Alexane Fierain

Aileen Mcgonigal

Stanislas Lagarde

Hélène Catenoix

Luc Valton

Sylvain Rheims

Anca Nica

Agnès Trebuchon-Da Fonseca

Romain Carron

Fabrice Bartolomei
email: fabrice.bartolomei@ap-hm.fr

Rheims Sylvain

Prof Bartolomei Fabrice

______________________________________________

Stereoelectroencephalography (SEEG) and epilepsy surgery in posttraumatic epilepsy: A multicenter retrospective study

Keywords: focal epilepsy, epilepsy surgery, traumatic brain injury, SEEG post-traumatic epilepsy, TBI: traumatic brain injury, SEEG: stereoelectroencephalography, MRI: magnetic resonance imaging, SOZ: seizure onset zone, SOP: seizure onset pattern, EI: epileptogenicity index, MTS: mesial temporal sclerosis, TLE: temporal lobe epilepsy, FLE: frontal lobe epilepsy

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

A c c e p t e d m a n u s c r i p t

Introduction

Post-traumatic epilepsy (PTE) is a common cause of structural epilepsy, defined by the occurrence of one or more late unprovoked seizures associated with a known traumatic brain injury (TBI) [START_REF] Szaflarski | Post-traumatic epilepsy: current and emerging treatment options[END_REF][START_REF] Scheffer | ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology[END_REF]. Several risk factors for developing PTE have been identified, including duration of loss of consciousness, missile injuries, intracerebral hemorrhage, diffuse cerebral contusions, acute subdural hematoma with surgical evacuation, early post-traumatic seizures and depressed skull fracture [START_REF] Agrawal | Post-traumatic epilepsy: an overview[END_REF]. Estimated relative risks of seizure occurrence have been estimated to be 1.5 after mild TBI (loss of consciousness or amnesia less than 30 minutes), 2.9 after moderate TBI (loss of consciousness from 30 min to 1 day or a skull fracture) and 17.2 after severe TBI (loss of consciousness of more than 1 day, SDH or brain contusion) [START_REF] Agrawal | Post-traumatic epilepsy: an overview[END_REF][START_REF] Annegers | The risks of epilepsy after traumatic brain injury[END_REF]. PTE can occur even decades after the initial TBI [START_REF] Hung | Treatment of post-traumatic epilepsy[END_REF] and accounts for 20% of symptomatic epilepsy in the general population [START_REF] Frey | Epidemiology of posttraumatic epilepsy: a critical review[END_REF]. Post-traumatic seizures can be difficult to control [START_REF] Diaz-Arrastia | Posttraumatic epilepsy: the endophenotypes of a human model of epileptogenesis[END_REF] and the diagnosis of PTE is present in 5% of all epilepsy patients referred to specialized epilepsy centers. Nevertheless, such epilepsy is not frequently assessed for epilepsy surgery, in particular using intracranial recordings. Indeed, PTE accounts for 2-3% of pre-surgical series using SEEG [START_REF] Lagarde | The repertoire of seizure onset patterns in human focal epilepsies: Determinants and prognostic values[END_REF][START_REF] Gonzalez-Martinez | Stereoelectroencephalography in the "difficult to localize" refractory focal epilepsy: early experience from a North American epilepsy center[END_REF]. Head injuries frequently cause bilateral or multifocal injuries not amenable to epilepsy surgery. There are also surgical challenges due to scar tissue and adhesions secondary to the TBI [START_REF] Mathern | Traumatic compared to nontraumatic clinical-pathologic associations in temporal lobe epilepsy[END_REF].

Therefore, presurgical evaluation of such epilepsies can be a challenging task in locating a limited seizure onset zone and guiding possible resective surgery.

Only a few series have studied surgical outcome of selected patients with PTE.

Outcome was found to be favorable in temporal cases associated with hippocampal sclerosis [START_REF] Diaz-Arrastia | Posttraumatic epilepsy: the endophenotypes of a human model of epileptogenesis[END_REF][START_REF] Marks | Seizure localization and pathology following head injury in patients with uncontrolled epilepsy[END_REF][START_REF] Hartzfeld | Characteristics and surgical outcomes for medial temporal post-traumatic epilepsy[END_REF][START_REF] Hitti | Surgical Outcomes in Post-Traumatic Epilepsy: A Single Institutional Experience[END_REF]but more uncertain in extra-temporal cases [START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF] [START_REF] Kazemi | Resection of frontal encephalomalacias for intractable epilepsy: outcome and prognostic factors[END_REF]. However, A c c e p t e d m a n u s c r i p t while cases localized to the temporal region are often associated with hippocampal sclerosis, they may exhibit more widespread injury on neuroimaging [START_REF] Marks | Seizure localization and pathology following head injury in patients with uncontrolled epilepsy[END_REF]. The MRI aspect is therefore not a reliable indicator of the potential seizure onset zone.

To our knowledge there have been no studies specifically dedicated to presurgical invasive evaluation using stereoelectroencephalography (SEEG) in PTE. The usefulness of intracranial explorations in these cases has however been highlighted, contributing to a better prognosis in series using subdural grid recordings [START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF]. Our objectives were to describe the SEEG-based characteristics of epileptogenicity in PTE and to correlate these data with surgical outcome.

Material and methods

2.1.Patients' selection

We selected 26 patients from four different French tertiary epilepsy centers (Marseille, Lyon, Toulouse and Rennes) with post-traumatic drug-resistant epilepsies who benefited from presurgical evaluation between 1998 and 2018. All of the patients suffered from severe traumatic brain injury (TBI), defined according to Annegers et al [START_REF] Annegers | The risks of epilepsy after traumatic brain injury[END_REF] : post-traumatic loss of consciousness/amnesia > 1 day and/or brain contusion/intra-cranial hematoma. The delay between the acute brain injury and the epilepsy onset was not restrictive, since it has been shown that brain-injured patients can develop epilepsy even more than 10 years after TBI [START_REF] Annegers | The risks of epilepsy after traumatic brain injury[END_REF][START_REF] Brady | Modelling traumatic brain injury and posttraumatic epilepsy in rodents[END_REF].

2.2.Pre-surgical evaluation

Prior to SEEG and/or surgery, all patients included underwent non-invasive assessment for drug-resistant focal epilepsy, including clinical history, neurological examination, long-term video-EEG monitoring, neuropsychological evaluation, 1.5 and/or 3 Tesla brain magnetic resonance imaging (MRI), interictal 18-A c c e p t e d m a n u s c r i p t fluorodeoxyglucose-positron emission tomography (18PET-FDG). Some of them also underwent ictal single photon emission computed tomography (SPECT), functional MRI and/or Wada test for language assessment. SEEG exploration was carried out during long term video-SEEG monitoring as part of patients' normal clinical care. The SEEG was decided on a case-by-case basis in order to clearly define the seizure-onset zone, in particular its relationship with the post-traumatic lesion. It was indicated only in cases where a reasonable hope of surgery was considered after the non-invasive phase and in accordance with French guidelines [START_REF] Isnard | French guidelines on stereoelectroencephalography (SEEG)[END_REF]. The targeting and number of electrodes was individually designed based on the data of the non-invasive pre-surgical assessment performed for each patient, according to hypotheses about the localization of the seizure onset zone (SOZ).

The institutional review board of the French Institute of Health approved this study and written consent from patients or legal tutors was obtained.

2.3.SEEG recordings

Recordings were performed using intracerebral multiple contact electrodes (Dixi Medical ™ or Alcis™) ; 5-15 contacts) placed intracranially according to stereotactic methodology [START_REF] Isnard | French guidelines on stereoelectroencephalography (SEEG)[END_REF]. The anatomical localization of the electrodes was defined using post-implantation 1.5T MRI or using a fusion of pre-implantation brain MRI and post-implantation brain computed tomography (CT). Signals were recorded on a Natus or Micromed System, sampled at 256 Hz, 512Hz or 1024Hz, depending on the time of recording and recorded on a hard disk (16bits/sample) using no digital filter. Two hardware filters were present in the acquisition process, one high-pass filter A c c e p t e d m a n u s c r i p t (cut-off frequency equal to 0.16 Hz) and one antialiasing low pass filter (cut-off frequency equal to 97Hz at 256Hz, 170Hz at 512Hz or 340Hz at 1024Hz).

2.4.Signal analysis

The seizure onset was defined as the first change of intracranial EEG signal within the context of a sustained rhythmic discharge and correlated with clinical signs.

Electroclinical seizures resulting from direct cortical stimulation were excluded.

Seizure-onset pattern (SOP) determination was assessed by two independent epileptologists (AF and FB), blinded to clinical data. SOP were described using visual analysis combined with time-frequency analysis and defined for the electrodes involved in the SOZ, within the first 5 seconds of the discharge. We categorized eight different SOP, as previously reported by our group [START_REF] Lagarde | The repertoire of seizure onset patterns in human focal epilepsies: Determinants and prognostic values[END_REF][START_REF] Lagarde | Seizure-onset patterns in focal cortical dysplasia and neurodevelopmental tumors: Relationship with surgical prognosis and neuropathologic subtypes[END_REF]. As previously discussed, four patterns were considered to be "fast SOP" (included ictal discharge above 12 Hz) and four were considered to be slow SOP, because of the absence of clear fast discharge (see Supplementary Figure ). If ictal recordings of a patient showed different types of SOP, the most prevalent type of SOP was selected. The SOZ was defined using visual analysis complemented by a systematic calculation of the epileptogenicity index (EI) [START_REF] Medina Villalon | EpiTools, A software suite for presurgical brain mapping in epilepsy: Intracerebral EEG[END_REF][START_REF] Bartolomei | Epileptogenicity of brain structures in human temporal lobe epilepsy: a quantified study from intracerebral EEG[END_REF]. The EI is used to determinate the epileptogenicity of analyzed brain structures, by quantifying two important features of the SEEG signal during the transformation from preictal to ictal activity: the energy ratio (ER) redistribution (decrease of low frequency below 12hz and increase of high frequency above 12 Hz) and its delay of occurrence (in regards of the first structure generating ictal discharge). The normalized EI value ranges from 0 (selected brain structure not involved in the ictal discharge) to 1 (maximum epileptogenicity, which means that the selected brain structure generates a rapid A c c e p t e d m a n u s c r i p t discharge with a minimal delay). The SOZ was defined without EI in case of SOP not including fast discharge. We used the methodology of previous studies of our group to characterize the SOZ [START_REF] Lagarde | Interictal stereotactic-EEG functional connectivity in refractory focal epilepsies[END_REF][START_REF] Aubert | Local and remote epileptogenicity in focal cortical dysplasias and neurodevelopmental tumours[END_REF] : the SOZ extension was classified as "focal" (one sub-lobe involved) or "network" (more than one sub-lobe involved) according to the number of distinct brain regions ("sub-lobes") involved, defined by a high nEI (≥0.4) or visual analysis (e.g. structures involved within the five first seconds of the ictal discharge) when the SOP was characterized by lower frequencies. The sublobes were distinguished as follows: mesial temporal, lateral temporal, insular, orbitofrontal, mesial prefrontal, lateral prefrontal, premotor, central (pre-and postcentral), operculum (frontal and Rolandic), mesial parietal, lateral parietal, mesial occipital and lateral occipital.

2.5.Surgical outcome

Surgical outcome was regularly assessed during the post-operative follow up, using the Engel classification [START_REF] Engel | Update on surgical treatment of the epilepsies. Summary of the Second International Palm Desert Conference on the Surgical Treatment of the Epilepsies (1992)[END_REF]. The latest surgical outcome was used in this study, with exclusion of patients with less than 12 months of follow-up.

2.6.Statistical analysis

Chi-squared (χ2) and Fisher's exact test and Mann Whitney U test were used to compare respectively categorical data or continuous data between some clinical parameters (localization of the SOZ, type of SOP (slow vs fast), organization of the SOZ (focal versus network), epilepsy duration and surgical outcome. In these analyses, p ≤ 0.05 was considered to be statistically significant.

Results

A c c e p t e d m a n u s c r i p t

3.1.Clinical and SEEG data

During the period under consideration, 1227 SEEG procedures were performed in the 4 centres. We reviewed 26 patients with drug-resistant PTE representing thus 2% of the total SEEG procedures. Three patients were excluded due to multiple etiologies (2 with TBI and focal cortical dysplasia type II) or incomplete medical records [START_REF] Szaflarski | Post-traumatic epilepsy: current and emerging treatment options[END_REF]. Furthermore, we excluded 2 patients because SEEG recordings were not available for analysis and 3 patients who did not benefit from SEEG recordings and who were operated based on non-invasive assessment (Fig1). Eighteen patients (5 women and 13 men) were thus selected for this study. Fourteen (74%) were righthanded and the mean age was 41 years (range 25-61). All of them suffered from severe TBI. The mean time between TBI and seizure onset was 4.5 years (range 0.2-25) and the mean age at seizure onset was 22.6 years old (range 5-38). All patients presented post-traumatic MRI lesions: 4/18 had hippocampal sclerosis and 17/18 had encephalomalacia/gliosis (8/18 focal and 9/18 multifocal, detailed in Table 1). Mean number of sublobes involved by post-traumatic lesions was 2.8 (range 1-7).

All the selected patients underwent SEEG exploration and recordings. The mean number of implanted electrodes per patient was 11.4 (range 7-14), with bilateral implantations in 55.5%. One patient (5%) experienced a surgical complication of intracranial electrode implantation (asymptomatic subdural hematoma). The mean number of lobes sampled per patient was 3.3 (where implantations sampled frontal, temporal, parietal and/or occipital lobes in 94, 100, 44 and 5.5% of the cases, respectively). The mean number of intra-lesional electrodes per patient was 3 (range A c c e p t e d m a n u s c r i p t 1-6). A total of 59 seizures were analyzed; mean number of seizures per patient was 3.3 (range 1-5).

3.2.Quantification of epileptogenicity and SOZ organization

Visual analysis and quantification led to the anatomical definition of the SOZ in all the patients. Finally, we could classify the involved SOZ as follows: temporal 8/18 (44%), temporal-plus (temporal and frontal) 4/18 (22%), bi-temporal 2/18(11%) and frontal 4/18 (22%). One example of a temporal "plus" epilepsy is represented in Fig 2 . Thus, taken as a whole, a majority of explored PTE included the temporal lobe region.

As described in the methods, SOZ extension and organization were defined as the number of sub-lobes displaying a high epileptogenicity index (≥0.4) or, for the cases with lower frequency patterns involved visually within the 5 first seconds of the epileptic discharge. SOZ displayed a focal organization in 44.4% (3 lateral temporal, 4 mesial temporal, 1 precentral) and a network organization in 63.6% (detailed in Table1). The SOZ was bilateral in 16% of cases and involved temporal lobe in 77.7% of the patients. Using the sub-lobar categorization, we compared the extent of the SOZ to the post-traumatic lesion (Fig 3B). Interestingly, SOZ was less extensive than the corresponding post-traumatic lesion in 50% of the patients, in terms of sublobes involved (detailed in Table 1).

3.3.Seizure-onset patterns

The SOP determination was the following: 6/18 slow wave or baseline shift followed by low voltage fast activity (LVFA) (pattern D according to [START_REF] Lagarde | The repertoire of seizure onset patterns in human focal epilepsies: Determinants and prognostic values[END_REF][START_REF] Lagarde | Seizure-onset patterns in focal cortical dysplasia and neurodevelopmental tumors: Relationship with surgical prognosis and neuropathologic subtypes[END_REF]; 4/18 sharp A c c e p t e d m a n u s c r i p t theta/alpha activity (pattern F) ; 2/18 delta-brush (pattern H) ; 1/18 beta sharp activity (pattern G) ; 1/18 LVFA (pattern A); 1/18 preictal spiking followed by LVFA (pattern B); 1/18 burst of polyspikes followed by LVFA (pattern C). Two patients showed 2 types of SOP (with only two available seizures per patient): delta brush (pattern H) and rhythmic slow spike (pattern E) for the first one; preictal spiking followed by LVFA (pattern B) in seizures emerging from the left side of the brain and rhythmic slow spikes (pattern E) in seizures emerging from the right side of the brain of the second patient. Examples of SOP seen in our study are represented in Fig 3A . Taken as a whole, we noticed that half of cases involved slow onset patterns without clear fast discharge. We did not find significant statistical association between slow versus fast patterns and type of epilepsy (temporal versus extra-temporal, p=0.33) or the network versus focal organization of the SOZ (p=0.9).

3.4.Surgical outcome

Thirteen patients (72%) benefited from surgical resection after SEEG. The mean duration of epilepsy before surgery was 18.6 years (SD 5.2). The 5 remaining patients were either contraindicated for surgery due to bilateral EZ (3/5) or refused to be operated (2/5).

Surgery was a tailored resection based on SOZ localization from SEEG recordings except one patient with a mesial temporal lobe epilepsy who underwent selective amygdalohippocampectomy with Gamma Knife radiosurgery (Table 1). Undesired side-effects were mild to moderate and only transient (dysarthria/facial palsy/hemiplegia worsening/headache/depression) in 5/13 patients. However, one patient died after resective surgery due to malignant brain edema. In the remaining 7 patients, no side effects were observed. Comparing seizure-free (n=8) with non-seizure-free (n=4) patients, we did not find significant association with the epilepsy duration before surgery (p=0.6) or age at onset (p=0.46), presence of hippocampal sclerosis (p=0.56) or type of SOZ (focal versus network, p=0.27). It should be noted, however, that the outcome seems to be better in focal cases than in network cases (3/4 of Engel III IV outcomes are network cases). Better outcome tended to be associated with pure temporal localization of the SOZ (Engel I, 6/7, 85%) versus extratemporal/temporal-plus (including bi-temporal) localization (Engel I, 1/5, 20%), though this was non-significant (Fisher test p=0.09).

Discussion

We report here the first retrospective study of SEEG-based analysis in PTE. We collected 18 cases from four epilepsy surgery centers in France over a period of twenty years, confirming that PTE is rare indication for patients undergoing presurgical evaluation. This is in agreement with the low rate of glial scars (potentially post-traumatic) in the European Brain Bank of epilepsy surgery specimens [START_REF] Blumcke | Histopathological Findings in Brain Tissue Obtained during Epilepsy Surgery[END_REF] and in agreement with previous SEEG series [START_REF] Lagarde | The repertoire of seizure onset patterns in human focal epilepsies: Determinants and prognostic values[END_REF][START_REF] Gonzalez-Martinez | Stereoelectroencephalography in the "difficult to localize" refractory focal epilepsy: early experience from a North American epilepsy center[END_REF].

A c c e p t e d m a n u s c r i p t

4.1.The seizure-onset zone in PTE

Our results showed that seizures arose mainly from temporal lobe (77.7% ; 44.4% temporal and 33.3% temporal "plus"), and frontal lobe (22.3%), consistent with previous findings [START_REF] Hitti | Surgical Outcomes in Post-Traumatic Epilepsy: A Single Institutional Experience[END_REF][START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF]. None of our patients suffered from parietal or occipital lobe epilepsy. In our sample, only 22.2% showed mesial temporal sclerosis (MTS), less than in previous studies [START_REF] Hitti | Surgical Outcomes in Post-Traumatic Epilepsy: A Single Institutional Experience[END_REF][START_REF] Gupta | Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features[END_REF]. Most of the patients (63.6%) showed a network organization of the SOZ, which was slightly more frequent in frontal lobe localization. Only 16% of our sample presented bilateral SOZ. By comparison, the proportion of cases with network organization of the SOZ appeared to be similar in other causes of drug-resistant epilepsy explored in SEEG, such as malformations of cortical development [START_REF] Aubert | Local and remote epileptogenicity in focal cortical dysplasias and neurodevelopmental tumours[END_REF] or tuberous sclerosis [START_REF] Neal | Epileptogenicity in tuberous sclerosis complex: A stereoelectroencephalographic study[END_REF] .

Slightly more than a third of the patients presented an SOZ smaller than the posttraumatic lesion (detailed in Table 1, example of patient 4 detailed in Fig 4). This is an important finding, since the main explanation for the lack of surgical data concerning PTE could be the belief that such epilepsies are less likely to be surgical candidates due to their "non-focal" nature and/or large lesion on MRI. Our findings highlight the interest of SEEG in this population, since in most cases tailored resection was possible, and was sometimes smaller than the visible MRI lesion.

Regarding the SOP, these were composed by relatively slow ictal discharge (below 12 Hz) in half of the cases, showing that fast discharges are not a necessary hallmark of seizure onset in such cases. It has already been proposed to distinguish patterns including LVFA (with or without preceding spikes) from patterns characterized by rhythmic slower activity (rhythmic theta-alpha waves or spikes). These slow SOP are found in a non-negligible proportion of SOP collected in a large SEEG retrospective study [START_REF] Lagarde | The repertoire of seizure onset patterns in human focal epilepsies: Determinants and prognostic values[END_REF].

A c c e p t e d m a n u s c r i p t

4.2.Surgical outcome

We reviewed the surgical series related to PTE, detailed in Table 2. Some series [START_REF] Gupta | Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features[END_REF], [START_REF] Hartzfeld | Characteristics and surgical outcomes for medial temporal post-traumatic epilepsy[END_REF] showed better surgical outcomes in temporal lobe epilepsy (TLE) (63 and 69% Engel I respectively) than in frontal lobe epilepsy(FLE) (30 and 33% Engel I respectively). These findings have been confirmed by another study [START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF] showing an Engel class I rate of 28% following resection of extra-temporal epileptic SOZ (mostly located in the frontal lobe) in PTE, highlighting the negative impact on outcome of absence of MRI lesions, absence of intracranial recordings prior to surgery and localization of the SOZ in the dominant hemisphere. Another study [START_REF] Schuh | Influence of head trauma on outcome following anterior temporal lobectomy[END_REF] found slightly better surgical outcomes in non-traumatic mesial TLE comparing with post-traumatic mesial TLE (40% and 50-69% of Engel class I, respectively), a tendency found in another study (78% and 63% of Engel class I, respectively), although non-statistically significant in this last study [START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF]. More recently, Hitti et al [START_REF] Hitti | Surgical Outcomes in Post-Traumatic Epilepsy: A Single Institutional Experience[END_REF] reviewed their institutional surgical experience in PTE and found an overall excellent surgical outcome (Engel Class I 68.8%), with a vast majority (82.6%) of mesial TLE. In our study, we confirmed previous findings showing an overall longterm surgical outcome that was good to excellent, with only one operated patient who did not experience worthwhile improvement of his epilepsy. Moreover, we found a globally better surgical outcome for TLE (Engel I 77%, Engel III 11.5%, Engel IV 11.5%) than for FLE (25% Engel I, 50% Engel III). As previously highlighted [START_REF] Hakimian | Long-term outcome of extratemporal resection in posttraumatic epilepsy[END_REF], intracranial electrode implantation during the presurgical assessment of drug-resistant PTE plays a crucial role in surgical outcome of such epilepsies.

Nevertheless, only a few patients included in previous studies have benefited from intracranial recordings, using mainly subdural electrodes [START_REF] Marks | Seizure localization and pathology following head injury in patients with uncontrolled epilepsy[END_REF][START_REF] Hitti | Surgical Outcomes in Post-Traumatic Epilepsy: A Single Institutional Experience[END_REF][START_REF] Gupta | Subtypes of post-traumatic epilepsy: clinical, electrophysiological, and imaging features[END_REF][START_REF] Schuh | Influence of head trauma on outcome following anterior temporal lobectomy[END_REF].

Implantation of invasive electrodes may indeed be surgically challenging due to

A c c e p t e d m a n u s c r i p t adhesions and scar tissue secondary to head trauma. On the contrary, SEEG electrode implantation is less affected by this issue [START_REF] Cardinale | Implantation of Stereoelectroencephalography Electrodes: A Systematic Review[END_REF] and only one patient in our group suffered from an asymptomatic post-SEEG complication (subdural hemorrhage).

To our knowledge, the present study is the first one carried out using SEEG prior to surgery for PTE. The good results obtained suggest that patient selection was satisfactory in the present series, and that PTE should not be considered per se to be a contraindication to pursing pre-surgical evaluation, including intracerebral recordings if necessary. Of note, about a third of our sample displayed a SOZ smaller than the MRI post-traumatic lesions observed. Therefore, SEEG data are crucial in this scenario to tailor the surgical resection, which can sometimes be smaller than the MRI lesion.

A

  c c e p t e d m a n u s c r i p t Surgical outcome was excellent in 8/12 (Engel I) (66%), slight improvement in 3/12 (Engel III, 2/3 FLE) and no worthwhile improvement in 1/12 (Engel IV). Mean follow-up duration was 39.5 months, with no significant difference between patients with Engel class Ia and the others (Engel class II-IV) (mean duration 36.5 and 45.5 months respectively, range 12-60 months). Post-operative MRI showed complete SOZ resection in 9/13, associated with excellent outcomes (Engel Ia) in 7/9. Incomplete resection of the SOZ (4/13), mainly due to close relationship with eloquent cortices, was associated with poorer outcomes (1/4 Engel I, 1/4 Engel III, 1/4 Engel IV).

Figure 1 .Figure 2 .

 12 Figure 1. Flow chart of patient selection. PTE = post-traumatic epilepsy; SEEG =

Figure 3 .A/

 3 Figure 3.

Figure 4 .

 4 Figure 4. Illustrative case of post-traumatic temporal lobe epilepsy in a patient

Table 1 .

 1 Clinical, neuroradiological and surgical outcome in the studied population. TBI: traumatic brain injury, SOZ: seizure onset zone determined on SEEG recordings, HS: hippocampal sclerosis, GK: Gamma Knife radiosurgery Table 2. Published surgical series on post-traumatic epilepsies. PTE = Post traumatic epilepsy ; TBI = traumatic brain injury ; vEEG = videoelectroencephalography ; SEEG = stereoelectroencephalography ; TLE = Temporal lobe epilepsy ; MTLE = Mesial temporal lobe epilepsy ; MTS = mesio-temporal sclerosis ; ATL = anterior temporal lobectomy ; FLE = frontal lobe epilepsy ; EcoG = electrocorticography ; PLE = parietal lobe epilepsy ; OLE = occipital lobe epilepsy ; VNS = vagal nerve stimulation ; RNS = responsive neurostimulat A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t ortic al epile psy 4/ 23 A c c e p t e d m a n u s c r i p t

 23

Table 1 A

 1

	a n u s c r i p t
	m
	c c e p t e d

Table 2

 2

	Authors	Numbe	Material/method	Population	Epil	Surgery	Outcome
	and date	r of		characteristics	epsy
		patient			type
		s
	Mathern et	26	Clinical and histopathological findings; no vEEG or	-Age : 0,2-29 years	TLE	Not detailed	Better outcome in post-traumatic TLE
	al, 1994 9		intracranial recordings	-TBI classification :			(80% Engel I) than TLE post birth
			Comparison with non PT-TLE (94)	15/26 severe, 6/26			brain injury (18% Engel I)
				moderate, 5/26 mild
	Marks et	25	Presurgical assessment of refractory epilepsy, using vEEG	-Mean age at TBI :	-	-MTLE : 14/17 ATL	-MTLE :
	al, 1995 10		in 25/25 and intracranial EEG in 21/25 (grids/strips	10.7 years	MTL	-Neocortical epilepsy : 3/8	-6/14 (42,8%) : seizure free,5/6
			mainly, depth electrodes only for MTLE)	-Mean age at epilepsy	E	neocortical cortectomy (2/3	with MTS
				onset : 13,6 years	17/2	partial frontal cortectomy +	-7/14 (50%) : no seizure
					5	callosotomy, 1/3 occipital	improvement (no MTS)
					-	cortectomy) ; 5/17	-1/14 (7,2%) : 50% reduction of
					"Neo	contraindicated	seizures (no MTS)
					corti		-Neocortical epilepsy : 3/3 seizure-
					cal"		free
					epile
					psy
					8/25

Kazemi et al, 1997 14

	Gupta et	123	Presurgical assessment of refractory epilepsy with vEEG	TBI and epilepsy -Mean age at TBI :	FLE	22/123 (procedures not	-TLE : Engel I 69%, Engel II 19%,
	Hartzfield et al, 2008 11 Hakimiane t al, 2012 13 al, 2014 24	57 21	-Presurgical assessment of refractory PTE, using vEEG and per-operative EcoG -Inclusion of operated patients -Presurgical assessment of refractory epilepsy with vEEG in 21/21, and intracranial recordings in 16/21 (mainly grids/strips) -Only patients with extra-temporal refractory PTE grids/strips) in 21/21, and intracranial recordings in 16/21 (mainly	m a n u s c r i p t onset : 1.6 years -Mean age at resection 36years -Average duration of epilepsy 18 years -MRI findings : 16 /57 MTS ; other 26/57 : gliosis (mostly in frontal lobes), focal atrophy, encephalomalacia ; --15/57 patients with normal MRI -MTL E 30/5 7 -Extra -hipp ocam pal 27/5 7 PTE group : -Medial temporal resection 30/57, with MTS in 53% -Extrahippocampal resection 20/57 -VNS 7/57 Non-PTE group : 31/31 with mesial temporal lobe resection PTE group : -Medial temporal lobe resection : Engel I 63%, Engel II 10%, -Extrahippocampal resections : Engel I 30%, Engel II 25%, -no statistical difference between PTE and non-PTE patients -Mean age at TBI 15,7years -Mean age at surgery : 34,7 years -Mean age at epilepsy onset : 15,7 years Severity of TBI : 8/21 -FLE 12/2 1 -Front al plus -Frontal lobectomy 12/21 -Frontal and temporal lobectomy 6/21 Posterior neocortical resection (parietal, posterior temporo-occipital and temporo-parietal) resections -Overall : Engel I 28%, Engel II 57%, -Risk factors of worst outcomes : MRI-negative PTE, no invasive monitoring assessment, resection in the dominant hemisphere (3/4 lesio nal) OLE m -Mean age at epilepsy onset : 23,1years 23 (32/4 0 lesio nal) TLE 66/1 23 (46/6 6 lesio nal, 29/4 MTS ) PLE : 4/12 3 a majority of MTS), others are FLE a n u s c r i p t 20years 40/1 detailed) ; mostly TLE (with -FLE : Engel I 33%, Engel II 17%,,
	Schuh et al, 1998 26 Diaz Arrastia et al, 2000 8 Hitti et al, 2019 12	17 29 33 23	Presurgical assessment of refractory epilepsy, using vEEG and per-operative EcoG -Presurgical assessment of refractory TLE, using intracranial EEG monitoring (depth electrodes or grids) in only 34/102 patients -Include only operated patients (anterior TL) -Comparison with non PT MTLE (93) Presurgical assessment of refractory epilepsy with vEEG in 23/33 patients who had refractory PTE A c c e p t e d -Post-traumatic encephalomalacia 8/17 -Others 9/17 -Mean age at epilepsy onset :12.1-14.9 years -Mean age at surgery : 31.9-34.2 years -PT Epilepsy -Mean age at TBI 22.9 years old -Mean time between mild, 12/21 moderate to severe ; one had both type of severity -MRI findings : encephalomalacia 12/21 Presurgical assessment of refractory epilepsy with vEEG in 23/23, and intracranial recordings in 8/23 -Mean age : 38 years -TBI classification : 60,9% moderate to severe, 30,1% mild A c c e p t e d	FLE MTL E MTL E 8/23 epile psy : 6/21 -"Post erior " epile psy : 3/21 : 4/12 3 (3/4 lesio nal) Mult ifoca l : 1/12 3 MTL E 19/2 3, all with MTS Neoc	Tailored frontal cortectomy (guided by MRI lesion findings) in all patients ATL in all post-traumatic MTLE ATL 2/8 3/21 ATL: 14 Tailored cortectomy: 2 VNS implantation 7 (multiple foci)	Engel I 59%, Engel II 11,5% Better outcome with complete resection of the encephalomalacia (71% Engel I vs 0% Engel I) Poorer outcome in post-traumatic MTLE (40% Engel I) vs no history of head trauma (50-69% Engel I) Engel I 50%, Engel II 50% -Resection group : Engel I 68,8%, Engel II 18,8%, -VNS group : average seizure frequency reduction 30,6%, leading to RNS 3/7 RNS implantation in 3/3 with more than 50% of seizure reduction

A c c e p t e d m a n u s c r i p t


Daniel Frankel
email: daniel.frankel@newcastle.ac.uk

Matthew Davies

Bharat Bhushan

Yavuz Kulaberoglu

Paulina Urriola-Munoz

Justine Bertrand-Michel

Melissa R Pergande

Andrew A Smith

Swapan Preet

Thomas J Park

Michele Vendruscolo

Kenneth Rankin

Stephanie M Cologna

Janet R Kumita

Nicolas Cenac
email: nicolas.cenac@inserm.fr

Ewan St

John Smith

Lipid membranes from naked mole-rat brain lipids are cholesterol-rich, highly phase-separated, and sensitive to amyloid-induced damage

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The naked mole-rat is a long-lived species having a life span that can exceed 30 years [START_REF] Smith | The naked mole-rat as an animal model in biomedical research: current perspectives[END_REF][START_REF] Ruby | Naked Mole-Rat mortality rates defy gompertzian laws by not increasing with age[END_REF] , which is nearly ten times longer than that of rodents of comparable size. Possessing many neotenic features [START_REF] Skulachev | Prolongation of Youth: From Naked Mole Rats to "Naked Apes" (Humans)[END_REF]4 and defying many of the signs of ageing [START_REF] Buffenstein | Negligible senescence in the longest living rodent, the naked molerat: insights from a successfully aging species[END_REF] , the naked mole-rat does not appear to develop neurodegenerative disease [START_REF] Edrey | Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease[END_REF][START_REF] Orr | Extended Postnatal Brain Development in the Longest-Lived Rodent: Prolonged Maintenance of Neotenous Traits in the Naked Mole-Rat Brain[END_REF] .

Considering that amyloid beta is an age-related pathogenic agent in Alzheimer's disease [START_REF] Van Der Kant | Amyloid-beta-independent regulators of tau pathology in Alzheimer disease[END_REF] , it was perhaps surprising that the naked mole-rat brain was observed to have high levels of amyloid beta, exceeding in particular those found in the commonly used 3´Tg transgenic mouse model of Alzheimer's disease, even in the brains of young animals [START_REF] Edrey | Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease[END_REF] . Somehow, therefore, naked mole-rats appear to be able to live for years with this neurotoxic peptide. Although the exact role of amyloid beta in Alzheimer's disease (AD) remains controversial [START_REF] Castellani | The amyloid cascade and Alzheimer's disease therapeutics: theory versus observation[END_REF] with numerous drugs targeting amyloid pathways failing in clinical trials, amyloid plaques are a hallmark of the diseased brain. It is remarkable that no such amyloid plaques are found in naked mole-rat brains despite the high levels of this peptide [START_REF] Edrey | Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease[END_REF] . Thus, one would like to understand why amyloid plaques do not form in naked mole-rat brains. This question is likely to have a complex answer as in comparing the human and naked mole-rat variants of amyloid beta, one finds only a single amino acid difference, which alters the aggregation kinetics, but not the degree of toxicity to mouse hippocampal neurones [START_REF] Edrey | Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease[END_REF] .

In this work we investigated whether the resistance to amyloid beta aggregation in the naked mole-rat can be traced back to specific properties of the lipid membranes of this animal, given that it is well established that lipid membranes can affect the aggregation of amyloid beta 10- 12,13 . For example, there is a strong link between cholesterol and Alzheimer's disease, which could be related to the interaction between amyloid beta with lipid membranes [START_REF] Wang | The relationship between cholesterol level and Alzheimer's diseaseassociated APP proteolysis/Abeta metabolism[END_REF][START_REF] Seghezza | Cholesterol drives abeta(1-42) interaction with lipid rafts in model membranes[END_REF][START_REF] Habchi | Cholesterol catalyses Abeta42 aggregation through a heterogeneous nucleation pathway in the presence of lipid membranes[END_REF] . Naked mole-rat lipids from brains and other tissue have been analysed in relation to their role in the membrane pacemaker theory of ageing, which states that long-lived species have membrane compositions that resist peroxidation, a proposed mechanism in resistance to ageing [START_REF] Hulbert | On the importance of fatty acid composition of membranes for aging[END_REF] . In naked mole-rat brains and within other tissues, it was observed that there was significantly less docosahexanoic acid (DHA), a fatty acid which is particularly susceptible to peroxidation, than that found in mice. In addition, it was reported that naked mole-rat membranes contain more vinyl ether linked phosphoplipids (plasmalogens) which are resistant to peroxidation. [START_REF] Hulbert | Oxidation-Resistant Membrane Phospholipids Can Explain Longevity Differences Among the Longest-Living Rodents and Similarly-Sized Mice[END_REF][START_REF] Mitchell | Membrane phospholipid composition may contribute to exceptional longevity of the naked mole-rat (Heterocephalus glaber): a comparative study using shotgun lipidomics[END_REF] Plasmalogens are highly enriched in lipid rafts/microdomains and contribute to their stability. [START_REF] Braverman | Functions of plasmalogen lipids in health and disease[END_REF] Thus, a high level of this ether-lipid may be an indicator of a high raft content, or increased raft stability. [START_REF] Gorgas | The ether lipid-deficient mouse: tracking down plasmalogen functions[END_REF] Here we used atomic force microscopy (AFM), as this method presents many advantages for examining lipid bilayer based systems, in particular its ability to quantify two dimensional ordering and to follow interactions of the membrane with peptides in real time [START_REF] Azouz | Membrane domain modulation of Abeta1-42 oligomer interactions with supported lipid bilayers: an atomic force microscopy investigation[END_REF][START_REF] Et-Thakafy | Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid-ordered phase, determined using AFM force spectroscopy[END_REF] . More specifically, we combined AFM with lipidomics to compare naked mole-rat and mouse brain lipids in terms of composition and two dimensional ordering. We then used AFM to follow the interactions of synthetic human amyloid beta with brain-derived lipid membranes from both mouse and naked mole-rat. The membrane pacemaker theory of ageing suggests that cell membranes of long-lived species will possess more peroxidation resistant species which may also be raft forming [START_REF] Hulbert | Oxidation-Resistant Membrane Phospholipids Can Explain Longevity Differences Among the Longest-Living Rodents and Similarly-Sized Mice[END_REF] . We thus hypothesise that naked mole-rat brain derived lipid bilayers show a high degree of phase separation (two-dimensional ordering) and that there may be a unique lipid signature that protects the membranes against amyloid beta induced damage.

Results

Comparison of the lipid membrane compositions of naked mole-rat and mouse brains.

Mass spectrometry of brain total lipid extract shows that there are key differences between naked mole-rat and mouse brains, which may have implications for amyloid beta assembly and membrane interactions. Firstly, naked mole-rat brains have significantly more cholesterol than mouse (Figure 1A). Also of note is the relatively low level of sphingomyelin in naked mole-rat brains relative to mouse (Figure 1B). Pettegrew et al. found that the levels of brain sphingomyelin positively correlate with amyloid beta plaques, however other studies show the opposite correlation between sphingomyelin and AD [START_REF] Pettegrew | Brain membrane phospholipid alterations in Alzheimer's disease[END_REF] . In contrast to the observations for cholesterol and sphingomyelin, the concentrations of the total phospholipids and ceramides were not different between naked mole-rat and mouse brains (Figure 1C,D). The relative abundances of these different lipid species were evaluated and presented as heatmaps (Figure 1E,F) and as tables (Supplementary Tables 123456).

We found that these lipidomic profiles were different between mouse and naked mole-rat brains, as illustrated by a clustering of mouse and naked mole-rat depending on the number of carbons of the phospholipids, or of the sphingomyelins and ceramides (Figure 1E,F, top dendrograms). Concerning the phospholipids, four major groups of lipids were identified: Cluster 1 contained lipids which were more abundant in naked mole-rat than mouse brains, Clusters 2 and 3 contained lipids which were not differentially expressed between mouse and naked mole-rat brains, and Cluster 4 contained lipids that were more abundant in mouse brains (Figure 1E). However, these four clusters did not reveal a distinct pattern of species. The ceramide and sphingolipids clustering highlighted 4 clusters: Cluster 1 was composed of lipids more abundant in mouse brains, Cluster 3 by lipids more abundant in naked mole-rat brains, and Clusters 2 and 4 being no different between the two species. Interestingly, the sphingomyelins and ceramides preferentially presented in naked mole-rat brains (Cluster 3) were the shortest lipids quantified. Thus, this lipid analysis revealed differences in the lipid composition of mouse and naked mole-rat brains.

With the finding that multiple lipids were different between the two species, we sought to investigate if this was supported at the protein level. Using an untargeted differential proteomic study, we compared protein abundance between mice and naked mole-rats (Figure 2). We observed decreased levels of acid ceramidase, the enzyme responsible for ceramide degradation into sphingosine -a primary constituent of sphingolipids -and free fatty acids, in naked mole-rat cerebellar tissue (Figure 2A), and ceramide synthase 2 (catalyses the formation of ceramide with selectivity towards very long, C22:0 -C24:0 chains) was also decreased in naked mole-rats both in hippocampal and cerebellar tissue lysate (Figure 2B,C). Sphingomyelinase (sphingomyelin phosphodiesterase 3), which catalyses the hydrolysis of sphingomyelin to ceramide and phosphocholine, is also decreased in naked mole-rat hippocampal tissue (Figure 2D) and the sphingosine-1-phosphate receptor 1 is also decreased in naked mole-rat cerebellum and cerebral cortex (Figure 2E,F). The decreased expression of these proteins establishes a connection for the decreased sphingomyelin levels in naked mole-rats and also the potential atlerations of chain-length specific ceramides.

Overall, naked mole-rat brain lipids contained a higher concentration of cholesterol and lower concentration of sphingomyelins, which were of shorter length than those present in mouse brains, but contained a higher percentage of saturated sphingomyelin (Supp. Figure 1). We completed the comparison of lipid composition of naked mole-rat and mouse brains by quantifying the concentration in total fatty acids (FA). No significant differences were observed between the concentrations of saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) in mouse and naked mole-rat brains (Figure 3A-C). However, the n-6/n-3 ratio of PUFAs was increased in naked mole-rat brains compared to mouse brains (Figure 3D). The relative abundances of these FA were evaluated and presented as a heatmap (Figure 3E) and as table (Supplementary Table 7). We found that the FA profile was different between mouse and naked mole-rat brains, illustrated by a clustering of mouse and naked mole-rat depending on the number of carbons within the FA (Figure 3E). Three major groups of FA were identified: Cluster 1 contained lipids which were more abundant in mouse brains, Cluster 2 contained FA which were not differentially expressed between mouse and naked mole-rat brains, and Cluster 3 contained lipids that were more abundant in naked mole-rat brains (Figure 3E). As observed for the ceramides and sphingomyelin, the SFA and MUFA abundant in naked mole-rat brains (Cluster 3) were of shorter length than those present in mouse brains. Supplementary Tables 1 -7 provide a detailed comparative overview of the percentage of different lipid species identified in mouse and naked mole-rat brain lipids.

Amyloid beta avoids gel phase domains in model lipid membranes.

Prior to an AFM examination of the interactions between human amyloid beta and the brain tissue derived lipids, we used a simple model system comprising of just two lipids, DOPC and DPPC. This system exhibits phase separation and provides a well-defined comparison for the more complex mixture of lipids present in brain derived lipids. When the binary system was mixed in a molar ratio of 3:1 of DOPC-to-DPPC, we observed the classic phase separated DPPC gel domains surrounded by the liquid disordered DOPC (Figure 4A). The origin of the islands of DPPC is due to the packing arrangement of DPPC chains, resulting in a height difference of around 1.5 nm to the surrounding DOPC (Figure 4B). Such spontaneous phase separation behaviour in model systems has been used as a rationale for the existence of lipid rafts in cells [START_REF] Bhatia | Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase[END_REF] . After 2 hours exposure to 8 µM of human amyloid beta the bilayer remained completely intact, but with clearly visible features in between the DPPC gel phase domains, that are absorbed on top of the DOPC liquid disordered phase (highlighted by blue circle in Figure 4C). These features are the amyloid beta peptide and it is to be noted that no peptide adsorbs on the gel phase domains. Upon closer inspection, the amyloid beta is observed to avoid the gel phase domains, such that the central domain is surrounded, but not covered, by the peptide (Figure 4D).

Lipid bilayers formed from naked mole-rat brain-derived total lipid extracts exhibit a high degree of phase separation.

Lipid bilayers formed from naked mole-rat brain-derived total lipid extracts were formed on a cleaved mica surface and were found to exhibit a high degree of phase separation, with the characteristic islands (Figure 5A). These islands could occupy an area greater than 80% of that of the whole lipid membrane. At higher magnification, it was sometimes possible to distinguish a two-tier raft (Figure 5B,C), but this was not seen in all preparations. However, the high degree of phase separation was observed in lipids extracted from all naked mole-rat brains studied. For comparison mouse brain lipids exhibited little to no phase separation, such that a lipid membrane formed with little to no deviation in height and structure (Figure 5D).

By indenting the conical AFM tip into the lipid bilayer it is possible to measure the breakthrough force [START_REF] Garcia-Manyes | Nanomechanics of lipid bilayers by force spectroscopy with AFM: a perspective[END_REF] , which is the force required to push through the soft thin film (Figure 6A,B), thereby providing an indication of the membrane fluidity. It also allows the measurement of membrane thickness, which is a necessary confirmation that a thin soft bilayer is being imaged. Although no difference was observed in the thickness of lipid bilayer membranes formed from mouse or naked mole-rat brain derived lipids (Figure 6C), the breakthrough force measurements for naked mole-rat membranes were significantly higher than mouse (Figure 6D), meaning that naked mole-rat lipid bilayer membranes are stiffer. The enhanced stiffness of naked mole-rat lipid bilayer membranes is most likely due to the increased cholesterol, which is known to increase membrane rigidity. 27

Amyloid beta disrupts lipid bilayers formed from naked mole-rat brain-derived total lipid extracts.

Prior to exposure to amyloid beta, the mouse brain derived lipid bilayer had a characteristic morphology exhibiting almost no phase separation with only the occasional, small domain being visible (Figure 7A). When exposed to 8 µM human amyloid beta for 2 hours, pores appeared in the mouse derived lipid bilayer (Figure 7B). The pores were not circular, and there was little sign of the amyloid beta that had caused them. A likely explanation is that the amyloid beta peptides have sunk into the membrane because the height profile through the pores shows that they are less deep than the thickness of the bilayer (Figure 7C,D). It is important to note that the integrity of the membrane, as a whole, is maintained.

By contrast to what was observed with mouse lipid bilayers, upon exposure of naked molerat lipid bilayers to human amyloid beta under the same conditions (8 µM for 2 hours), the naked mole-rat lipid bilayer membrane is disrupted (Figure 8). In contrast to the raft covered membrane observed under control conditions (Figure 8A), incubation with amyloid beta results in the presence of only fragments of lipid bilayer (Figure 8B,C), > 5 nm in height relative to the surface (consistent with the thickness of the bilayer rather than the height of a raft, Figure 8D). Membrane fragments after amyloid beta exposure were observed with lipid bilayers formed from lipids extracted from all 3 brains examined.

Discussion

Our lipidomic data show fatty acids and phospholipid trends between mouse and naked molerat that are consistent with the previous work of Mitchell et al [START_REF] Mitchell | Membrane phospholipid composition may contribute to exceptional longevity of the naked mole-rat (Heterocephalus glaber): a comparative study using shotgun lipidomics[END_REF] . However, in addition we have found that cholesterol levels in naked mole-rat brains are significantly higher than in mouse brains and that sphingomyelin levels are significantly lower and of a shorter chain lengths. These lipid findings are supported by proteomic analysis of brain lysates and critical lipid metabolism enzymes were also altered in naked mole-rats compared to mice. Using AFM, we found that naked mole-rat brain lipids have a high level of phase separation, which is consistent with predictions from the membrane pacemaker theory. Regardless of whether such domains exist in a living cell, the lipids that are able to phase separate could have a role in protection against ageing. Mechanical measurements show that the extra cholesterol results in a stiffer membrane, but surprisingly, these phase-separated and stiffer membranes offer limited protection against amyloid beta induced damage. The susceptibility of naked mole-rat membranes to amyloid beta induced damage was unexpected given the amyloid beta rich environment of the naked mole-rat brains and the lack of overt neurodegeneration in this species [START_REF] Edrey | Amyloid beta and the longest-lived rodent: the naked mole-rat as a model for natural protection from Alzheimer's disease[END_REF] .

Cell membranes consist of more than just phospholipids, with cytoskeleton, proteins and carbohydrates all contributing to membrane stability. However, the relative difference in response between mouse and naked mole-rat lipids bilayers could be a significant contributor to the amyloid beta response in each species. It should also be noted that the total lipid extract from brain tissue will include not just lipids from cell membranes but also from mitochondria and other organelles. Interestingly, the same susceptibility of naked mole-rat membrane lipids to damage was found when using naked mole-rat amyloid beta peptide (Supplementary Figures 2345). This is despite the single amino acid difference in sequence whereby histidine is replaced by arginine in naked mole rat amyloid beta. We find that naked mole rat amyloid beta readily forms fibres and fibrils (Supplementary Figures 2 and3) as is the case with human amyloid beta. Again with mouse brain lipids, the naked mole rat amyloid beta sinks into the bilayer, leaving the bilayer intact but with holes (Supplementary material Figure 4). Upon exposure to naked mole rat amyloid beta the naked mole rat brain derived supported bilayers are broken up into membrane fragments (Supplementary material Figure 5). This consistency in behaviour between human and naked mole rat amyloid beta with respect to membrane interactions strengthens the argument that brain lipid composition may be critical to the physiology of neurodegeneration in naked mole rats.

In summary, naked mole-rat brain lipids have high cholesterol and low sphingomyelin levels compared to mouse brain lipids and the bilayers formed from naked mole-rat brain lipids are unusual in terms of a high degree of phase separation. By contrast, both mouse from our data and pig brain lipids [START_REF] Mari | Reversible Cation-Selective Attachment and Self-Assembly of Human Tau on Supported Brain Lipid Membranes[END_REF] show no or little signs of such two dimensional ordering. The comparative sensitivity of naked mole-rat lipid bilayers to amyloid beta damage suggests that the naked mole-rat has evolved neuroprotective mechanisms based more on the resistance to oxidative processes than mechanical resistance to support a healthy brain within an amyloid beta rich environment.

Materials and Methods

Animals

Experiments were performed on lipids and proteins extracted from C57BL6/J mice and nonbreeding naked mole-rats. A mixture of males and females were used of both species, all adults for lipid studies. For proteomic studies, mice (N=8) were euthanized at 2-weeks (N=4) and 6 months (N=4) of age. Similarly, NMRs were euthanized at 2-4 months (N=4) and 3 years (N=4) of age. Cerebellum, cortex and hippocampus were collected and flash frozen, as previously described [START_REF] Browe | The naked mole-rat has a functional purinergic pain pathway despite having a non-functional peptidergic pain pathway[END_REF] . Mice were conventionally housed with nesting material and a red plastic shelter in temperature-controlled rooms at 21 °C, with a 12 h light/dark cycle and access to food and water ad libitum. Naked mole-rats were bred in-house and maintained in an inter-connected network of cages in a humidified (~55 %) temperature-controlled room at 28 °C, with red lighting (08:00-16:00) and had access to food ad libitum. In addition, a heat cable provided extra warmth under 2-3 cages/colony. Mice were humanely killed by cervical dislocation of the neck and cessation of circulation, whereas naked mole-rats were killed by CO2 exposure followed by decapitation. Experiments were conducted under the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 under a Project License (P7EBFC1B1) granted to E. St. J. Smith by the Home Office and approved by the University of Cambridge Animal Welfare Ethical Review Body. Experiments performed for proteomic analysis were done so in accordance with institutionally-approved protocols at the University of Illinois at Chicago (PI -T.J. Park).

Lipid extraction

Lipid extraction was performed according to a modified chloroform/methanol (2:1, v/v) procedure [START_REF] Petkovic | Comparison of Different Procedures for the Lipid Extraction from HL-60 Cells:A MALDI-TOF Mass Spectrometric Study[END_REF] . Briefly, frozen brain tissue from naked mole-rat or mouse was crushed into small pieces by razor in a petri dish, then the sample was transferred into a 15 mL tube and 2 mL of chloroform/methanol (2:1, v/v) was added; chloroform (Sigma 288306-1L), methanol (Fisher 10675112). The suspension was vortexed and incubated at room temperature with agitation for 20 min. After the incubation, 400 µL of 0.9% NaCl was added to the mixture and was then vortexed and centrifuged at 500 x g for 10 min to separate the organic phase from the aqueous phase. The chloroform (lower) layer was transferred into a new 1.5 mL tube and was centrifuged at 500 x g for 10 min. Finally, the supernatant was collected without touching the pellet and was transferred into a new 1.5 mL tube. The sample was stored at -80 °C until the analysis.

Brain neutral lipids analysis

Internal standards: glyceryl trinonadecanoate, stigmasterol, and cholesteryl heptadecanoate (Sigma) were added to 45 µL of lipid extract. Triglycerides, cholesterol, and cholesterol esters were analysed with gas-liquid chromatography on a Focus Thermo Electron system equipped with a Zebron-1 Phenomenex fused-silica capillary column (5 m, 0.25 mm i.d., 0.25 mm film thickness). Oven temperature was programmed to increase from 200 to 350 °C at 5 °C/min, and the carrier gas was hydrogen (0.5 bar). Injector and detector temperatures were 315 °C and 345 °C, respectively.

Brain phospholipid, ceramide and sphingolipid analysis

Internal standards (Cer d18:1/15:0, 16 ng; PE 12:0/12:0, 180 ng; PC 13:0/13:0, 16 ng; SM d18:1/12:0, 16 ng; PI 16:0/17:0, 30 ng; PS 12:0/12:0, 156.25 ng; all from Avanti Polar Lipids) were added to 45 µL of lipid extract. Sample solutions were analysed using an Agilent 1290 UPLC system coupled to a G6460 triple quadripole spectrometer (Agilent Technologies). MassHunter software was used for data acquisition and analysis. A Kinetex HILIC column (Phenomenex, 50 × 4.6 mm, 2.6 μm) was used for LC separations. The column temperature was maintained at 40 °C. Mobile phase A was acetonitrile and B was 10 mM ammonium formate in water at pH 3.2. The gradient was as follows: from 10% to 30% B in 10 min, 100% B from 10 to 12 min, and then back to 10% B at 13 min for 1 min to re-equilibrate prior to the next injection. The flow rate of the mobile phase was 0.3 ml/min, and the injection volume was 5 μl. An electrospray source was employed in positive (for Cer, PE, PC, and SM analysis) or negative ion mode (for PI and PS analysis). The collision gas was nitrogen. Needle voltage was set at +4000 V. Several scan modes were used. First, to obtain the naturally different masses of different species, we analysed cell lipid extracts with a precursor ion scan at 184 m/z, 241 m/z, and 264 m/z for PC/SM, PI, and Cer, respectively. We performed a neutral loss scan at 141 and 87 m/z for PE and PS, respectively. The collision energy optimums for Cer, PE, PC, SM, PI, and PS were 25 eV, 20 eV, 30 eV, 25 eV, 45 eV, and 22 eV, respectively. The corresponding SRM transitions were used to quantify different phospholipid species for each class. Two MRM acquisitions were necessary, due to important differences between phospholipid classes. Data were treated with QqQ Quantitative (vB.05.00) and Qualitative analysis software (vB.04.00).

Brain proteomic analysis

Protein extracts for each tissue (cerebellum, cortex and hippocampus) and animal were prepared and analyzed via liquid chromatography-mass spectrometry (LC-MS) analysis. Protein extracts were obtained by lysis in RIPA buffer (50mM Tris pH 8, 150 mM NaCl, 1% Triton-X 100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate and 0.25% NP-40) and protein concentration determined via the BCA assay (Pierce-Thermo, San Jose, CA, USA). Peptide digests of 100μg of protein were prepared for each of the tissue lysates using the filter aided sample preparation (FASP) after spiking with recombinant green fluorescent protein (GFP) into each sample at a concentration of 100fmol per 1µg protein extract as previously described [START_REF] Pergande | Label-Free Proteomics in the Symptomatic Niemann-Pick, Type C1 Mouse Model Using Standard Flow Liquid Chromatography and Thermal Focusing Electrospray Ionization[END_REF] . Samples were desalted via C18 reverse phase chromatography and dried. All samples were resuspended in 100µL 0.1% (v/v) formic acid prior to LC-MS analysis.

LC-MS analysis

One microliter of each sample was injected for label-free, quantitative LC-MS analysis. Mass detection was carried out with a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an Agilent HPLC system. Chromatographic separation of peptides was accomplished using a Zorbax 300SB-C18 column (3.5µm i.d. x 150mm, particle size 5µm, pore size 100Å, Agilent Technologies, Wilminton, DE). Peptides were loaded onto a Zorbax 300SB-C18 trap cartridge at a flow rate of 2µl per minute for 10 min. After washing with 0.1% formic the peptides were eluted using a 5-60% B gradient for 180 min at a flow rate of 250 nl min -1 acid (mobile phase A= 0.1% formic acid; mobile phase B =0.1% formic acid in acetonitrile). The mass spectrometer was operated in a Top 10 data-dependent mode with automatic switching between MS and MS/MS with HCD fragmentation. Source ionization parameters were as follows: spray voltage, 1.5 kV; capillary temperature, 280 °C; and s-lens level, 50.0. Full-scan MS mode (m/z 300-1700) was operated at a resolution of 70 000 with automatic gain control (AGC), target of 1 × 10 6 ions and a maximum ion transfer (IT) of 250 milliseconds. Ions selected for MS/MS were subjected to the following parameters: resolution, 17, 500; AGC, 1 × 10 5 ions; maximum IT, 100 milliseconds; 1.5 m/z isolation window; normalized collision energy 27.0, and dynamic exclusion, 30 seconds.

Protein identification and label-free quantification

Raw data was searched against both the SwissProt Mus musculus (20,239 sequences) and an in house GFP database using the Proteome Discoverer software (v2.3, Thermo Fisher, Carlsbad, CA). Trypsin was set as the protease with two missed cleavages and searches were performed with precursor and fragment mass error tolerances set to 10 ppm and 0.02 Da, respectively, where only peptides precursors of +2, +3 and +4 were considered. Peptide variable modifications allowed during the search were: oxidation (M) and deamination (NQ), whereas carbamidomethyl (C) was set as fixed modifications. Label-free relative quantitation analysis was performed across tissue types using the average of the top 3 peptides for each protein where normalization was performed across all samples and all tissues using the intensity of the GFP internal standard. Differentially expressed proteins for NMR relative to mice for each tissue type was determined by applying a Permutation test (p < 0.05). Raw data is available at: ftp://massive.ucsd.edu/MSV000085798/raw/.

Brain fatty acid analysis

To measure the totalbrain molecular species derivatized with a methyl ester (FAME), internal standard, glyceryl triheptadecanoate (2 μg), was added to 40 µL of lipid extract. The lipid extract was transmethylated with 1 ml BF3 in methanol (14% solution; Sigma) and 1 ml heptane for 60 min at 80 °C and evaporated to dryness. The FAMEs were extracted with heptane/water (2:1). The organic phase was evaporated to dryness and dissolved in 50 μl ethyl acetate. A sample (1 μl) of total FAME was analysed with gas-liquid chromatography (Clarus 600 Perkin Elmer system, with Famewax RESTEK fused silica capillary columns, 30-m × 0.32-mm i.d., 0.25-μm film thickness). Oven temperature was programmed to increase from 110 °C to 220 °C at a rate of 2 °C/min, and the carrier gas was hydrogen (7.25 psi). Injector and detector temperatures were 225 °C and 245 °C, respectively.

Atomic force microscopy

AFM experiments were performed on an Agilent 5500 with closed loop scanners using a liquid cell for imaging of supported lipid bilayers. All experiments were carried out at 20 °C. Tapping mode imaging was performed using aluminium coated cantilevers (PPP-NCSTR, Apex Probes, UK) and all images were obtained in PBS buffer. Contact mode images were obtained using contact mode aluminium coated silicon cantilevers (PPP-CONTR, Apex Probes, UK) having nominal spring constants of between 0.02 and 0.77 N m -1 and typical tip radius of curvatures of less than 7 nm.

Amyloid beta preparation

Beta-amyloid (1-42), human (Anaspec) was reconstituted with 1.0% NH4OH and aliquoted before freezing. Before use the aliquot was defrosted and made up to the desired concentration with PBS buffer. This solution was then vortexed for 2 minutes before being added to the supported lipid bilayer. For all experiments 200 µl of 8 µM amyloid beta solution was added to the supported bilayer and left for 2 hr. This was removed by gentle pipetting and the bilayer was rinsed gently three times in PBS buffer to remove any free floating amyloid beta.

Preparation of supported lipid bilayers

2-Dipalmitoyl-sn-glycero-3-phosphocholine (DOPC) and 1, 2-dioleoyl-sn-glycero-3phosphocholine (DPPC) (Avanti Polar Lipids) were dissolved in chloroform in a 3 to 1 molar ratio and dried under nitrogen. These lipids mixtures were then re-suspended in ultrapure water to a final concentration of 1 mg/ml and intensively vortexed at room temperature. For brain derived lipids the lipids were dried down to form a film and then resuspended in ultrapure water. Uni-lamellar vesicles were formed via ten cycles of freeze-thawing followed by extrusion ten times) through a polycarbonate membrane with pores sizes of 100 nm using a mini-extruder (Avanti Polar Lipids) kept 50 °C on a hotplate. 200 µl of solution containing the extruded vesicles was pipetted onto freshly cleaved muscovite mica and this was then placed on a thin metal disk that itself was placed on a hotplate. The hotplate was heated up to 50 °C and the sample was annealed for 15 minutes. The sample was then loaded into the AFM liquid cell ready for imaging.

Breakthrough force measurements

Indentation measurements were made using contact mode aluminium coated silicon cantilevers (PPP-CONTR, Apex Probes, UK) having nominal spring constants of between 0.02 and 0.77 N m -1 and typical tip radius of curvatures of less than 7 nm. The spring constants were calibrated using the equipartition theorem (Thermal K). Actual measured spring constant values were between 0.05 and 0.18 Nm -1 . Force volume indentation grids of 256 points (16´16) were taken at no less than three different areas across the bilayer. Indentation rate was 1µm/s which was the optimum rate for measuring breakthrough events. Force distance curves were analyzed using a Scanning Probe Image Processor (Image Metrology, Lyngby, Denmark) and in house developed batch analysis code based on the methodology proposed by Li et al [START_REF] Li | Atomic force microscopy force mapping in the study of supported lipid bilayers[END_REF] .

Statistical analysis

Data are presented as means ± standard error of the mean (SEM). Analyses were performed using GraphPad Prism 6.0 software (GraphPad, San Diego, CA). Comparisons between groups were performed by the Mann-Whitney test. Statistical significance was set at P < 0.05. The heatmaps were created with the R software (www.r-project.org) with R package, Marray. Ward's algorithm, modified by Murtagh and Legendre, was used as the clustering method. Figure 2. Differential proteins in mouse compared to naked mole-rats. (A) Differential levels of acid ceramidase in cerebellar tissue. Ceramide synthase 2 is decreased in naked mole-rat tissues in both the cerebellum (B) and hippocampus (C). Sphingosine phosphodiesterase 3, a member of the sphingomyelinase family is decreased in naked mole-rat hippocampus (D) where sphingosine-1phosphate receptor 1 is decreased in the cerebellum (E) and the cerebral cortex. ND indicates not detected or quantifiable. The homology between the two species, relative to mouse, was 81% (A), 90% (B), 91.3% (C) and 95% (D).

Figure 1 .

 1 Figure 1. Mass Spectrometry of brain derived total lipid extract. (A-D) Quantification of cholesterol (A), sphingomyelin (B), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) (C), and ceramides (D) in lipid extract from mouse (white bar) or naked mole-rat (black bar) brains. Data are expressed as mean ± SEM. Statistical

Figure 3 .

 3 Figure 3. Mass Spectrometry of brain derived total fatty acids. Lipids were taken from the brains of 7 naked mole-rats and 6 mice (2 independent experiments). Quantification of (A) saturated fatty acids, (B) monounsaturated fatty acids, (C) polyunsaturated fatty acids (PUFA), and (D) n-6 PUFA/n-3 PUFA ratio extract from mouse (white bar) or naked mole-rat (black bar) brains. Data are expressed as mean ± SEM. Statistical analysis was performed using Mann-Whitney test. **p<0.01, significantly different from mouse group. (E) Heat map of the different fatty acids (FA) quantified by LC-MS/MS. Data are shown in a matrix format: each row represents a single FA, and each column represents the lipid extract of the brain of one animal (6 mice and 7 naked mole-rat). Each colour patch represents the normalized quantity of FA (row) in a single animal brain (column), with a continuum of quantity from bright green (lowest) to bright red (highest). The pattern and length of the branches in the left dendrogram reflect the relatedness of the FA. The dashed red line is the dendrogram distance used to cluster the FA. The pattern and length of the branches in the top dendrogram reflect the relatedness of the different animals.

Figure 4 .

 4 Figure 4. AFM contact mode images in PBS of DOPC/DPPC model supported lipid bilayers and their interactions with human amyloid beta peptide. (A) Supported bilayer made from DOPC to DPPC in a molar ratio of 3:1. Typical phase separation is observed with the DPPC gel phase islands surrounded by the liquid disordered DOPC phase. (B) Proposed origin of height difference of the gel phase relative to liquid disordered phase. (C) DOPC/DPPC bilayer after exposure for 2 hours to 8 µM of human amyloid beta. The amyloid beta has adsorbed exclusively onto the DOPC liquid disordered phase, the DPPC gel phase remaining untouched. (D) A zoom in of the area enclosed in the circle in (C)., the amyloid beta being seen to avoid the DPPC gel domains, even the small central gel domain remaining

Figure 5 .

 5 Figure 5. AFM tapping mode images of brain-derived supported lipid bilayers. (A) Supported bilayer made from naked mole-rat brain-derived lipids in PBS. More than 80% of the membrane is made up of phase separated islands of ~1.5 nm in height relative to the surrounding liquid disordered phase. (B,C) Supported bilayer made of naked mole-rat brain lipids showing a two-tier raft structure, the horizontal blue line corresponding to the height profile in C, where the two-tier raft structure is evident from the peaks having a shoulder, the first at ~0.75 nm, the second at ~1.25 nm, indicated by arrows in the figure. (D) Supported lipid bilayer made from mouse brain derived lipids. In contrast to the naked mole-rat bilayers there is little evidence of phase separation.

Figure 6 .

 6 Figure 6. AFM results from mouse samples (n=1220) and naked mole-rat samples (n=2859) lipid bilayer indentations. (A,B) Example indentation on a naked mole-rat bilayer (A), where multiple breakthrough events were detected only the event closest to the mica (B) was recorded. The points indicated by the dashed and solid arrow in panel B show the start and end of the breakthrough event respectively. Thickness was recorded as the difference in separation between the points and breakthrough force was recorded at the start of the breakthrough indicated by the dashed arrow. (C) Thickness values of 3.1 ± 0.4 nm and 2.8 ± 0.6 nm were recorded for the naked mole-rat and mouse lipid bilayers respectively. (D) Breakthrough force values of 8.1 ± 0.4 nN and 1.2 ± 0.2 nN were recorded for the naked mole-rat and mouse lipid bilayers respectively. Data are expressed as mean ±

Figure 7 .

 7 Figure 7. AFM tapping mode images of mouse brain tissue derived supported lipid bilayers in PBS exposed to 8 µM of human amyloid beta for 2 hours. (A) Large scale image showing a sparse amount of phase separation prior to amyloid beta adsorption. (B) Mouse brain derived lipid bilayer after exposure for 2 hours to 8 µM of human amyloid beta. Pits appear in the membrane, with some structure visible within the pits. (C) A close up of the pits showing that they do not break through the entire bilayer, i.e. they are not holes. (D) Line profile corresponding to the horizontal blue line in (C).The pits are between 2 and 2.5 nm deep, too shallow to be holes in the bilayer, which is ~3.1 nm thick.

Figure 8 .

 8 Figure 8. AFM tapping mode images of naked mole-rat brain derived lipid bilayers exposed to 8 µM of human amyloid beta for 2 hours. (A) The naked mole-rat brain derived bilayer before amyloid beta adsorption in PBS exhibiting the high degree of phase separation characteristic of naked mole-rat bilayers. (B,C) Naked mole-rat brain derived bilayer exposed to 8 µM of human amyloid beta for 3 hours. (D) A height profile corresponding to the blue horizontal line in (C), confirming the height of fragments to be of bilayer thickness of 4~nm.

Acknoweldgements

The authors would like to thank the University of Illinois at Chicago, Department of Chemistry and College of Liberal Arts and Science for support of this work. Funding was also received from: the National Science Foundation (Award #1655494 to T.J.P. and S.M.C.) and the UIC DFI Fellowship to M.R.P.; Cancer Resarch UK/RCUK (Award C56829/A22053 to D.F., K.R. and E.St.J.S.); BBSRC-DTP studentship (A.A.S.), Centre for Misfolding Diseases (A.A.S, S.P, M.V., J.R.K.); Agence Nationale de la Recherche (ANR-18-CE14-0039-01 to N.C). We thank Thu T.A. Nguyen for technical assistance.