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Flanker task-elicited Event Related Potential sources reflect human recombinant Erythropoietin differential effects on Parkinson’s patients

We used EEG source analysis to identify which cortical areas were involved in the automatic and controlled processes of inhibitory control on a flanker task and compared the potential efficacy of recombinant-human erythropoietin (rHuEPO) on the performance of Parkinson’ s Disease patients. The samples were 18 medicated PD patients (nine of them received rHuEPO in addition to their usual anti-PD medication through random allocation and the other nine patients were on their regular anti-PD medication only) and 9 age and education-matched healthy controls (HCs) who completed the flanker task with simultaneous EEG recordings. N1 and N2 event-related potential (ERP) components were identified and a low-resolution tomography (LORETA) inverse solution was employed to localize the neural generators. Reaction times and errors were increased for the incongruent flankers for PD patients compared to controls. EEG source analysis identified an effect of rHuEPO on the lingual gyri for the early N1 component. N2-related sources in middle cingulate and precuneus were associated with the inhibition of automatic responses evoked by incongruent stimuli differentiated PD and HCs. From our results rHuEPO, seems to mediate an effect on N1 sources in lingual gyri but not on behavioural performance. N2-related sources in middle cingulate and precuneus evoked by incongruent stimuli differentiated PD and HCs.

The samples were 18 medicated PD patients (nine of them received rHuEPO in addition to 21 their usual anti-PD medication through random allocation and the other nine patients were 22 on their regular anti-PD medication only) and 9 age and education-matched healthy controls 23 (HCs) who completed the flanker task with simultaneous EEG recordings. N1 and N2 event-24 related potential (ERP) components were identified and a low-resolution tomography 25 (LORETA) inverse solution was employed to localize the neural generators. 26 Reaction times and errors were increased for the incongruent flankers for PD patients 27 compared to controls. EEG source analysis identified an effect of rHuEPO on the lingual gyri 28 for the early N1 component. N2-related sources in middle cingulate and precuneus were 29 associated with the inhibition of automatic responses evoked by incongruent stimuli 30 differentiated PD and HCs. 31 From our results rHuEPO, seems to mediate an effect on N1 sources in lingual gyri but not on 32 behavioural performance. N2-related sources in middle cingulate and precuneus evoked by 33 incongruent stimuli differentiated PD and HCs. 34 Discovering neuroprotective agents to slow down the progression of Parkinson's Disease (PD) 36 and, importantly, to improve cognitive deficits is an active area of research (Athauda & 37 Foltynie, 2015). The search for agents to supplement usual dopaminergic treatments directed 38 towards motor symptoms is not surprising since the characteristic motor impairment of 39 patients is usually accompanied by cognitive deficits (Kehagia, Barker, & Robbins, 2010). Since 40 cognitive dysfunction has a negative impact on the quality of life of patients (Schrag,41 Jahanshahi, & Quinn, 2000); finding effective therapies that target cognition in PD is of 42 paramount importance. As an example, we found that human recombinant erythropoietin 43 (rHuEPO) (Pedroso et al., 2012) improved general measures of cognition in chronically 44 medicated PD patients, an additional benefit to that obtained on their usual medical 45 treatment. This result extends to PD the evidence for neuroprotective properties of rHuEPO 46 already described in other neurologic diseases (Brines & Cerami, 2005) and is supported by 47 the anti-apoptotic, anti-inflammatory and cytoprotective effects of EPO in PD animal models 48 (Sirén, Faßhauer, Bartels, & Ehrenreich, 2009) (Xue, Zhao, & Guo, 2007). This promising result 49 suggested the need to further study the effect of rHuEPO on cognition in PD. 50 We believe that to further understand the effect of rHuEPO on cognition in PD patients we 51 need to examine its effect on specific stages of information processing. This is because the 52 overt behavioural measures used in our previous study: a) do not have temporal sensitivity, 53 being the end outcome of many sequential processes, and b) do not reflect localized neural 54 activity. Consequently, and as a first objective, we zeroed in on very early automatic neural 55 processes involved in inhibitory control, the lack of which is so common in non-demented PD 56 patients. This early lack of inhibitory control is easily measured in a number of tasks such as 57 the Stop signal, go no-go, Stroop, Hayling Sentence Completion task and the Simon task 58 described in (Obeso et al., 2011) and ( during "on" (mean 6.3, SD1.1) and "off" medication (mean 21.7, SD 4.3) states. 113 For the purpose of comparisons, 9 healthy controls (HCs) matched in age (mean 51.2, SD 3.9 114 years) and educational level were recruited at the same clinic. The PD patients were tested 115 on their usual anti-parkinsonism medication. The patients and controls signed an informed 116 consent to participate in this study as a complement of the clinical trial following the CIREN 117 ethics committee regulations. 118

Eriksen´s Flanker Task 119
All participants completed the Eriksen´s Flanker task, while the EEG was simultaneously 120 recorded. Each trial of the task consisted of the presentation of a set of 5 ordered letters 121 (HHHHH or SSSSS) for the congruent condition and 5 letters with H or S at the centre and 122 different laterals or flankers (SSHSS or HHSHH) for the incongruent condition. Participants 123 were instructed to respond to the central letter, whether H or S, by pressing a key with the 124 index finger of the right or left hand respectively. Participants were instructed to respond as 125 fast and as accurately as possible. A total of 480 trials in two blocks, each lasting 8 minutes 126 were completed. In each block 80 stimuli were shown for the congruent condition and 160 127 for the incongruent. Only the correct responses with reaction times (RTs) >150 and <800 128 msec. were selected for analysis. 129 The physical characteristics of the stimuli were black letters on a white frame with a height = 130 1.5 cm. and Lenght= 7 cm., under 6 0 a visual angle. The distance of the participant to the 131 computer monitor was 60 cm. Each stimulus was presented at the centre of the screen for 132 190 msec., followed by a fixed interstimulus interval (ITI) of 1735 msec. A training block of 40 133 stimuli was designed to ensure task instructions were understood. 134

ERP measurement 135
The Electroencephalogram (EEG) was continuously recorded at a sampling rate of 512 Hz from 136 64 electrodes located at standard positions of the International 10/20 System using a Brain 137 Vision system (https://www.brainproducts.com/products_by_apps.php?aid=5) (Jasper, 138 1958). Linked ears were used as on-line reference and the front as earth. To monitor eye 139 movement artefacts, the electro-oculogram (EOG, horizontal and vertical) was recorded from 140 electrodes placed 1 cm to the left and right of the external canthi, and from an electrode 141 beneath the right eye. 142 Data were filtered using 1-30 Hz and a notch filter to eliminate the 60Hz powerline artefact. 143 All data were referenced using an average reference to all the channels. The baseline was 144 corrected between -200 to -0 msec. Epochs with electric activity exceeding baseline activity 145 by 100 µV were considered as artefacts and were automatically rejected from further 146 processing (15% of epochs related to hits and 11% of the epochs related to errors). For the 147 analysis, several electrodes were excluded (EOG, ECG, TP9 and TP10). 148 ERPs were obtained from the EEG recordings for each participant for all the electrodes 149 within the two experimental conditions and averaged over the two groups using Analyzer 150 software (https://www.brainproducts.com/productdetails.php?id=17 The statistical analyses performed were: 174 a) Reaction Times and errors were analysed using a two-way repeated measure ANOVA 175 with the with Group (HCs, PD Control and PD rHuEPO) as the between group factor 176 and the experimental condition (incongruent versus congruent) as the within-subject 177 repeated measures factor. We report the F statistic and the p value for tests of the 178 main effect and the interaction. The Greenhouse-Geisser adjustment was applied 179 since lack of sphericity was observed. These analyses were completed with STATISTICA 180 7.0. 181 b) An exploratory analysis of the differences in ERP amplitude topographies between the 182 HCs and PD Control +PD rHuEPO groups was carried out by means of a multivariate t 183 test that corrects for multiple comparisons by means of a permutation technique. The 184 permutation test has the following advantages: the tests are distribution free that 185 control the experiment-wise error for the simultaneous univariate comparisons, no 186 assumptions of an underlying correlation structure are required, and they provide 187 exact p-values valid for any number of subjects, timepoints and all 60 electrodes. The 188 overall significance level was selected to be 0.05. The method is described in (Galán,189 Biscay

229
When using the contrast comparing all PD patients and HCs (

Exploratory results of ERPs 237
As mentioned in the Methods, the multivariate t tests corrected for multiple comparisons 238 with permutation tests provides exact p-values, valid for any number of participants, 239 timepoints and recording sites yielded as significant the ERP components in the midline at the 240 0.05 level. Within this group the most significant ERP was Oz for N1 and Cz for N2 as described 241 in the literature. We will therefore concentrate on these electrode sets henceforth since they 242 all are significant above the globally valid significance threshold. 243 The same procedure allows, additionally, to select the time windows and which factor 244 (Condition or Group) to be further analyzed. Figure 1 illustrates, for one derivation, the 245 statistics shown above the red line, the latencies with significance for each factor (Group or 246 Condition) in all the time window for analysis. The interaction between them was not 247 significant at any time. The exploratory analysis between experimental conditions did not 248 reflect significant differences in the time range for the early ERP components N1 and N2 249 (around 100 and 200 msec. respectively). 250 Note that the significant differences for Condition are in the range of the P300 or later, not in 251 the scope of our study. For that reason, we focus all the further analysis on the incongruent 252 condition, which is the condition which elicits inhibitory control. Nevertheless, henceforth we 253 continue to report the full two-way analysis (Group x Condition), though concentrating on the 254 Group Factor analyses. 255

Analysis of the N1 component: 260
We tested the N1 amplitudes with the repeated measures rMANOVA (Group X Condition), 261 and examined the main effects and the interaction between them. The interaction and the 262 factor Condition were not significant (p=0.23  F4, FC2, FC4, FC6, C2, C4

275
The localization of the differences between the two Parkinson groups of this component are 276 localized anatomically by means of the randomized nonparametric test for LORETA. This 277 showed that the PD rHuEPO had a larger N1 component than the PD Control group at the p< 278 0.01 level (corrected for multiple comparisons) at the lingual gyri (See right side of Figure 2). 279

Analysis of the N2 component: 280
We tested the N2 amplitudes with the repeated measures rMANOVA (Group X Condition), 281 and examined the interaction and the main effects. The interaction was not significant with a 282 Wilk's Lambda=0.43, F(6,44)=2.97 and the factor Condition was also not significant (p=0.323). 283 The main effect of Group (comparing three groups) was significant, F(2,24)=6.14, p=0. 006 Note that the N2 grand average at the Cz electrode followed an opposite pattern than N1 292 (See Figure 4): the amplitude of the PD Control group (-2.10 µV) and healthy controls (-2.46 293 µV) was not different statistically. On the other hand, the amplitude of the PD rHuEPO group 294 (-0.67 µV) was significantly lower than both of them. See Table 3 for details of amplitude and  295 latencies of N2 in Cz. 296

304
The source analysis of the differences (comparing the three groups), for the N2 component, 305 was localized anatomically by means of the LORETA randomized nonparametric test (p< 0.01 306 level corrected for multiple comparisons) at the the middle cingulum and precuneus 307 bilaterally. See Figure 3, right side. 308 In order to know if the errors were related to the N2 amplitude, we select a linear mixed 309 effect model, and carried out a repeated measures ANOVA log(errors) x Group x amplitudeN2. 310 But the results were not significant for the interaction of log(errors) with the N2 amplitude, 315

316
The current study was designed to examine if the novel rHuEPO neuroprotective compound, 317 given to Parkinson patients in addition to their usual medication changed the amplitude of 318 ERP components during an inhibitory control task. 319 The behavioural results were consisted with previous studies in PD patients in both the 320 rHuEPO and PD Control groups. Both groups showed significantly increased reaction times 321 and a higher number of errors to the incongruent stimuli during the performance of the 322 flanker task as compared to age and education matched HCs. These higher error It should be noted that we did not find the expected beneficial effect of rHuEPO on 330 behavioural performance (RT and accuracy) in PD patients who received the 331 neuroprotective agent as compared to those that only received the usual treatment. Rather, 332 the differences between groups of patients were found in the ERP components. This is in 333 accordance with our hypothesis that an overall behavioural response might be noisier than This is what we found: the generators of N1, both in the scalp topography and using LORETA,352 in the visual areas of the occipital lobe of both hemispheres. The activation of the source for 353 the PD patients who received rHuEPO was much larger that of the PD group who did not 354 receive it. In fact, the response of the rHuEPO group became statistically indistinguishable 355 from that of the HCs, suggestive of a possible neuroprotective effect of rHuEPO on the lingual 356 gyrus, a region associated with the early and automatic processing of visual stimuli. In 357 summary, our findings suggested an effect of rHuEPO on the visual attentional window in the 358 early information-processing stage, thus enhancing the automatic processing of flankers 359 regardless of their compatibility. 360 Regarding the N2 component. The second component N2 has been found in several studies 361 of inhibition using the Flanker task and its amplitude and latency was unaltered in medicated 362 PD patients (for a review see (Seer et al., 2016) diminished with respect to the other two groups. These effects were topographically located, 372 as expected, in the fronto-central areas, with neural generators of these differences localised 373 to the posteromedial portion of the parietal lobe, the precuneus, a structure involved in the 374 processing of perceptual ambiguities of stimuli (Cavanna & Trimble, 2006)  However, in our study the striking decrease of the N2 produced by rHuEPO needs further 382 research to find an adequate explanation. 383

Behaviour versus ERPs 384
Contrary to our expectation, rHuEPO was not associated with a significant improvement in 385 behavioural performance and did not influence the neural generators of the N2. 386 The ERP allows neural activity tracking on a millisecond time scale and represents a 387 continuous measure of information processing, for that reason we selected the ERP to study 388 a more refined measure of the process of inhibitory control. 389 This apparent contradiction between behavioural and electrophysiological results could be 390 related to their different temporal course. Note that the inhibition is a complex process that 391 can be automatically initiated in the first 100 msec. post-stimulus and extend its action 392 through both automatic and controlled processes until 800 msec. Reaction time, on the other 393 hand, started much later >400 milliseconds after the stimulus presentation, with a strong 394 motor component to complete the response. 395 Therefore, the aim of our study is to use a flanker task to identify if rHuEPO improves 396 automatic and controlled inhibitory control in PD patients and to locate the neural generators 397 in these processes. This could be a first step in identifying an ERP biomarker for this type of 398 cognitive process to be used in clinical trials. 399 Limitations 400 Since this study was completed as part of a safety trial, the samples and the doses employed 401 were small. This might also explain the lack of clear correlations with behaviour, for example 402 reaction time with N2 amplitude. Thus, the results require confirmation with larger samples 403 in future studies. However, the results highlighted the role of EEG source analysis and 404 advantages of electrophysiology with its high temporal resolution and insensitivity to placebo 405 effects, in identifying brain changes after an intervention such as rHuEPO. 406 Conclusions 407 -We found that rHuEPO improved automatic inhibitory control in PD patients but did not 408 improve behavioural performance. 409 -The differences between PD rHuEPO and PD Control groups was in the N1 component at the 410 lingual gyrus. The differences between PD and healthy controls was on the N2 component in 411 the cingulate and precuneus. 412 -Electrophysiology is potentially a useful tool for identifying effects of neuroprotective 413 compounds on different stages of information processing. 414 -The components N1 and N2 as well as others like P3 should be further studied as possible 415 biomarkers for the evaluation of neuroprotective drugs in Parkinson's Disease. 416

417
The tables with the behavioural performance (reaction time, hits and errors) and the N2 418 amplitude for the averaged time window (200-300 milliseconds) of the samples was 419 submitted in the supplementary material 1. The raw and pre-processing EEG recordings in 420 BrainVision format with all the individual and grand average potentials for group and 421 condition can be available by request to maria.bringas@neuroinformatics-collaboratory.org 422