MD, PhD Thomas Desmidt
email: t.desmidt@chu-tours.fr

MD, PhD Julien Delrieu

MD, PhD Thibaud Lebouvier

MD, PhD Gabriel Robert

MD, PhD Renaud David

MD Anna-Chloé Balageas

PhD Alexandre Surget

PhD Catherine Belzung

MD, PhD Nicolas Arlicot

MD, PhD Maria-Joao Ribeiro

MD, PhD Pierre Payoux

MD, PhD Bruno Vellas

MD, PhD Wissam El-Hage

PhD Elsa Tavernier

MD, PhD Vincent Camus

Benzodiazepine use and brain amyloid load in nondemented older individuals a florbetapir PET study in the Multidomain Alzheimer Preventive Trial cohort

Keywords: Benzodiazepine, GABA-A, Amyloid, Florbetapir, Positron Emission Tomography, Alzheimer's disease

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

INTRODUCTION

Benzodiazepines (BZDs) are widely used among older adults. Estimates indicate that more than 25% of older adults chronically use BZDs or hypnotic gamma-aminobutyric acid (GABA)ergic non-BZDs in various countries, including France [START_REF] Breining | Exposure to psychotropics in the French older population living with dementia: a nationwide population-based study[END_REF]. BZDs are relatively safe and efficacious for treating symptoms such as anxiety and insomnia if the guidelines for BZD prescription are observed, which include a short duration of use, usually less than 1 month for hypnotics and 3 months for anxiolytics. In contrast, chronic use of BZDs has been associated with multiple side effects, including sedation, fall risk, and functional and cognitive impairment [START_REF] Andrieu | Benzodiazepine Misuse in the Elderly: Risk Factors, Consequences, and Management[END_REF]. Moreover, concerns have recently been raised regarding the increased risk of major neurocognitive disorders (MNCDs), especially Alzheimer's disease (AD), in chronic BZD users. Indeed, several epidemiological studies have found an increased prevalence of MNCDs in older adults chronically using BZDs [START_REF] Chan | Association between high cumulative dose of benzodiazepine in Chinese patients and risk of dementia: a preliminary retrospective casecontrol study[END_REF] [START_REF] Zhong | Association between Benzodiazepine Use and Dementia: A Meta-Analysis[END_REF]. A recent meta-analysis of 8 studies found an increased risk of having diagnostic criteria for dementia in BZD users compared to BZD nonusers, with a pooled summary odds ratio of 1.78 (95% CI 1.33-2.38) [START_REF] Islam | Benzodiazepine Use and Risk of Dementia in the Elderly Population: A Systematic Review and Meta-Analysis[END_REF].

The likelihood of having MNCD diagnostic criteria may be particularly high with long-acting BZDs, long-term use and high dosage (Billioti de [START_REF] Billioti De Gage | Is there really a link between benzodiazepine use and the risk of dementia?[END_REF]. However, not all epidemiological studies have found an increased risk of MNCDs with BZDs, especially some large longitudinal studies [START_REF] Gray | Benzodiazepine use and risk of incident dementia or cognitive decline: prospective population based study[END_REF] that found no association between BZD use and MNCDs, and it remains unclear whether BZDs constitute a risk factor for MNCDs.

The identification of pathophysiological mechanisms linking BZDs to MNCDs could provide further information on the putative risk of MNCDs in BZD users. However, few studies have investigated the impact of BZDs on brain changes related to MNCDs, and only one small pilot study [START_REF] Chung | Benzodiazepine Use Attenuates Cortical β-Amyloid and is Not Associated with Progressive Cognitive Decline in Nondemented Elderly Adults: A Pilot Study Using F18-Florbetapir Positron Emission Tomography[END_REF] has investigated the association between chronic BZD use and amyloid deposition, a hallmark of the pathophysiology of AD; the latter study examined a group of nondemented older adults from the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort.

The authors of this pilot study found, unexpectedly, that the fifteen individuals who were chronically using BZDs had a decreased amyloid load as assessed with F 18 -florbetapir positron emission tomography (PET) compared to matched BZD nonusers, which challenges the view of BZDs as a risk factor for AD. Nevertheless, this result is consistent with animal studies that have identified neuroprotective properties of BZDs. Indeed, diazepam was found to be protective against hippocampal cell death caused by amyloid in mice. Furthermore, mice treated with diazepam [START_REF] Tampellini | Effects of synaptic modulation on beta-amyloid, synaptophysin, and memory performance in Alzheimer's disease transgenic mice[END_REF] [START_REF] Quiroga | Effects of repetitive exposure to anesthetics and analgesics in the Tg2576 mouse Alzheimer's model[END_REF] or midazolam [START_REF] Yamamoto | Midazolam inhibits the formation of amyloid fibrils and GM1 ganglioside-rich microdomains in presynaptic membranes through the gamma-aminobutyric acid A receptor[END_REF] show lower amyloid deposition than control mice. Finally, there may be a contradiction between epidemiological studies, on the one hand, which found an increased risk or no change in the risk of dementia with BZDs, and pathophysiological studies, on the other hand, which suggest neuroprotective effects of BZDs. However, no large pathophysiological study is available in a clinical population, and the few available data in humans require further replication in larger samples.

In our study, we investigated the association between chronic BZD use and amyloid load as assessed by F 18 -florbetapir PET in a population of 268 nondemented older adults from the Multidomain Alzheimer Preventive Trial (MAPT) study. The primary goal was to test the efficacy of a multidomain intervention and/or omega-3 polyunsaturated fatty acid supplementation on cognitive decline. Based on results from animal studies and a pilot study, we hypothesized that individuals with BZDs would exhibit lower amyloid loads than matched BZD nonusers would. In addition, we investigated whether certain characteristics of BZD use, including dose, duration of use and short-versus long-acting BZDs, specifically influenced amyloid load.

Participants

The participants were part of the MAPT study (registration: NCT00672685), which was a large 3-year, multicenter, randomized, placebo-controlled superiority trial with four parallel groups.

The methods and primary results of the MAPT study have been extensively described elsewhere [START_REF] Andrieu | Effect of long-term omega 3 polyunsaturated fatty acid supplementation with or without multidomain intervention on cognitive function in elderly adults with memory complaints (MAPT): a randomised, placebo-controlled trial[END_REF]. Briefly, 1680 nondemented older adults with a memory complaint, limitation in at least one instrumental activity of daily living or slow gait speed were randomly assigned (1:1:1:1) to the multidomain intervention plus omega-3 polyunsaturated fatty acids, the multidomain intervention plus placebo, omega-3 polyunsaturated fatty acids alone, or placebo alone. The primary goal was to assess the efficacy of the interventions in slowing cognitive decline in older adults at risk of MNCDs. A number of participants also underwent florbetapir PET scanning, which was performed as an ancillary study for amyloid assessment. Among the 1680 participants in the MAPT study, n=1539 were assessed for eligibility at one of the centers with PET imaging facilities during the clinical follow-up (Figure 1). A total of 1268 participants were excluded from the MAPT F 18 -florbetapir ancillary study for the following reasons, as shown in Figure 1: 705 participants were out of the MAPT study at the time of the implementation of the MAPT F 18 -florbetapir ancillary study (end of follow-up or early discontinuation); 414 participants refused to participate; 81 participants did not attend the PET visit (due to technical problems or personal reasons); and 68 participants were included in another MAPT substudy for PET-FGD assessment. Ultimately, a total of 271 participants participated in the MAPT F 18 -florbetapir ancillary study.

At baseline, the participants were community-dwelling men and women without dementia, aged ≥70, who met at least one of the following criteria: spontaneous memory complaints, limitation in executing ≥1 instrumental activity of daily living or slow gait speed (<0.8 meters/sec).

Participants with a Mini-Mental State Examination (MMSE) score lower than 24, those with a diagnosis of dementia, and those with any difficulty in basic activities of daily living were excluded, as were those taking polyunsaturated fatty acid supplements at baseline. In the present analysis, 2 participants were excluded because of missing data regarding BZD use duration, and 1 participant was excluded because of a Clinical Dementia Rating (CDR) score of 1 at the time of the PET scan. Thus, a total of 268 participants were included in the analyses (Figure 1). Both the MAPT and the F 18 -florbetapir PET study were approved by the French Ethical Committee in Toulouse (CPP SOOM II), and written consent was obtained from all participants.

Benzodiazepine use and clinical assessments

Participants were identified as chronic BZD users if they had used any type of BZD for at least 1 year at any time before the PET scan. The duration of BZD use was calculated as follows: end date of use (or date of PET scan, if still on medication at PET scan date) minus starting date of use. The BZD dose was standardized by converting the various BZD doses to diazepam dose equivalents. We also distinguished between short-(half-life ≤ 20 hours) and long-acting (half-life > 20 hours) BZDs. We defined this BZD classification according to the official definition recommended by the French National Agency for Drug Safety1 because previous epidemiological studies used this classification and found a significant effect of long-acting BZDs but not shortacting BZDs on AD incidence [START_REF] Shash | Benzodiazepine, psychotropic medication, and dementia: A population-based cohort study[END_REF].

Clinical assessment included age, sex, educational level, cognitive and dementia status assessed with the MMSE and the CDR, MAPT group allocation (four groups: placebo, multidomain intervention, n-3 PUFA supplementation and multidomain intervention + n-3 PUFA supplementation), depressive symptoms assessed with the 15-item Geriatric Depression Scale (GDS), history of major depressive episodes, history of antidepressant intake, and apolipoprotein E ε4 (ApoE ε4) genotype (carriers of at least one ε4 allele versus noncarriers).

F 18 -florbetapir PET analysis PET scans were realized as close as possible to a clinical visit during the 3 years of follow up of each patient, as previously described [START_REF] Vellas | MAPT STUDY: A MULTIDOMAIN APPROACH FOR PREVENTING ALZHEIMER'S DISEASE: DESIGN AND BASELINE DATA[END_REF][START_REF] Del Campo | Relationship of regional brain β-amyloid to gait speed[END_REF].

Participants were examined using 5 different hybrid PET-CT scanners, including one PET CT 690 (GE Healthcare), one Discovery RX VCT (General Electric), 2 True Point HiRez (Siemens Medical Solutions) and one Biograph 4 Emission Duo LSO (Siemens Medical Solutions). All tomographs operated in 3D detection mode. All PET sinograms were reconstructed with an iterative algorithm, with corrections for randomness, scatter, photon attenuation and decay, which produced images with an isotropic voxel of 2x2x2 mm 3 and a spatial resolution of approximately 5-mm full width at a half maximum at the field of view center. The acquisition data were processed using the standard package delivered with each acquisition system. All cerebral emission scans began 50 min after a mean injection of 4 MBq/kg weight of F 18 -florbetapir. For each subject, 10 or 15 minute frames were acquired to ensure movement-free image acquisition.

A semi-automated quantitative analysis (cortical to cerebellar regional mean standardized uptake values, SUVR) was applied using the mean signal of six predefined anatomically relevant cortical regions of interest (frontal, temporal, parietal, precuneus, anterior cingulate and posterior cingulate) with the whole cerebellum used as the reference region as previously described (Clark et al., 2011;[START_REF] Fleisher | Using Positron Emission Tomography and Florbetapir F 18 to Image Cortical Amyloid in Patients With Mild Cognitive Impairment or Dementia Due to Alzheimer Disease[END_REF]. In this procedure, the F18-florbetapir PET images were coregistered to the F18-florbetapir template provided by AVID company. Quality control based on the semi-quantification process was also provided by AVID Lab.

In addition to the SUVR calculation, the PET scans were visually analyzed in the MAPT cohort and classified as either amyloid positive or negative. The methods applied for the visual analyses have been described in detail elsewhere [START_REF] Payoux | Cognitive and functional patterns of nondemented subjects with equivocal visual amyloid PET findings[END_REF], but briefly, the F18florbetapir PET images were visually assessed by a panel of 3 independent observers, who were specialists in molecular imaging and blinded to all clinical and diagnostic information; the observers used a binary scale to classify each scan as 'negative' if there was no significant F18-florbetapir cortical retention or 'positive' if there was some significant F18-florbetapir cortical retention; the final consensus allowed each PET scan to be classified as either positive or negative.

The mean (standard deviation) SUVR value associated with a positive PET scan was 1.35 (0.17), indicating that SUVR values associated with positive PET scans were generally higher than the threshold found in the literature (1.17) [START_REF] Clark | Use of Florbetapir-PET for Imaging beta-Amyloid Pathology[END_REF][START_REF] Fleisher | Using Positron Emission Tomography and Florbetapir F 18 to Image Cortical Amyloid in Patients With Mild Cognitive Impairment or Dementia Due to Alzheimer Disease[END_REF], whereas the mean (standard deviation) SUVR in the negative group was 1.08 (0.1). In our report, we also compared the proportions of positive and negative PET scans between the BZD users and nonusers.

Statistical analysis

Because the MAPT F 18 -florbetapir ancillary study was implemented secondarily in the MAPT cohort, the PET scans may have been performed at different time points during the participants' follow-up. Analyses were therefore performed using clinical scores from the follow-up visit closest to the PET scan. Clinical variables were described and compared according to the use of BZDs. Quantitative variables were described as the means and standard deviations and compared used Student's t-test. Qualitative variables were described as counts and percentages and compared using the chi-squared test.

We used multiple linear regression to estimate the effect of BZD use (the independent variable) on SUVR (the dependent variable). The model included adjustments for age, sex, educational level, MMSE, GDS, history of major depressive episodes, history of antidepressant intake, ApoE ε4 genotype, group allocation in the trial and duration from baseline to PET scan to account for the potential confounding effects of these factors. In case of missing data on the adjustment covariates, we performed multivariate imputation by chained equations (van Buuren and Groothuis-Oudshoorn, 2011), with 5 imputations. The same analysis was used for each cortical region: frontal, temporal, parietal, anterior cingulate, and posterior cingulate. The use of BZDs was first considered as a binary variable (BZD users versus BZD nonusers), then as a 3-category variable (short versus long-acting BZDs versus BZD nonuse). BZD nonusers were used to define the reference level of the variables. In the subgroup of BZD users, we used univariate linear regressions to estimate the link between SUVR and BZD dosage and then between SUVR and BZD duration. Analyses were performed using R 3.2.3 (R Development Core Team. R: A language and environment for statistical computing).

RESULTS

Sample characteristics

The clinical and demographic characteristics of the study participants are shown in Table 1.

Forty-seven participants out of 268 were identified as chronic BZD users, and the BZDs included in our analyses were as follows: bromazepam (n=12), clonazepam (n=6) and prazepam (n=1) as longacting GABAergic BZD derivatives; alprazolam (n=3), lorazepam (n=3), lormetazepam (n=4) and oxazepam (n=1) as short-acting GABAergic BZD derivatives; zolpidem (n=9) and zopiclone (n=8) as short-acting GABAergic non-BZD derivatives.

There was no difference between chronic BZD users and BZD nonusers in age, sex, educational level, MMSE scores, CDR score, ApoE ε4 status and group allocation. The GDS score, history of major depressive episode (MDE) and antidepressant intake were increased in chronic BZD users.

Associations between benzodiazepine use and brain amyloid load

The results of multiple linear regression analyses are illustrated in Figures 2 and3. The first model (Figure 2) includes amyloid SUVR in the total cortex and in 5 brain regions as the dependent variables and chronic BZD use, age, sex, educational level, MMSE, CDR, GDS, history of MDE, antidepressant intake, ApoE ε4 status, group allocation and duration from baseline to PET scan as explicative variables. In the regression analysis of total cortex SUVR, we found significant effect of chronic BZD use (beta = -0.06, p=0.023), ApoE ε4 status (beta = 0.11, p<0.001) and multidomain intervention (beta = -0.09, p<0.001). SUVRs were significantly lower in BZD users than in nonusers in the total cortex and all the studied brain regions, except the frontal lobe.

In the second model (Figure 3), where we distinguished short-and long-acting BZDs, we found significant effects of short-acting BZDs on amyloid load (beta = -0.10, p=0.005 for the total cortex SUVR), whereas we found no significant effect of long-acting BZDs (beta = -0.02, p=0.6 for total cortex SUVR). SUVRs were significantly lower in short-acting BZD users compared to for the parietal cortex and the anterior cingulate.

In addition, we found that the proportion of negative PET scans in the BZD users was significantly greater than that in the nonusers (n=37, 79% versus n=140, 64%, respectively, χ 2 1=4.085, p=0.043), and notably, 23 (85%) negative PET scans were found in the short-acting BZD group.

Effect of dose and duration of benzodiazepine use on brain amyloid load

We found no significant associations between SUVR in the total cortex and dose (beta = 0, p = 0.896) or duration of BZD use (beta = 0, p = 0.625) in the subgroup of BZD users.

DISCUSSION

We found that nondemented older adults who chronically use BZDs had a reduced brain amyloid load compared to BZD nonusers, independent of potential confounding factors such as cognitive impairment, history of depression and antidepressant intake. In addition, we found that short-acting BZDs showed the strongest effect, whereas we found no effect of dose or duration of BZD use on brain amyloid load. Our results are consistent with a previously published pilot study [START_REF] Chung | Benzodiazepine Use Attenuates Cortical β-Amyloid and is Not Associated with Progressive Cognitive Decline in Nondemented Elderly Adults: A Pilot Study Using F18-Florbetapir Positron Emission Tomography[END_REF] that found reduced brain amyloid in chronic BZD users from the ADNI cohort.

In addition, our results are consistent with animal studies showing that administration of BZDs reduces brain amyloid deposition.

BZDs act as positive allosteric modulators of GABA-A receptors and depresses neuronal activity via the increase in intracellular chlorine ions through chloride channels, hyperpolarizing the cell and decreasing its probability of firing. Neurodegeneration and reduction in neuronal and synaptic activity are key features of the later stages of AD. In contrast, recent findings tend to suggest that neuronal hyperactivity is one of the earliest dysfunctions in the pathophysiological cascade of AD, with neuronal hyperactivity preceding the formation of amyloid plaque in animal studies [START_REF] Busche | Impairments of neural circuit function in Alzheimer's disease[END_REF]. Indeed, a positive longitudinal correlation has been found between neuronal activity and amyloid accumulation, which suggests that regulation of neuronal activity may modulate amyloid production [START_REF] Bero | Neuronal activity regulates the regional vulnerability to amyloid-β deposition[END_REF] [START_REF] Cirrito | Endocytosis is required for synaptic activity-dependent release of amyloidbeta in vivo[END_REF]. Interestingly, while neuronal hyperactivity is associated with increased amyloid accumulation, a reduction in neuronal activity results in decreased amyloid production, as well as reduced axonal dystrophy and synaptic loss in areas near amyloid plaques [START_REF] Yuan | Attenuation of β-Amyloid Deposition and Neurotoxicity by Chemogenetic Modulation of Neural Activity[END_REF]. In agreement with this concept, [START_REF] Cirrito | Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo[END_REF] found that a greater neuronal firing rate increased extracellular amyloid deposition in mice, whereas inhibition of neuronal activity decreased the amyloid load. Consistent results have been found in vitro using hippocampal slices, in which increased neuronal activity has been found to promote amyloid deposition by modulating betasecretase processing of amyloid precursor protein (APP) [START_REF] Tampellini | Synaptic activity reduces intraneuronal Abeta, promotes APP transport to synapses, and protects against Abeta-related synaptic alterations[END_REF]. Preclinical findings from sleep induction [START_REF] Boespflug | The Emerging Relationship Between Interstitial Fluid-Cerebrospinal Fluid Exchange, Amyloid-β, and Sleep[END_REF] and from epilepsy [START_REF] Sen | Cognition and dementia in older patients with epilepsy[END_REF]) also suggest a similar relationship between neuronal activity and amyloid production. Some human studies tend to be consistent with this relationship, especially a recent study that found that cognitively normal older adults with the greatest activation on fMRI during a memory task showed the highest accumulation of amyloid plaque two to six years later [START_REF] Leal | Hippocampal activation is associated with longitudinal amyloid accumulation and cognitive decline[END_REF]. These results suggest that individuals who require additional neuronal activation to perform in the memory task may be at an increased risk of amyloid accumulation, whereas those with low neuronal activation in the memory task, although performing well, may have a lower risk of amyloid production. Based on these accumulating findings, some authors have proposed that targeting neuronal hyperactivity with pharmacological treatment such as antiepileptic drugs may attenuate amyloid progression and ultimately prevent the development of AD [START_REF] Haberman | Targeting Neural Hyperactivity as a Treatment to Stem Progression of Late-Onset Alzheimer's Disease[END_REF]. Consistently, the reduced amyloid load observed in the BZD users in our study may be related to the GABAergic effect of BZDs by which BZDs decrease neuronal excitability.

The other possible explanations of our findings are related to the effect of BZDs on translocator protein 18 kDa (TSPO), which was formerly known as peripheral BZD receptor, and mechanisms related to neuroinflammation as a potential risk factor for cognitive impairment. TSPO is an outer mitochondrial membrane protein involved in steroid metabolism as well as other mitochondrial functions, including cell proliferation and differentiation, mitochondrial respiration and regulation of cytochrome C release, caspase activation and apoptosis [START_REF] Veenman | Channel-like functions of the 18-kDa translocator protein (TSPO): regulation of apoptosis and steroidogenesis as part of the host-defense response[END_REF].

Ligands of TSPO elicit pleiotropic neuroprotective effects including reduction of amyloid accumulation (see [START_REF] Arbo | Therapeutic actions of translocator protein (18 kDa) ligands in experimental models of psychiatric disorders and neurodegenerative diseases[END_REF] for review). Specifically, the effect of BZDs on amyloid as ligands of TSPO may be mediated by modulation of neuroinflammation via the synthesis of neurosteroids, which has been found to modulate amyloid pathology [START_REF] Minter | The contribution of neuroinflammation to amyloid toxicity in Alzheimer's disease[END_REF].

We found that users of short-acting BZDs but not users of long-acting BZDs had lower amyloid loads than BZD nonusers. Epidemiological studies tend to find that long-acting BZDs but not short-acting BZDs are associated with an elevated risk of MNCDs(Billioti de [START_REF] Billioti De Gage | Is there really a link between benzodiazepine use and the risk of dementia?[END_REF], although no clear explanation has been found yet. Pharmacological differences between short-(especially Z-drugs) and long-acting BZDs include differences in alpha 1 receptor affinity, with short-acting BZDs showing higher alpha 1 affinity, which results in a stronger soporific effect [START_REF] Nutt | Searching for perfect sleep: the continuing evolution of GABAA receptor modulators as hypnotics[END_REF]. Interestingly, recent evidence suggests that poor sleep quality promotes amyloid pathology, and conversely, individuals with good sleep quality may be at reduced risk of brain amyloid accumulation [START_REF] Boespflug | The Emerging Relationship Between Interstitial Fluid-Cerebrospinal Fluid Exchange, Amyloid-β, and Sleep[END_REF]. Therefore, in our study, the reduced amyloid load in short-acting BZD users may be explained by superior sleep quality in these individuals.

However, only a few participants in the MAPT cohort were assessed for sleep quality, and we were unable to reliably test this hypothesis.

The most robust region-specific associations were observed in our study for the anterior cingulate and the parietal cortex, similar to findings from the previously published pilot study [START_REF] Chung | Benzodiazepine Use Attenuates Cortical β-Amyloid and is Not Associated with Progressive Cognitive Decline in Nondemented Elderly Adults: A Pilot Study Using F18-Florbetapir Positron Emission Tomography[END_REF]. However, because we found that the total amyloid load was smaller in BZD users, it is likely that BZDs have a global effect on brain amyloid. The most significant differences observed in the anterior cingulate and parietal cortex may be related to recent findings showing that early accumulation of amyloid occurs primarily in these regions [START_REF] Grothe | Alzheimer's Disease Neuroimaging Initiative[END_REF]. Indeed, the nondemented individuals in our study are likely to show early progression of amyloid pathology, and differences between BZD users and nonusers may be at a maximum in these regions of early amyloid deposition, whereas amyloid deposition may be less significant in other brain regions with smaller differences between BZD users and nonusers.

We found no association between the amyloid load and dosage or duration of BZD use, which is consistent with a previously published pilot study [START_REF] Chung | Benzodiazepine Use Attenuates Cortical β-Amyloid and is Not Associated with Progressive Cognitive Decline in Nondemented Elderly Adults: A Pilot Study Using F18-Florbetapir Positron Emission Tomography[END_REF]. A possible explanation of this result is related to the potential ceiling effect of BZDs on amyloid, suggesting that after a certain dose or time of use, no additional benefit in lowering the amyloid load is achieved with BZDs. Another complementary explanation could be that the maximum lowering effect of BZDs occurs with a relatively short usage and that longer usage has no further benefit on the amyloid load. This hypothesis is consistent with preclinical studies showing that the in vitro effects of BZDs on amyloid formation are rapid and occur within hours of exposure [START_REF] Yamamoto | Midazolam inhibits the formation of amyloid fibrils and GM1 ganglioside-rich microdomains in presynaptic membranes through the gamma-aminobutyric acid A receptor[END_REF]. In our study, we have limited the scope of the investigation to chronic BZD use lasting longer than one year, and it remains unclear whether a shorter duration of use could also have an impact on amyloid load, which may be investigated in further studies.

We found no significant difference in the MMSE score between the BZD users and nonusers despite the lower amyloid load in the BZD users. A greater amyloid load has been associated with lower cognitive performance, and individuals with a lower amyloid load are expected to exhibit greater cognitive performance. However, previous studies have suggested that the MMSE may be inappropriately sensitive in reflecting cognitive deficits related to amyloid deposition, especially in non-demented individuals with high MMSE scores and low inter-individual variations, such as those included in our study [START_REF] Lim | Sensitivity of composite scores to amyloid burden in preclinical Alzheimer's disease: Introducing the Z-scores of Attention, Verbal fluency, and Episodic memory for Nondemented older adults composite score[END_REF]. In addition, we may hypothesize that the greater cognitive performance observed in the BZD user group due to the lower amyloid load could have been counteracted by the well-known negative effect of BZDs on cognitive performance, resulting in a similar cognitive impairment to that observed in the BZD nonusers with a higher amyloid load.

Limitations of our study include that the MAPT study was not primarily designed to assess the effect of BZD use on the brain amyloid load, and our results were derived from a secondary analysis, which may limit the strength of our conclusions. Moreover, the use of a cross-sectional design to examine the association between BZD use and amyloid load prevents inference of causality; the possible causal relationship between reduced amyloid load and BZDs remains to be confirmed. In addition, no measure of anxiety was available in the MAPT study, and only a few participants had sleep quality assessments. These potential confounding factors may have influenced our results, since anxiety and sleep quality have been previously associated with changes in amyloid load. Participants also had PET scans at different time points during follow-up, which may constitute a limitation. However, controlling for duration from baseline to PET scans in our regression models did not influence the associations between BZD use and amyloid load. Regarding the distinction between short-and long-acting BZDs in our analyses, we had relatively small sample sizes; thus, the predominant effect of short-acting BZDs on brain amyloid should be considered with caution. Finally, the effect of BZDs on the amyloid load was relatively small in our study, and we could not exclude the possibility that the observed differences were derived from a subgroup of individuals among the BZD nonusers with a total SUVR greater than 1.5. Nevertheless, most BZD users had SUVRs located within the SUVR values of the visually rated negative PET scans, and compared to the nonusers, we found a greater proportion of negative PET scans among the BZD users, suggesting a potentially clinically relevant effect of BZDs on brain amyloid.

In conclusion, our results do not support the view of BZDs as risk factors for AD and instead suggest that pharmacological mechanisms related to reduction in neuronal activity, neuroinflammation and/or sleep quality may be involved in the reduction of amyloid pathology. In fact, and although epidemiological studies found conflicting results, a recent well-controlled study found that, after controlling for multiple confounding factors including psychiatric disorders, BZD use was associated with a reduced incidence of AD [START_REF] Imfeld | Benzodiazepine Use and Risk of Developing Alzheimer's Disease or Vascular Dementia: A Case-Control Analysis[END_REF]. However, we do not intend to suggest that BZDs should be used to prevent AD specifically because the chronic use of BZDs has several side effects that certainly overcome the potential benefits on amyloid pathology.

Guidelines for BZD prescription should be carefully observed, including the duration of use, which may not exceed 1 month as a hypnotic and 3 months as an anxiolytic. Moreover, reducing amyloid pathology does not necessarily lead to a decreased incidence of AD, which involves multiple other pathophysiological mechanisms, such as tau pathology and vascular disorders. Interestingly, a recent animal study found that lowering neuronal activity may indeed prevent amyloid formation but simultaneously promotes tau pathology and causes detrimental effects on synapses in AD transgenic mice [START_REF] Akwa | Synaptic activity protects against AD and FTD-like pathology via autophagic-lysosomal degradation[END_REF]. Nevertheless, our paper suggests that further investigations of GABA-and/or TSPO-related mechanisms involved in neuronal excitability, neuroinflammation and sleep quality may allow the identification of novel pathophysiological pathways in AD and provide pharmacological targets to reduce amyloid formation. The amyloid load was significantly lower in short-acting BZD users compared to BZD nonusers in the total cortex and all the studied regions after controlling for age, sex, educational level, MMSE, CDR, GDS, history of MDE, antidepressant intake, ApoE ε4 status, group allocation and duration

FIGURE LEGEND Figure 1 -

 LEGEND1 FIGURE LEGENDFigure1-Flowchart of the MAPT F 18 -florbetapir substudy.

Figure 2 -

 2 Figure 2 -Multiple linear regressions of the associations between benzodiazepine use

Figure 3 -

 3 Figure 3 -Multiple linear regressions of the associations between benzodiazepine use, divided

Figure 4 -

 4 Figure 4 -Scatterplots of the individual total SUVR in BZD users and nonusers (left graph)

Table 1 -

 1 Demographic and clinical characteristics of the population

	BZDs users	BZDs nonusers	p
	(n=47)	(n=221)	value

Depression Scale; MDE: major depressive episode; n-3 PUFA: omega-3 polyunsaturated fatty acid

https://www.ameli.fr/fileadmin/user_upload/documents/FEGENOR_PIS_RI_Avis1_CT13342.pdf

CONTRIBUTORS:

BV designed the MAPT study. PP supervised the MAPT Amyloid ancillary study. BV, PP and JD gathered data, which were analyzed and interpreted by ET and TD. TD wrote the Article, which was critically reviewed by all authors.

ACKNOWLEDGEMENTS:

The MAPT study was supported by grants from the Ge ŕontopôle of Toulouse, the French Ministry of Health (PHRC 2008(PHRC , 2009))


MD Didier Samuel
email: didier.samuel@aphp.fr

Use of HCV organs in HCV-negative recipients: yes but with good tools

Since the discovery of the hepatitis C virus (HCV) in 1989, the HCV epidemic has become a major health concern with 1-3% of the global population infected. HCV is characterised by transmission mainly through transfusion of infected blood, parenteral exposures (infected needles, syringes, piercings, tatoos, etc) and a low sexual transmission risk. HCV infection evolves in 80% of cases to chronic hepatitis with a risk of liver cirrhosis in 30-40% of the patients at 20-30 years after infection and with a 2-4% annual incidence of Hepatocellular carcinoma in patients who develop cirrhosis. Among patients who have received an organ transplant, the progression to liver cirrhosis is strongly accelerated. Therefore, until recently it appeared important to avoid HCV infection after organ transplantation by not transfusing HCV-infected blood and not transplanting HCV-infected organs. In some countries such as USA, the percentage of organ donors infected with HCV was 5.58% (18% in high risk donors)

(1) and the potential use of these organs may increase the pool of donors. Transplantation of HCV-positive organs to anti-HCV-positive recipients with HCV viremia was possible after approval by health authorities and, signed informed consent by the recipient. When using HCV viremic donors, the risk of HCV transmission was constant with a viral population coming both from the donor and the recipient. Before the advent of direct antiviral agents (DAA), IFN-based therapy failed to control HCV infection in most organ recipients and was associated to a risk of organ rejection.

The advent of effective IFN-free DAA anti HCV treatments has dramatically changed the face of the problem (2,3). First, these DAA agents do not seem to increase the risk of organ rejection [START_REF] Coilly | Optimum timing of treatment for hepatitis C infection relative to liver transplantation[END_REF][START_REF] Coilly | Pros and Cons: Usage of organs from donors infected with hepatitis C virus -Revision in the direct-acting antiviral era[END_REF]. Second, these agents are increasingly potent and the most recent generation of DAAs have pangenotypic activity and are either a combination of Sofosbuvir, an anti-NS5b agent, and velpatasvir, an anti NS5A agent (2) or a combination of glecaprevir, an anti NS3-NS4 agent, and pibrentasvir, an anti-NS5A agent [START_REF] Zeuzem | glecaprevir-Pibrentavir for 8-12 weeks in HCV genotype 1 or 3 infection[END_REF]. The potency of these combinations is very high, achieving sustained virologic response (SVR) in 97-100% of HCV infected patients. Third, it is now possible in many countries to obtain nucleic acid testing (NAT) for HCV RNA within few hours in potential organs donors and, accordingly, to match donor and recipients.

For all these reasons, some teams have advocated the transplantation of HCV-infected organs not only in HCV-positive recipients but also in HCV-negative recipients. Several teams, particularly in the USA, have used HCV-infected organs in HCV-negative heart, lung, kidney and liver recipients [START_REF] Wooley | Heart and lung transplants from HCV-infected donors to uninfected recipients[END_REF][START_REF] Trotter | Use of organs from hepatitis C viruspositive donors for uninfected recipients: a potential cost-effective approach to save lives[END_REF][START_REF] Cotter | Increasing utilization and Excellent initial outcomes following liver transplant of hepatitis C Virus (HCV)-Viremic donors into HCV-negative recipients: outcomes following liver transplant of HCV viremic donors[END_REF][START_REF] Mclean | Transplanting hepatitis C virus infected hearts into uninfected recipients: a single arm trial[END_REF]. The use of infected liver grafts raised another issue, which is the potential presence of HCV-related graft liver disease, which may impact the results of liver transplantation [START_REF] Coilly | Pros and Cons: Usage of organs from donors infected with hepatitis C virus -Revision in the direct-acting antiviral era[END_REF]. For the recipients of organs other than the liver, the question is the risk of HCV transmitted infection in the recipient and the possibility to eradicate the transmission of HCV. In the Lancet Gastroenterology & Hepatology, a group of USA-based authors descrive the outcome of preemptive pangenotypic DAA administration in HCVnegative heart recipients receiving HCV-positive hearts [START_REF] Bethea | Preemptive pangenotypic direct antiviral agent in donor HCV positive to recipient HCV-negative cardiac transplantation[END_REF]. 25 patients receive HCV-positive hearts, in 5 of them the donor was HCV RNA-negative and those recipients did not receive any treatment and no HCV transmission was observed. In contrast, the other 20 recipients who received a heart from an HCV RNA-positive donor, developed immediate HCV infection, and, according to the design of the trial, received preemptive DAA therapy using a combination of glecaprevir/pibrentasvir started in the immediate preoperative period for an 8 weeks duration. In most patients, serum HCV RNA was detected during the first posttransplant hours, confirming the high infection rate. In these 20 patients, HCV RNA became undetectable at a maximum of day 17, and SVR was obtained in all patients. The tolerance of the DAA was good, with no increase in graft rejection. To avoid drug-drug interaction (DDI), patients did not receive IPP and all were on Tacrolimus. These results are in line with a recent study in 44 uninfected transplant recipients (8 heart, 36 lung) receiving organs from HCV viremic patients, the recipients were preemptively treated with a combination of sofosbuvir-velpatasvir for 4 weeks started few hours after transplantation. 95% of the patients had detectable viral load after transplant and the first 35 patients with an adequate follow-up achieved all SVR, with a comparable rate of rejection to heart and lung transplant recipients receiving HCV-negative organs. These excellent results are the witness of the high potency of DAA and of the absence of residual virus in patients who achieved SVR.

Using HCV-infected organs in uninfected recipients may increase access to transplantation to patients on waiting lists, particularly in countries where the prevalence of HCV-infected donors is high. Health authorities should support this strategy. Several points of attention are remaining: first, transplant recipients received many drugs in addition to immunosuppressive drugs and drug-drug interaction should be prevented; Second, the cost of transplantation is significantly increased due to the use of DAA in the recipients; third, recipients of such organs should give their informed consent; Fourth, SVR seems as high if DAA are started before or immediately after transplantation with 4 or 8 weeks of preemptive therapy; fifth, HCV infection is almost constant and rapid after transplantation of infected organs and replication is involving numerous strains from the recipient and from the donor [START_REF] Zahid | High multiplicity of infection following transplantation of hepatitis C virus-positive organs[END_REF]. There is a possibility to face a resistant strain as shown recently (9), which will require modification of the DAA therapy [START_REF] Bourliere | Sofosbuvir, velpatasvir and voxilaprevir for previously treated HCV infection[END_REF] and expert virological support. The teams using such organs should not be too confident, they should have expert virologic support, identify the viral strains involved and ensure the obtention of full SVR in the recipients.