, RAP1GAP, ATP1A2, HSP90AA1, SLC1A2, UBA1, SYN2, RAB6A, SLC4A10 Nervous System 1.81E-04- AP2B1, STMN1, RAB3A, CDK5, 1.28E-06- NCDN, NPTN, PACSIN1, CADPS2, Development and 4.81E-02 CALB2, S100B, THY1, KIF5A, 2.93E-02 SH3GL2, AP2A2, NPTX1, SLC12A5, Function ANP32A, GMFB, MFN2 TUBB2B, NSF, Camk2b, MTOR, BRSK1, PTPRZ1, DLG4, CNTNAP1, AP2B1, NGEF, ADD2, ATL1, GLS, CANX, TRIM2, L1CAM, ITPKA, ITPR1, NFASC, DNM1, COL5A3, TUBA1A, NF1, RAP1GAP, TNR, ATP1A2, HSP90AA1, SLC1A2, STXBP5, SYN2, ATP2B4, CAMK2G, SLC4A10 Cell-To-Cell Signaling and Interaction

2.89E-03- AP2B1, STMN1, TOLLIP, 4.85E-02 RAB3A, CDK5, CALB2, S100B, PDE1B, THY1, KIF5A

1.28E-06- CADM3, NCDN , NPTN, CADPS2, 2.93E-02 NPTX1, SLC12A5, NSF, MTOR, CAMK2B, BRSK1, DLG4, CNTNAP1, AP2B1, ADD2, GLS, L1CAM, ITPKA, ITPR1, NFASC, DNM1, NF1,TNR, RAP1GAP, ATP1A2, SLC1A2, ACTN4, STXBP5, SYN2, ATP2B4, CAMK2G



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure Legends Figure 1. Weight and diet during the experiment, prior to the first day of treatment (PT), through the 26-day treatment period from treatment day 1 (T1), average weight to the end of each week (W1, W2 and W3), on the last day of treatment (LDT), the average during the treatment free period (WO) and final day of the experiment saline challenge (SC). Panel A shows weight (B) grams of chow consumed (C) volume of treatment fluid. Open circles represent control and black circles represent CAF treated animals. SEM shown as error bars, n=8 for both groups. Figure 2. Locomotor activity during treatment (12 hr). Panel A and B show the number of beam breaks in each hour of the first and last 12 hours of treatment during the dark active period, respectively. Black circles represent CAF treated animals and open circles represent control. SEM shown as error bars, n=8 for both groups (* p < 0.05 ** p < 0.01). Figure 3. Locomotor activity post-treatment. Panel A shows total activity on the procedural habituation and saline challenge days. Open bars represent control and black CAF. Seconds active over time across the 15 min before and 60 min post the i.p. saline challenge injection is shown for the procedural habituation day in panel B and final saline challenge day in panel C. The dotted line at 15 min indicates i.p. injection. Black circles and the solid line represent CAF treated animals and open circles and a dashed line represents control. SEM shown as error bars, n=7 for both groups (* p < 0.05 ** p < 0.01 *** p < 0.0001). Figure 4. Venn diagram showing the proteins differentially expressed by CAF that were linked by IPA to neurological disease. All the proteins in white in the purple oval have been implicated in the aetiology of Alzheimer’s disease though none are uniquely linked to this disorder. Proteins listed in the grey area that is not overlapping another area were uniquely


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


linked to Dementia, in the red area to Tauopathy, in the purple to Parkinson’s disease (PD) and in the blue to Schizophrenia. Where an area overlaps another the proteins contain within the black outline of both shapes are common to both disorders. For example IPA linked TUBA1A to PD and Tauopathy. The two proteins in the gold area play a role in the pathophysiology associated with PD and Schizophrenia. Figure 5. Neurological disease, biological functions and key proteins changed by CAF. This figure shows the three key biological functions used to explore the relationship between CAF exposure and the development of neurological disease. The proteins discussed are grouped, where appropriate, into coloured ovals, one for each biological function, the red box above the protein label indicates the protein may be altered as a consequence of the upstream regulation of ADORA2A. A white circle with a + or – sign indicates the direction of change, with the shape of protein label indicating the type of protein (see the key in the figure).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Supporting Information.

Table S1: All reproducible proteins identified in CAF and control. Table S2: Complete IPA upstream regulator list for proteins changed by CAF.


Page 46 of 52

Figure 1

A

Page 47 of 52

contol CAF

400 300 200

SC

WO

LDT

W3

W2

W1

0

T1

100 PT

Weight grams

500

Experiment day

B control CAF

40 30 20

SC

WO

LDT

W3

W2

W1

0

T1

10 PT

Chow grams

50

Experiment day

C

50

control CAF

40 30 20


Experiment day

SC

WO

LDT

W3

W2

W1

0

T1

10 PT

Treatment ml

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

A

1250

First 12 hours treatment beam breaks (1hr)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


* **

Page 48 of 52

** *

control CAF

1000 750 500 250 0

1

2

3

4

5

6

7

8

9 10 11 12

Hours (dark active)

B Last 12 hours treatment beam breaks (1hr)

1250

*

1000 750 500 250 0

1

2

3

4

5

6

7

8

Hours (dark active)

9 10 11 12


control CAF

Figure 3

A

Page 49 of 52


control

1500

CAF

**

* ***

1000 500 0

Procedural Habituation

Saline Challenge Day

B

Procedural habituation Seconds active (5 min)

250 200

**

* ***

**

control CAF

150 100 50 0

5 10 15 5 10 15 20 25 30 35 40 45 50 55 60 after saline challenge (min) IP

Time

C

250

* *** Saline challenge day seconds active (5 min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

seconds active (75 min)

2000

**

control CAF

** *

200 150 100 50 0

5 10 15 5 10 15 20 25 30 35 40 45 50 55 60 after saline challenge (min) IP ACS Paragon Plus Environment

Time


Page 50 of 52

Figure 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Dementia 23 D 8.04E-04 24 Deme Dementia e a entia 25 13 Proteins 8.04E-04 8.04E E E-04 26 GLS Proteins otteins 27 13 Pro GLS G L LS S 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Alzheimer's Disease 1.92E-03 12 Proteins P t i

Tauopathy

ANP32A ANP32A ANP32A CADPS2 CADPS2 CADPS2 CANX CANX CANX CDK5 CDK5 CDK5 MFN2 MFN2 MFN2 RAB6A RAB6A RAB6A TUBB TUBB TUBB

1.22E-06 18 Proteins

Parkinson’s Disease

Schizophrenia

SH3GL2 THY1

MTOR NPTX1 SLC1A2

9.06E-05 9 Proteins


TUBA1A

CSE1L EIF4G1 STMN1 TUBB2B

ABCB1 ABCB1 SYN2 SYN2

NPTN ATP1A2 CADM3 CNTNAP1 DLG4 FXYD6 NSF S100B SLC12A5


PIN1 TUBA4A TUBB2A TUBB3, TUBB B3, TUBB4B TUBB B4B TUBA1A TUBA A1A

Figure 5

Page 51 of 52


NEUROLOGICAL DISEASE Alzheimer’s Disease 1 1 2 Dementia 2 3 3 Tauopathy 4 Parkinson’s Disease 4 5 Schizophrenia5

TYPE Enzyme Ion Channel Kinase Other Peptidase

Mitochondrial and Endoplasmic Reticular Function and Interaction

6 7 8 MFN21,2,3, ADORA2A Upstream regulation 9 Microtubule ER 10 Tethering Ca2+ 11 Regulation Protein Folding 12 Control 13 Ca2+ pump 14 15 2 3 CANX 11,2,3 ITPR1 16 17 Ca2+ importer 18 Endoplasmic 19 Reticulum 20 Mitochondria 21 22 23 24 25 GABA A Microtubule Receptors 26 K Kinesin 27 Dopamine P Protein 28 KIF5A 29 30 A2AR 31 32 33 Cytoskeletal Proteins 34 A2AR A 35 A2AR 36 USP5 P5 4 STMN1 37 DSTN2 DNA Repair epair i 38 39 A Actin depolymerisation Disassembly A2AR 40 of microtubules RAB6A 41 42 Vesicular Actin Filaments 43 trafficking in 44 the Golgi 45 TAU Hyperphosphorylation Multiple processes 46 and Amyloid plaque formation Microtubules Micro otubules including actin filament a 47 dynamicss and vesicle 48 docking docking g 49 50 51 α ANP32A1,2,3 A2AR 52 β 53 Microtubule RAB3A MTOR1 Tubulin 54 Dynamics. CDK51,2,3 Proteins 55 Tau Localization of A2 R 56Hyperphosphorylation A2AR A pathology Golgi, 57 of tau that can result 3 Mitochondria and TUBB2A TUBB3 T UBB33 58 in the neurofibrillary other organelles o 59 tangles TUBA1A3 TUBB4B3 60 Ca2+ pump

Ca2+ importer

K

Do

Do

pam

KIF5

ine

pa

IF

mi

5A

ne

GA Re BA ce A pto rs

A

A BA GA ceptors Re

GABA A Receptors

KIF5A

Nucleus

Mitochondria


TUBA4A3 T

TUBB2B4 T

Dopamine

Golgi Appara Apparatus


TOC Graphic only

Page 52 of 52

Figure 4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Dementia 23 D 8.04E-04 24 Deme Dementia e a entia 25 13 Proteins 8.04E-04 8.04E E E-04 26 GLS Proteins otteins 27 13 Pro GLS G L LS S 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Proteins differentially expressed by caffeine exposure that are linked to neurological disease

Alzheimer's Disease 1.92E-03 12 Proteins P t i

Tauopathy

ANP32A ANP32A ANP32A CADPS2 CADPS2 CADPS2 CANX CANX CANX CDK5 CDK5 CDK5 MFN2 MFN2 MFN2 RAB6A RAB6A RAB6A TUBB TUBB TUBB


Parkinson’s Disease

Schizophrenia

SH3GL2 THY1

MTOR NPTX1 SLC1A2

9.06E-05 9 Proteins


TUBA1A

CSE1L EIF4G1 STMN1 TUBB2B

ABCB1 ABCB1 SYN2 SYN2

NPTN ATP1A2 CADM3 CNTNAP1 DLG4 FXYD6 NSF S100B SLC12A5


PIN1 TUBA4A TUBB2A TUBB3, TUBB B3, TUBB4B TUBB B4B TUBA1A TUBA A1A

Quantitative Proteomic Analysis of the Orbital Frontal Cortex in Rats

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