Melatonin is a hormone produced by the pineal gland and is available over the counter for treating sleep problems in the pediatric population. We conducted a systematic review of randomized clinical trials (RCTs) on MEDLINE and included six studies that met our inclusion criteria. RCTs were conducted in patients from two to 18 years of age with a diagnostic and statistical manual of mental disorders (DSM)-IV diagnosis of autism spectrum disease (ASD) and/or attention-deficit hyperactivity disorder (ADHD) in both short-term and long-term RCTs ranging from eight-week to 52-week studies. The mean difference in the children’s sleep disorder showed statistically significant improvement in sleep duration and sleep latency onset compared to the placebo. Overall, a high response rate was observed in the melatonin group compared to the placebo in treating sleep problems in children. Melatonin is a well-tolerated and safe medication in the dose range of 2-10 mg/day in the child and adolescent population. Sleep problems are increasingly becoming prevalent in children, as recent data found that about 50% of children from four to 12 years old have different sleep issues .A higher proportion of patients with ASD have difficulty in sleep onset 53%), restless sleep 40%), nighttime awakening (34%), and difficulty in arousal from sleep (32%) [0%, night3%, restlComorbid sleep problems have an impact on the daily functionality of children by an increase in externalizing and internalizing behaviors and an increase in autistic-like behaviors. Also, an increase in emotional and hyperactive symptoms can lead to impairment in academic and social functioning and maintaining relationships ,9-10. AsBased on past studies, non-pharmacologic treatment for sleep problems in children with psychiatric disorders includes behavioral therapy, like graduated extinction, bedtime routine, scheduled awakening, positive routine, and parent education -13. AmonIn our systematic review, we will do a qualitative synthesis of past randomized controlled trials (RCTs) conducted on melatonin in the pediatric population and to evaluate the overall efficacy and safety of melatonin for managing insomnia in ASD and neurodevelopmental disorder.Study search strategy and selectionThe MEDLINE database was used to identify clinical trials, clinical studies, multicenter studies, or observational studies published in English from April 19, 2010 to April 15, 2020 with MeSH terms "child" or "adolescent". The search strings were "melatonin" and "autism" or "autistic spectrum" or "developmental disorders" that yielded 79 studies. The screening was done independently by two authors  using the Preferred Reporting Items for Systematic Reviews and Meta-analyses Statement (PRISMA) guidelines. The titles and abstracts were screened, based on the purpose of our study objective, and finally included seven studies. After reading the full text, a total of six studies met the criteria for our systematic review and were included as shown in Figure Mechanism of actionMelatonin or 5-methoxy N-acetyltryptamine is synthesized by the pineal gland from amino acid tryptophan by the first hydroxylation and then decarboxylation to form serotonin . MelatonMelatonin acts via MT1 G-protein coupled receptor that is pertussis toxin (PTX)-sensitive (Gi) and PTX insensitive (Gq). Activation of PTX-sensitive receptors inhibits cAMP, protein kinase A (PKA), and cAMP-responsive element-binding formation (CREB) . SCN melEfficacyFive studies conducted in an outpatient setting evaluated the efficacy of melatonin on sleep patterns in ASD -25. DoubCortesi et al. included 66 children  with ASD in their DB-RCT and found that patients on both cognitive-behavioral therapy (CBT) and melatonin improved significantly compared to those in the melatonin-only group in terms of reduction in sleep onset latency and wakefulness after sleep onset, and increase in total sleep duration . SchrodeDB-RCT conducted by Mohammadi et al. in children with ADHD and ASD (N = 50) showed that melatonin did not show statistically significant improvement in total duration of sleep compared to control groups that did not receive melatonin (P = 0.19) . Only onAdverse eventsMelatonin is safe and widely used for sleep disorders. As per a 52-week long-term study, the most common side effects with melatonin include fatigue (18.9%), vomiting (16.8%), mood swings (13.7%), and upper respiratory infection (10.5%) .Limitations of studiesThe studies mentioned in our systematic review had some limitations. RCT by Malow et al. did not compare the results of the melatonin group with placebo . DB-RCT Patients with ASD and/or ADHD showed improvement in total sleep time, sleep latency and improved uninterrupted sleep with melatonin, and augmented by combination management with melatonin and CBT. There was an improvement in externalizing behavior and subsequent improvement in the caregiver’s quality of life. Melatonin is a well-tolerated and safe medication in the dose range of 2-10 mg/day in the child and adolescent population. The efficacy and safety were proved by both short-term and long-term DB-RCTs.


The aim of this study is to investigate clinical characteristics and fatal outcomes of hypertension as well as the role of angiotensin-converting enzyme inhibitors/angiotensin receptor blockers (ACEI/ARB) use in patients with severe coronavirus disease 2019 (COVID-19). A total of 220  patients with severe COVID-19 were included. The mean age of included patients was 59.5 years and 70 (31.8%) patients had a history of hypertension. There were 23 patients (32.9%) receiving ACEI/ARB therapy. Patients with hypertension were older and had more comorbidities, and were more likely to suffer from severe inflammatory response and acute cardiac injury. Moreover, patients with hypertension were associated with significantly higher risk of in-hospital mortality than patients without hypertension. After adjustment of potential confounders, the independent correlation was still observed. In addition, ACEI/ARB users were associated with lower level of high-sensitivity cardiac troponin I and creatinine kinase–myocardial band, and lower risk of acute cardiac injury than ACEI/ARB non-users. In conclusion, patients with hypertension were more likely to suffer from severe inflammatory response, acute cardiac injury and had high risk of in-hospital mortality in severe COVID-19. The use of ACEI/ARB may protect patients with COVID-19 from acute cardiac injury. The coronavirus disease 2019 (COVID-19) has become a worldwide pandemic . Some stNotably, hypertension may frequently be treated with angiotensin converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARB) –7. But tUp to date, there are limited clinical studies evaluating the safety of ACEI/ARB in the treatment of COVID-19 with hypertension. The purpose of this study was to investigate clinical characteristics and outcomes of hypertension as well as the role of ACEI/ARB in severe COVID-19.The study initially enrolled 237  consecutive patients with severe COVID-19. We excluded 14 discharged patients and 3 patients who died because of incomplete data, leaving 220 patients for final analysis. The median age of included patients was 59.5 years (range 24 to 94 years). Sixty-two (31.8%) patients coexisted with hypertension. Patients with severe COVID-19 with hypertension were more likely to be older  and to have previous coronary artery disease , and were more likely to be exhibit dyspnea  and have a higher systolic blood pressure  at admission . There wIn terms of laboratory findings, patients with hypertension compared with patients without hypertension showed higher median creatinine kinase–myocardial band , high-sensitivity cardiac troponin I , N-terminal pro-B-type natriuretic peptide , white blood cell count , and levels of D-dimer , creatinine  and lower estimated glomerular filtration rate (eGFR)  at admission .In this cohort, patients with hypertension vs those without hypertension had a similar durations from symptom onset to admission  patients died during hospitalization. The risk of in-hospital mortality was higher among patients with vs without hypertension  . To deteOf the 70 patients with hypertension, 23 (32.9%) patients received ACEI/ARB  treatment and 47 (67.7%) did not receive ACEI/ARB treatment. Eighteen of 21 patients taken ACEI/ARB before admission and continued during hospitalization, and 2 patients administrated ARB at admission. Most of patients (38 [80.9%]) taken calcium channel blockers in control group. As presented in As shown in The major findings of our study are the following: (i) hypertension is highly prevalent among patients with severe COVID-19; (ii) patients with hypertension were more likely to suffer from severe inflammatory and acute cardiac injury; (iii) hypertension is an independent risk factor of in-hospital mortality; (iv) the use of ACEI/ARB may protect COVID-19 from acute cardiac injury.Several epidemiological studies have reported that hypertension is one of the most distinctive comorbidities in patients with severe COVID-19 (approximately 23-56.6%) –5, 11. CIt might be explained by the reason that the proportion of patients with coronary artery disease in hypertension was higher than patients without hypertension, and more potential cardiac dysfunction might preexist in patients with hypertension, such as left ventricular hypertrophy and heart failure. This may also be one of the reasons why dyspnea was more common in patients with hypertension. Recently, some studies reported that cardiac injury was a frequent condition among hospitalized patients with COVID-19, and it was associated with a higher risk of in-hospital mortality .We have also observed that patients with hypertension suffered from more severe inflammation than cases without hypertension. The expression of ACE2 is relatively insufficient in hypertension. Persistent severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection and replication contribute to reduced ACE2 expression –14. The Although hypertension has become a major comorbidity in patients with COVID-19, it is unclear whether hypertension is directly related to an increased risk of fatal outcomes. In our study, we found that hypertension was associated with a significantly high risk of in-hospital mortality. Hypertension plays a key role in the development of chronic target organ damage (mainly heart and kidney) and may be exacerbated after SARS-CoV-2 infection, just like other infections. These conditions may lead to higher hospital mortality.Even if the HR value was weakened after adjustment for multiple potential confounders, including sex, age, cardiac injury, inflammation makers and emerging risk factors of renal dysfunction and D-dimer –18, hypeA previous study reported that ACEI/ARB chronic therapy, through their beneficial effects on the cardiovascular system, had a positive effect on survival in very elderly hospitalized patients . AlthougFang et al suggested that hypertension with ACE2-stimulating drugs was associated with an increased risk of developing severe and fatal COVID-19 , 25, 26.This study has several limitations that must be acknowledged. First, as the data on echocardiography was waived in light of the urgent medical condition, the underlying cardiac function of severe COVID-19 cases was unclear. We could not entirely rule out the possibility of residual confounders by these unmeasured parameters. Second, data were extracted from a pre-existing electronic medical records, which subjects it to potential selection bias. But it could be minimized by conducting multivariate regression analyses. Third, for the effect of ACEI/ARB on severe COVID-19 with hypertension, the sample size of this study was small and our findings still need to be further confirmed.In summary, patients with COVID-19 with hypertension was more likely to suffer from severe cardiac injury, inflammatory response and poor prognosis. Considering the high risk of death, incremental caution and in-hospital discussion of anticipated risk in hypertension are completely necessary. In addition, administration of ACEI/ARB may contribute to low risk of acute cardiac injury in severe COVID-19. Our findings supported that ACEI/ARB can continue to be used in patients with severe COVID-19.https://clinicaltrials.gov/ identifier NCT04292964. Consecutive patients with severe COVID-19 admitted to hospitals in Hubei and Chongqing from January 11st, 2020 to February 20th, 2020, were collected by national medical team. Case definitions of confirmed human infection with COVID-19 conformed to the interim guidance from the World Health Organization. All patients were diagnosed as severe cases according to the 7th edition guideline issued by China’s National Health Commission. This study was approved by the institutional ethics board of the First Affiliated Hospital of Chongqing Medical University . The study was also registered on Chinese medical research registration information system.This was a retrospective, observational study registry with Information on symptoms, signs, underlying comorbidities, laboratory findings, blood pressure, medical history , treatments and clinical outcomes were obtained from electronic medical records. All the data were collected by two investigators independently and double checked by other investigators. If the information on medical history were not available from electronic medical records, we directly communicated with patients or their families to obtain further data.2) is ≤ 93%; 3) partial pressure of oxygen (PaO2)/Fraction of inspiration O2 (FiO2) ≤ 300mmHg. ACEI/ARB treatment was defined as a patient taking ACEI/ARB during hospitalization irrespective of dose and whether taken before admission or not. The primary endpoint was in-hospital mortality, defined as any death occurring within 15 days after hospitalization. The secondary endpoints included acute cardiac injury, ARDS and acute kidney injury. Acute cardiac injury was defined as blood levels of cardiac biomarkers (hs-cTnI) above the 99th-percentile upper reference limit.Severe COVID-19 was defined as meeting at least one of the following criteria: 1) respiratory rate ≥30/min; 2) at rest, oxygen saturation  and 95% confidence intervals (CIs). All P values were two-tailed, and significance was set at P < 0.05. Statistical analyses were performed using SPSS software (version 22.0).Continuous measurements described as mean (standard deviation: [SD]) if they are normally distributed or median (interquartile range: [IQR)] if they are not. Categorical variables were described as frequency rates and percentages. Test for differences in categorical variables among the two groups used XSupplementary Tables


Lower expression levels of these miRNAs were associated having greater depressive symptoms. Furthermore, lower levels of miR-484 and miR-197-3p were associated with faster decline of cognition over time. Moreover, lower miR-484 level was associated with higher probability of having Alzheimer’s dementia. Importantly, the associations between miR-484 and depressive symptoms and Alzheimer’s dementia, respectively, were replicated in an independent cohort. Lastly, the predicted targets of miR-484 were enriched in a brain protein co-expression module involving synaptic transmission and regulation of synaptic plasticity. This study identified four brain microRNAs associated with late-life depressive symptoms assessed longitudinally. In addition, we found a molecular connection between late-life depression and dementia through miR-484.Late-life depression is associated with an increased risk for dementia but we have limited knowledge of the molecular mechanisms underlying this association. Here we investigated whether brain microRNAs, important posttranscriptional regulators of gene expression, contribute to this association. Late-life depressive symptoms were assessed annually in 300 participants of the Religious Orders Study and Rush Memory and Aging Project for a mean of 7 years. Participants underwent annual cognitive testing, clinical assessment of cognitive status, and uniform neuropathologic examination after death. microRNAs were profiled from the prefrontal cortex using NanoString platform in the discovery cohort and small RNA sequencing in the replication cohort. A global microRNA association study of late-life depressive symptoms was performed using linear mixed model adjusting for the potential confounding factors. Four brain microRNAs were associated with late-life depressive symptoms at adjusted  We found four miRNAs significantly associated with late-life depressive symptoms. Next, we examined these four miRNAs in relation to the dementia-related traits, including rate of decline of cognitive performance over time, clinical diagnosis of mild cognitive impairment (MCI) or dementia, and eight measured neuropathologies. Two miRNAs were found to be associated with these dementia features (miR-484 and miR-197-3p). Then we examined the targets of these two miRNAs in a published protein co-expression networks and found that predicted targets of miR-484 were enriched in the protein co-expression module involved in synaptic transmission and regulation of long-term synaptic plasticity. Lastly, we performed a biological replication of our findings using small RNA sequencing technology and replicated the associations between miR-484 and (i) late-life depressive symptoms and (ii) clinical diagnosis of AD in an independent cohort. Together, these findings point to a molecular link between late-life depression and elevated risk for AD dementia through miR-484 for further mechanistic studies.p = 0.90). There was higher education level in ROS  and higher average depression score in MAP . We have adjusted for these differences by including sex and study (ROS vs. MAP) as the covariates in all analyses.A total of 300 non-demented ROS/MAP subjects were included in the global brain miRNA analysis of depressive symptoms assessed longitudinally. Table p value < 0.05 after accounting for sex, age, global AD pathology, cell type, postmortem interval (PMI), RNA integrity number (RIN), and study . Together, these findings suggest that these miRNAs are associated with depressive symptoms independent of AD pathology.We found four miRNAs significantly associated with late-life depressive symptoms at an adjusted udy Fig. . All fouudy Fig. . When we2 we examined associations between these four depressive symptom-associated miRNAs and the features of dementia to test whether these miRNAs share a common molecular mechanism with dementia. Interestingly, two miRNAs (miR-484 and miR-197-3p) were associated with the rate of cognitive decline over time after adjusting for sex; age at death; education; PMI; RIN; proportions of neurons, oligodendrocytes, astrocytes, and microglia; and study . Both miR-197-3p and miR-484 were downregulated in individuals with faster cognitive decline, as well as with greater depressive symptoms, consistent with the known detrimental effects of depression on cognitive aging.Given the observation that late-life depression is associated with increased incidence of dementia in several epidemiological studies,β = −0.15, p = 0.009, N = 505, Supplementary Fig. Regarding the clinical diagnosis of dementia, miR-484 was significantly associated with clinical diagnosis of MCI or AD after adjusting for sex, age at death, education, PMI, RIN, cell-type proportions, and study , atherosclerosis, and Lewy bodies in relation to these four miRNAs. However, we did not see a significant association between any of the four depressive symptom-associated miRNAs and any of the dementia pathologies. These findings are consistent with the observation that depressive symptoms are not directly associated with dementia pathologies.β = 0.023, p = 0.016, N = 390). This suggests that miR-197-3p contributes to cognitive decline via mechanisms independent of the known dementia pathologies and may be a target for further studies of cognitive resilience.Since cognitive decline can be due to neurodegenerative pathologies or independent of them, we examined the associations between miR-484 and miR-197-3p and cognitive decline adjusting for all the measured neurodegenerative pathologies  and sex, age at death, PMI, RIN, study, and cell-type proportions. Using this model, we found that only miR-197-3p was significantly associated with cognitive decline  adjusted p = 0.034; Fig. 15 The CWCS is an estimate of how effective the miRNA binds to and represses the expression of that gene. The more negative the CWCS, the more effective the miRNA can suppress the expression of that target.15 Filtering targets of miR-484 and miR-197 to include only targets with CWCS < −0.1 for this enrichment analysis led to recapitulating our initial results but with a greater degree of significance, i.e., targets of miR-484 were enriched in the M1 turquoise module involved in synaptic transmission and regulation of synaptic plasticity  cohort.β = −1.04 [0.46], p = 0.024, N = 160; Supplementary Fig. β = 0.03 [0.58], p = 0.96).An independent set of 160 ROS/MAP subjects was available as a biological replication cohort and their characteristics are presented in Table β = −2.35 [0.86]; p = 0.0060; N = 160).Furthermore, we found that lower expression of miR-484 was associated with MCI or AD in this biological replication cohort after adjusting for sex, age at death, PMI, RIN, and education, consistent with what we found in the discovery cohort . Among these depressive symptom-associated miRNAs, miR-484 and miR-197-3p were also associated with faster decline of cognition over time and miR-484 with higher probability for having a clinical diagnosis of MCI or AD dementia. These findings suggest that miR-484 and miR-197-3p may play a significant role in the association between late-life depression and elevated risk for AD and vascular dementia. Importantly, the association between miR-484 expression level and depressive symptoms was replicated in an independent ROS/MAP cohort of 160 participants through small RNA sequencing technology. This biological replication via a different miRNA profiling technology increases the confidence of the importance of miR-484 in late-life depression. Furthermore, we replicated the association between lower miR-484 expression and MCI or AD dementia in a biological replication cohort. Of note, our finding of lower miR-484 expression being associated with AD dementia is consistent with another study that found decreased level of temporal cortex miR-484 in AD compared with no cognitive impairment.18 thus altered miR-484 expression can lead to altered mitochondrial fission. Mitochondrial fission and fusion refer to dynamic interactions among mitochondria to establish organelle shape, size, number, and allow for mixing of mitochondrial contents.19 From our findings, it can be inferred that depression may increase risk for AD dementia through altered mitochondrial fission. The connection between mitochondrial fission and AD dementia is consistent with prior studies showing that altered balance of mitochondrial fission and fusion is an important mechanism leading to neuronal dysfunction in AD dementia brain.21 Furthermore, mitochondria in neurons are crucial for maintaining synaptic function because they regulate synaptic transmission by generating ATP to power the process and by modulating presynaptic calcium level, which determines the release of neurotransmitters.24 Moreover, we found that the predicted targets of miR-484 were enriched in the protein co-expression module involving synaptic transmission and regulation of long-term neuronal synaptic plasticity. We hypothesize that miR-484 acts through its effects on synaptic transmission and neuronal synaptic plasticity in elevating risk for AD and vascular dementia, which should be tested in model systems.miR-484 regulates mitochondrial fission;25 Bipolar disorder and depression share at least two features. First, individuals with bipolar disorder are depressed the majority (70–81%) of their ill-time.26 Second, there is a significant shared heritability between depressive symptoms and bipolar disorder (r = 0.35).27 Our finding of the connection between late-life depression and AD dementia through miR-484 is complementary to the finding that polygenic risk score (PRS) of major depression predicted the conversion of non-depressed amnestic MCI to AD dementia.28 The effect of the PRS of major depression on the conversion of MCI to AD was mediated through left hippocampal volume.28 Consistently, another study found different patterns of hippocampal atrophy among participants with AD, amnestic MCI, and late-life depression.29Our finding that prefrontal cortex miR-484 was down-regulated in individuals with greater depressive symptoms is consistent with results from two prior studies of bipolar disorder, a syndrome predominated by severe depressive symptoms, that observed downregulation of prefrontal cortex miR-484 in bipolar disorder.30 miR-135, however, was not associated with depressive symptoms in the ROS/MAP cohorts (p = 0.06). This is likely due to two reasons. First, Issler and colleagues examined the brainstem and not the dlPFC. Furthermore, Issler and colleagues examined miR-135 in five different subnuclei of the raphe. Differential expression of miR-135 in depression was only detected in two subnuclei, the dorsal raphe and raphe magnus, reflecting the brain region-specific expression of this miR-135. Second, the association between miR-135 and depression was found by Issler and colleagues in depressed suicide victims, which typically reflects severe major depression, while we studied late-life depressive symptoms in a community-based cohort with mild depressive symptoms; very few of our participants had major depression. Thus it is plausible that the severity of depression may influence the abundance of miR-135. In addition, miR-132 was found to be associated with AD and pathologies by Lau and colleagues and also by our group.32 However, we did not find an association between miR-132 and depressive symptoms here (p = 0.39).In a prior study, miR-135 was found to be lower in the brain of 11 depressed suicide human subjects by Issler and colleagues.34 Both depression and AD dementia are heterogeneous etiologically and clinically.33 Furthermore, the question of whether depression is a risk factor for dementia or part of its prodrome has not been resolved. In other word, the neurodegenerative process of developing AD dementia may contribute to the development of late-life depression or vice versa.33 Hence, we here examined brain miRNA signature of depressive symptoms assessed longitudinally in subjects with either normal cognition or MCI to identify the molecular basis behind the association between late-life depression and higher risk for AD dementia. One limitation of our study is that it has miRNA profile from only one region of the brain, the dlPFC. Examining miRNA profiles from different regions of the brain would provide a more comprehensive picture of the association between late-life depression and brain miRNA changes. However, the dPFC is a region that is relatively less affected in AD and thus more likely to show more subtle changes compared to areas where pathologies occur early . Another limitation of the study is that this is a postmortem brain study, therefore it is cross-sectional by nature and no causal relationship can be established.Depression and AD dementia are relatively common conditions in older age and are frequently comorbid.Our study has several strengths. First, it is the first global brain miRNA association study of late-life depressive symptoms to the best of our knowledge. Second, in our study, late-life depressive symptoms were assessed annually and longitudinally over a mean of 7 follow-up years. Third, this is the first study to observe a link between late-life depressive symptoms and dementia at the molecular level, via miRNAs. Fourth, we rigorously controlled for potential confounding factors in our analyses including postmortem brain interval and tissue heterogeneity. Fifth, we performed a biological validation of our initial findings using next-generation small RNA sequencing in a separately ascertained collection of samples.35 may be partially explained by miR-484 through its effects on synaptic transmission, synaptic plasticity, and mitochondrial fission. Further mechanistic studies are needed to confirm these observations.In conclusion, we show that late-life depressive symptoms are linked to the downregulation of prefrontal cortex miR-484. Furthermore, the association between late-life depression and elevated risk for dementia observed in 23 prospective epidemiological studies36 ROS is comprised of older Catholic priests, nuns, and monks throughout the USA. MAP recruits older lay persons from the greater Chicago area. Both studies involve detailed annual cognitive and clinical evaluations and brain autopsy. Participants provided informed consent, signed an Anatomic Gift Act, and a repository consent to allow their data and biospecimens to be repurposed. The studies were approved by the Institutional Review Board of Rush University Medical Center. To be included in our global miRNA association study of depressive symptoms, participants must not carry a final clinical diagnosis of dementia.Participants were from two longitudinal clinical–pathologic cohort studies of aging and AD—ROS and MAP.37 The CES-D captures the major symptoms of depression as identified in clinical and factor analytical studies, i.e., depressed affect, positive affect, somatic complaints, and interpersonal problems, with a reliability of 0.8.37 The possible score range for the CES-D is 0–10, with higher score indicating more depressive symptoms.Depressive symptoms were assessed annually using the 10-item version of the Center for Epidemiological Studies Depression scale (CES-D).38 Case conferences including one or more neurologists and a neuropsychologist were used for consensus as necessary. Clinical diagnoses of cognitive status can include no cognitive impairment, MCI, or AD.Final clinical diagnosis of AD dementia is determined at the time of death by a neurologist with expertise in dementia using all available clinical data but blinded to postmortem data. The diagnosis was based on the recommendation of the National Institute of Neurological and Communicative Disorders and Stroke and the AD and Related Disorders Association.Z score with respect to the mean and standard deviation of the cohort at the baseline visit. These Z scores were averaged to create the composite annual global cognitive score. Rate of cognitive change is the random slope with respect to follow-up years in the mixed linear model in which the annual global cognitive performance is the longitudinal outcome; follow-up year is the independent variable with random effect per subject; and age at recruitment, sex, and years of education are the covariates.Rate of decline of cognitive performance is the individual rate of decline of global cognitive performance over time. Annually, 21 cognitive tests were administered to each ROS/MAP participant with 19 in common. The raw scores from 19 cognitive tests were standardized to a 40 Nine brain regions of interest  were dissected and stained for assessment of pathology. Global AD pathology  was visualized in five cortical regions using a modified Bielschowsky silver stain. Counts of silver-stained neuritic plaques, diffuse plaques, and neurofibrillary tangles were used to create a continuous measure of AD global pathology. The square root of this global pathology measure was used in our analyses to improve its normal distribution. Chronic gross infarcts were identified visually by examining slabs and pictures from both hemispheres and confirmed histologically and was treated as a dichotomous variable (present vs. absent) in our analyses. Microinfarcts were those that were not visible to the naked eye but were identified under microscope using hematoxylin and eosin stain in a minimum of nine regions, including six cortical regions, two subcortical regions, and midbrain. Microinfarcts were treated as present or absent in our analyses. Lewy body pathology was assessed using antibodies to α-synuclein in six regions, including substantia nigra, limbic, and neocortices, and treated as present or absent in our analyses. Hippocampal sclerosis was identified as severe neuronal loss and gliosis in hippocampus or subiculum using hematoxylin and eosin stain and treated as present or absent in analyses.41 TDP-43 cytoplasmic inclusions were assessed in six regions using antibodies to phosphorylated TDP-43. Inclusions in each region were rated on a six-point scale and the mean of the regional scores was created.42 TDP-43 scores were dichotomized into absent  or present (mean score >0) in our analyses. Cerebral atherosclerosis was assessed by visual inspection of vessels in the circle of Willis and rated as absent, mild, moderate, or severe and treated as a semiquantitative variable in our analyses.43 CAA was assessed using amyloid-β immunostaining in four regions . In each region, meningeal and parenchymal vessels were assessed for amyloid deposition and scored from 0 to 4, where 0 indicates no deposition, 1 refers to scattered segmental but no circumferential deposition, 2 means circumferential deposition up to 10 vessels, 3 reflects circumferential deposition in >10 vessels and up to 75% of the vessels, and 4 indicates circumferential deposition in >75% of the vessels.44 The score for each region was the maximum of the meningeal and parenchymal scores, and a continuous summary score was created by averaging scores across the regions.44Brain autopsy was performed by examiners who were unaware of deceased participants’ clinical information and have been described in detail before.32 Briefly, total RNA was extracted from the full-thickness cortex with dissection of the underlying white matter of the dlPFC in the Brodmann areas 9 and 46 (BA 9/46). Then small RNA was isolated from total RNA and profiled using the Nanostring platform. RIN ranged from 5 to 10. Samples were randomized with respect to clinical diagnosis of cognitive status to minimize batch effects. miRNAs from the Nanostring RCC files were re-annotated using miRBase v19. After correcting for the probe-specific backgrounds, a three-step filtering of miRNA expression levels was performed. First, miRNA with missing expression levels in >95% of samples were removed. Second, samples with >95% of miRNAs with missing expression were removed. Third, all miRNA whose absolute values were <15 in at least 50% of the samples were removed to eliminate miRNA that had negligible expression in brain samples. After the miRNA and sample filtering, the dataset consisted of 292 miRNAs. Combat was used to remove batch effects and quantile normalization was performed.45 All miRNA data are available at https://www.synapse.org/#!Synapse/syn3219045.Quantification and quality control of miRNA profiles were described in detail before.46 Hence, we used the transcriptomes derived from RNA sequencing for these subjects to estimate the proportions of neurons, astrocytes, oligodendrocytes, and microglia for the dlPFC brain tissue from which we profiled miRNAs. We used the proportions of these four cell types as covariates in all our miRNA analyses to control for the heterogeneity of cell types in brain tissues. We used CIBERSORT pipeline47 and Darmanis’ single-cell RNA sequencing expression profiles from human brain tissue samples as the reference 48 to estimate brain cell-type composition.The total RNA extracted from postmortem brain dlPFC in ROS/MAP subjects was used for miRNA profiling as well as for RNA sequencing as described in detail before.p < 0.05.49Using linear mixed model, we examined the association between longitudinally assessed depressive symptoms and global miRNA profile, adjusting for sex; age at visit; global AD pathology burden; proportions of neurons, oligodendrocytes, astrocytes, and microglia; PMI; RIN; and study (ROS or MAP). Specifically, depression score was modeled as the outcome and miRNA as a predictor, with a random intercept and random slope for each subject with respect to the follow-up year, adjusting for the above-mentioned covariates. In this linear mixed model, we tested whether the miRNA abundance had a non-zero effect on the depression scores. By using a mixed effect on follow-up year (a covariate in this model), we accounted for the individual-specific change in depression score from year to year in the association test. To address multiple testing, we used BH method to control the false discovery rate and declared significantly associated miRNAs as those with an adjusted We examined associations between depressive symptom-associated miRNAs from the global miRNA analysis above and the dementia-related features. These dementia features include (i) rate of decline of cognitive performance; (ii) clinical diagnosis of dementia; and (iii) neurodegenerative pathologies, including global AD pathology, Lewy bodies, TDP-43, cerebral atherosclerosis, gross infarcts, chronic microinfarcts, CAA, and hippocampal sclerosis. We included all ROS/MAP participants with miRNA profiles in this analysis, hence the sample size for these analyses is >300. We used general linear regression model in which specific dementia feature was the outcome, miRNA was the predictor, and sex; age at death; education; PMI; RIN; proportions of neurons, oligodendrocytes, astrocytes, and microglia; and study are the covariates.14 Since an miRNA represses the expression of its target genes by either destabilizing the transcripts or repressing the translation of transcripts to proteins, the effects of miRNAs ought to be most apparent at the protein level. Hence, we asked whether the targets of miR-484 or miR-197 would be enriched in any of these protein co-expression modules. To that end, predicted targets of miR-484 and miR-197 were identified from TargetScan database v7.2.15 Next, we asked whether these targets were enriched in any of the 16 modules of co-expressed proteins mentioned above using gene set enrichment analysis and adjusting for multiple testing with BH method.A published study identified 16 modules of co-expression proteins in the BLSA.For a biological replication cohort, we used postmortem brain samples from the dlPFC BA 9/46 of 160 independent ROS/MAP subjects who were not part of the discovery cohort. Total RNA was extracted from the cortically dissected dlPFC with TRIzol  following the manufacturer’s protocol. Total RNA was used to create small RNA libraries using New England Biolabs’ NEBNext® Multiplex Small RNA Library Prep kit following the manufacturer’s protocol. Small RNA libraries were sequenced single-end read on the Illumina HiSeq3000. To avoid batch effects, samples were prepared for sequencing in batches of 8 that were randomized based on sex and cognitive diagnosis for RNA extraction, and by sex, cognitive diagnosis, RIN, PMI, pathologies, depression score, and study for small RNA library preparation and sequencing.50 Trimmed reads were aligned to the miRBase v.2251 with SHRIMP aligner.52 Counts of aligned miRNAs were normalized with variance stabilization transformation, which performs log2 transformation, normalizes for library size, and transforms the counts to approximately homoscedastic.53 After variance stabilization transformation, miR-484 and miR-197-3p were extracted for association analysis with longitudinal depressive symptoms using linear mixed model adjusting for the same covariates as was done in the discovery dataset.Quality of the sequencing data was inspected with FastQC. Adapters were trimmed with Trimmomatic.Further information on research design is available in the Supplementary InformationReporting Summary


Heat- and cold-related mortality risks are highly variable across different geographies, suggesting a differential distribution of vulnerability factors between and within countries, which could partly be driven by urban-to-rural disparities. Identifying these drivers of risk is crucial to characterize local vulnerability and design tailored public health interventions to improve adaptation of populations to climate change. We aimed to assess how heat- and cold-mortality risks change across urban, peri-urban and rural areas in Switzerland and to identify and compare the factors associated with increased vulnerability within and between different area typologies.We estimated the heat- and cold-related mortality association using the case time-series design and distributed lag non-linear models over daily mean temperature and all-cause mortality series between 1990-2017 in each municipality in Switzerland. Then, through multivariate meta-regression, we derived pooled heat and cold-mortality associations by typology  and assessed potential vulnerability factors among a wealth of demographic, socioeconomic, topographic, climatic, land use and other environmental data.2.5) drove differences in heat-mortality association, while for peri-urban/rural clusters socio-economic variables were also important. For cold, socio-economic variables drove changes in vulnerability across all typologies, while environmental factors and ageing were other important drivers of larger vulnerability in peri-urban/rural clusters, with heterogeneity in the direction of the association.Urban clusters reported larger pooled heat-related mortality risk ) , and rural 1.03 ), but similar cold-mortality risk  , vs rural 1.28 and peri-urban 1.39 (1.27-1.53)) clusters. We found different sets of vulnerability factors explaining the differential risk patterns across typologies. In urban clusters, mainly environmental factors (i.e. PMOur findings suggest that urban populations in Switzerland may be more vulnerable to heat, compared to rural locations, and different sets of vulnerability factors may drive these associations in each typology. Thus, future public health adaptation strategies should consider local and more tailored interventions rather than a one-size fits all approach. size fits all approach. Currently, non-optimal temperatures are associated with approximately 9.4% of the total mortality burden globally, which corresponds to 74 deaths per 100,000 people, of which the largest part can be attributed to cold (8.5% versus 0.9% for heat)2. Evidence suggests that climate change is already substantially affecting populations leading to an additional heat-mortality burden which is likely to increase further in the future and even overtake the current cold-related mortality under various climate change scenarios 4. Even with full implementation of the Paris agreement and reaching net-zero carbon emissions by 2050, the inherent inertia of the climate system will continue to increase temperatures for several more decades after6, yielding a substantial additional health burden 8. Thus, accelerated adaptation to non-optimal temperature is essential to reduce the heat-related mortality burden 10. Moreover, besides the expected increase in heat-related mortality, cold-related mortality is likely to further increase due to population ageing, showing the necessity to identify further adaptation strategies and vulnerability factors 12There is a well-established relationship between exposure to non-optimal temperatures and a wide range of adverse health outcomes 22, which are driven by small area characteristics such as access to air conditioning (AC), ageing, greenness and socioeconomic level amongst others25. However, most of the existing evidence of temperature-mortality risks and corresponding vulnerability factors rely on impact assessments conducted in urban locations alone, since smaller cities and rural locations have barely been assessed due to a lack of valid exposure data 27, low statistical power 29, or have been considered as part of larger regions lacking the local dimension of the risks.A large body of literature has shown that the temperature-mortality association can substantially vary across different geographical units and population sub-groups33. Moreover, the association between urbanicity and heat-vulnerability has been hypothesised to follow a U-shape curve, with larger risks in extremely urban or extremely rural regions17, while for cold, rural regions tend to be more vulnerable due to lower access to health care, lower baseline health or poverty amongst others 16. Although there is agreement on spatial variability of the temperature-mortality association, little is known regarding differential drivers of temperature-vulnerability between typologies. Moreover, in Switzerland, previous studies have observed large variation in the heat- and cold-related mortality impacts between cities and cantons with larger heat-mortality impacts in urban regions , and larger cold impacts in rural regions. However, thus far the underlying mechanisms for this large spatial variation has remained unexplored and it is not known which social , biological  or environmental vulnerability factors  explain the variation of non-optimal temperature-mortality impacts between regions in Switzerland. Understanding the mechanisms and factors driving vulnerability in urban/rural locations can help to identify the most vulnerable populations and aid the design of tailored public health interventions to modulate heat and cold-related vulnerability.Even though urban areas tend to be warmer than rural regions due to the Urban Heat Island effect (UHI), rural regions have found to be at least equally vulnerable to temperature and climate change34, using a wealth of sociodemographic and environmental data available at high resolution.In this assessment, we hypothesise that vulnerability to heat and cold vary across urban and rural locations driven by different sets of vulnerability factors. First, vulnerability to non-optimal temperature is usually dependent on small-area level characteristics of the population and environment, which are highly heterogeneous within and between urban and rural regions. Second, these characteristics or factors can have different effects in each type of area . Therefore, we aimed to assess how heat- and cold-mortality risks differ across urban, peri-urban and rural regions between 1990 and 2017 in Switzerland, and to explore what factors are associated with increased vulnerability to non-optimal temperatures in each type of region. The novelty of this assessment is the application of a recently developed statistical framework to study the effect modification of individual variables in a complex multivariable regression model 22.1Switzerland is a country with a particularly sparse population which is unevenly distributed throughout the country . In part2.235. The use of such high-resolution temperature grid cells has shown to be a valid alternative to monitor stations to assess temperature-mortality impacts. It also has many advantages as opposed to monitor stations, as it allows us to assign an exposure to remote areas regardless the presence or not of weather stations .36We collected daily time series data on all-cause mortality and temperature for all 2,054 municipalities in Switzerland between the 1st of January 1990 and the 31st of December 2017. The individual mortality data was provided by the Swiss Federal Bureau of Statics (BFS). We obtained the daily mean temperature on a 1.6 x 2.3 km grid across the full Swiss geographic extent from a gridded climate dataset (MeteoSwiss-grid-product) developed by Meteoswiss. We then derived the corresponding population-weighted average temperature series for each municipality, as described in a previous study2.32.5 annual concentration, percentage of water area). The full list of variables with the corresponding definition and source is provided in We initially compiled an integrated dataset of 42 variables characterizing the population and environment for each municipality which we believed could modulate the vulnerability to non-optimal temperatures. We included several socioeconomic variables , percentage of new houses, ageing index), topographic variables , climatic variables  as well as land use and environmental data , PMSince many of the 42 variables showed a large degree of multi-collinearity , we redu2.437. The Ward-like algorithm is a constrained hierarchical clustering method that aims to optimize a convex combination using two dissimilarity matrices and a mixing parameter to create a new higher agglomerative layer consisting of municipalities that are both similar and proximal to each other  defined by BFS, and also more appropriate than using an ad hoc definition based on clustering variables as used for the Ward-like hierarchical clustering method . In particular, we believe that the differential effects of vulnerability factors by typology on the temperature-mortality association could be diluted as the Swiss orography, population characteristics  and distribution are highly heterogeneous within districts. Using the proposed approach, the municipalities included in the derived high agglomeration clusters are more homogeneous, thus allowing for a better characterisation of the vulnerability of the population and it would help to better capture the signal of potential effect modification of vulnerability factors.We defined a set of urban, peri-urban and rural clusters by combining all of the 2,054 municipalities into 94 higher agglomerative clusters using the Ward-like hierarchical clustering method with geographical constraints using municipality-level information on several vulnerability variablesch other 37. Metho2.538. The case time series design allowed us to estimate the exposure-response function within a cluster, but still use the high-resolution municipality level data, therefore, reducing exposure misclassification and increasing the precision of the estimates. This design also controls for temporal trends using a matching stratum defined by year, month and day of the week by municipality. We modelled the cluster-specific temperature-mortality association using the distributed lag non-linear framework, a flexible technique to model non-linear exposure-response associations and lagged dependencies 39. We defined a quadratic B-spline with three internal knots placed at the 10th, 75th and 90th percentile of the cluster-specific temperature distribution  summarizing the 10 cluster-level selected variables in the meta-regression model. We assessed the heterogeneity using the likelihood-ratio test and the Cochran Q-test and the I2 statistic  and 95th percentile  value for each of the 9 selected district-specific meta vulnerability factors. Thus, for each typology  we aimed to compare the heat- and cold-related mortality association for the hypothetical high and low levels of the vulnerability factor and subsequently calculated the corresponding p-value between “high” and “low” exposure for each vulnerability factor using the Wald-test. For example, “high” level of exposure to travel time to health care means longer travel time to health care in that specific cluster (corresponding to the 95th percentile), whilst “low” exposure represents short travel time to healthcare for a given cluster by typology (5th percentile). Similarly, exposure to “high” ageing represents a higher ratio of population aged over 65 years compared to the 20-64 age group present in a cluster compared “low” exposure to ageing for a given typology, which has a smaller proportion of people aged over 65 compared to the 20-64 age group, and thus can be characterised as a younger population. We did this for all vulnerability factors. Then, we extracted the RR at the 1st percentile of the temperature distribution for cold and the 99th percentile for heat for each variable with the corresponding 95% confidence interval. Lastly, to ease interpretability, we computed for each vulnerability factor the absolute relative risk difference between “high” and “low” exposure of the RR estimate.To assess vulnerability factors across Switzerland by typology, we applied the same multivariate meta-regression framework used before but separately for each typology and by including each of the 9 vulnerability factors as predictors in univariable models 32), as well as slightly elevated annual levels of PM2.5  versus 9.9μg/m3) and shorter time to health care  versus 6.8 minutes ).th percentile of the temperature distribution) with a RR of 1.17  compared to peri-urban and rural clusters  and 1.03 , respectively). For cold, urban and peri-urban clusters show a similar risk  and 1.39 , respectively), while rural clusters show signs of a slightly lower risk ), although the confidence intervals partly overlap. There is some evidence for differential patterns of overall non-optimal temperature-mortality association across urban, peri-urban and rural clusters based on the Wald test .2.5 concentration  using the univariable meta-regression model . Urban clusters with high annual mean PM2.5 concentrations show a larger heat-related mortality risk  indicated with the red vertical dashed line) compared to clusters with low PM2.5 ), which is associated with a lower heat-mortality risk. A similar pattern can be observed for rural locations, while for peri-urban clusters no differences were found in the heat tail. Instead, for peri-urban clusters with high PM2.5 concentrations we observed a larger cold-mortality risk ) versus 1.15 ) for low levels of mean annual PM2.5 concentration, while similar risks can be observed for urban and rural clusters for cold.For illustrative purposes, th percentile) and high (95th percentile) levels for the selected vulnerability factors, respectively. The full exposure-response functions for each vulnerability factor  versus low 1.14 ), while in rural clusters the association is reversed , versus low 1.20 ). In peri-urban clusters also factors such as high population density somewhat increase the risk , versus low 1.19 ), whilst high density yields lower risk in rural clusters.2.5, temperature and temperature range are somewhat associated with a higher risk  versus low ) ) . Also, s42.5), however, for peri-urban and rural regions other factors also modify the association such as socio-economic factors as well as population ageing. For cold, across all urban/peri-urban/rural clusters social factors  modify the cold-mortality association, while for peri-urban and rural clusters also environmental factors and biological factors affected the cold-mortality association, with heterogeneity in the direction of the association between typologies. Although not all identified vulnerability factors such as biological factors are modifiable, this study can help identify vulnerable subpopulations in Switzerland in specific tasks like vulnerability mapping41. Moreover, future public health adaptation strategies which aim to attenuate heat and cold-related health impacts should account for heterogeneity and implement more tailored interventions according to the local characteristics of the population.This nationwide study aimed to assess how vulnerability to heat and cold varies across urban, peri-urban and rural clusters, and more importantly, to identify which factors are driving such vulnerability patterns. Our results suggest that urban clusters are at increased vulnerability to non-optimal temperatures, mainly to heat, compared to rural and peri-urban clusters in Switzerland. Therefore, health impacts derived at national or large regional level may be under- or overestimated if ignoring differential vulnerability between urban and rural regions. This may be relevant for the evaluation of historical and future health impacts of climate change. More importantly, our findings challenge the assumption that urban/peri-urban/rural regions share similar vulnerability drivers in terms of characteristics of the population, geographic and socio-economic factors. The main driver for the heat-mortality association across all urban/peri-urban/rural clusters are environmental factors  were associated with increased vulnerability which is in accordance with literature49. However, we cannot disentangle the contribution of the UHI and/or any of the other drivers due to the complex correlation between them and the lack of specific UHI metrics50. Regarding social vulnerability, previous assessments found that low SES, social isolation and population ageing could increase vulnerability in urban areas, for the former we observed good evidence for effect modification for both heat (peri-urban clusters) and cold , where higher SES is associated with a reduction in risk53. Despite increased risk for heat mortality with lower SES, we did not observe patterns in urban heat exposure and climate injustice between clusters, which usually is present on an intra-city level56. For peri-urban clusters, where besides environmental factors, also social  and biological factors  were found to be important effect modifiers. In contrast to many previous studies, we did not observe evidence for greenness as an effect modifier for the heat-mortality risk 58. This may be explained by the limited variability in the EVI values across urban and peri-urban clusters in Switzerland . Although for urban clusters we found a negative association between greenness and temperature, for peri-urban and rural clusters, temperature was positively correlated with greenness   , illustr21. Our findings suggest that cold-related vulnerability in urban clusters was mainly driven by socio-economic factors  as well as population density, consistent with previous studies 22, while more relevant drivers were identified in peri-urban and rural clusters such as % of new houses, PM2.5, ageing and population density. To note, although many previous studies found that low-housing quality exacerbates the risk of heat 52, this is one of the more recent studies that also report housing as an effect modifier for cold-related mortality, particularly in peri-urban regions 60. It has been hypothesised that the reason for the existing inconclusive findings or complex patterns on cold vulnerability might be due to the more complex pathways of how cold exposure can affect health . Future research should aim to study cold-related vulnerability factors and clarify the links between factors and mechanisms driving increased risks.Unlike heat vulnerability, evidence on cold-related vulnerability factors remains limited and inconclusive in the literature2.5 concentration and mean annual temperature), as well as population density, are negatively associated with cold-related mortality in rural clusters while positively associated in peri-urban clusters. Meaning that peri-urban clusters that are more similar to urban clusters have higher risks for cold-related mortality than peri-urban clusters which are more similar to rural clusters. Moreover, rural clusters with higher temperatures, higher PM2.5 concentrations and higher population density that are generally associated with increased urbanisation have a lower risk than rural clusters with low temperatures and low population density. Therefore, we believe that for cold-related mortality the lowest risk can be observed in rural/low-density peri-urban areas, a finding shared by a recent study that observed this association for heat17. For heat, however, we only observed increased vulnerability for urban clusters, which might be due to the urban heat island, while in peri-urban and rural no differences were found, possibly because outside of the main cities, Switzerland is very sparsely populated . Finally, to assess the effect modification of vulnerability factors we applied a novel extended second-stage time series which allowed us to test vulnerability factors in the complex non-linear mixed meta regression which has not been applied to nationwide data before34. Then, to assess vulnerability factors in both urban and rural areas we used gridded climate datasets which allowed us to assign temperature exposure at municipality level, which is considered an unprecedented high resolution in many ecological studies investigating temperature and mortality36. Lastly, using the Ward-like hierarchical clustering with geographical constraints 37, we defined ad hoc clusters of municipalities with similar characteristics, as an alternative to the administrative district boundaries  defined by the BFS . The Swiss orography and population distribution are highly heterogeneous, with large variation in demographic and environmental variables within administrative clusters and thus the effect modification of vulnerability factors by cluster typology can be diluted if heterogenous municipalities are grouped in the same cluster.This study has several strengths. First, we used advanced statistical methods recently developed in climate epidemiology to maximize the power of the available data and increase the precision of our estimates and the reliability of our conclusions. In particular, we applied the novel case time series design which allowed us to use temperature and mortality data at a high resolution and thereby increase the precision of the risk estimates 65. Lastly, this is an ecological assessment conducted at the municipality level. Thus, our results would not necessarily correlate with evidence on vulnerability factors driving differences at a finer resolution within municipalities .Some limitations should be acknowledged. First, our findings suggest vulnerability patterns according to levels of specific vulnerability factors but do not remove effects from correlated variables. That is, risks at different levels of the vulnerability factor were derived using univariate models, thus, not accounting for other (correlated) factors that might partly explain differences in the temperature-mortality association within typologies . Second, the low statistical power in rural clusters hindered the assessment of vulnerability factors. Additionally, we observed limited variability for some effect modifiers by urban/peri-urban/rural typologies, which could have limited the power to detect effect modification by variable. Then, we did not include humidity, influenza and air pollution concentrations as confounding variables, as we believe that their impact would be, if present, minimal, as their role as an confounding variable remain debated 5Our findings suggest larger temperature vulnerability in urban clusters, particularly for heat compared to rural regions, while cold-related vulnerability was similar across typologies. More importantly, this study has shown that drivers of temperature vulnerability can considerably vary by urban-rural typology in Switzerland. Therefore, future public health adaptation strategies aimed at mitigating the adverse impacts of climate change on population health should consider tailored interventions according to the characteristics of the target population.Supplementary file


Glucose is a major energy source consumed by proliferating mammalian cells. Therefore, in general, proliferating cells have the preference of high glucose contents in extracellular environment. Here, we showed that high glucose concentrations impede the proliferation of satellite cells, which are muscle-specific stem cells, under adherent culture conditions. We found that the proliferation activity of satellite cells was higher in glucose-free DMEM growth medium (low-glucose medium with a glucose concentration of 2 mM) than in standard glucose DMEM (high-glucose medium with a glucose concentration of 19 mM). Satellite cells cultured in the high-glucose medium showed a decreased population of reserve cells, identified by staining for Pax7 expression, suggesting that glucose concentration affects cell fate determination. In conclusion, glucose is a factor that decides the cell fate of skeletal muscle-specific stem cells. Due to this unique feature of satellite cells, hyperglycemia may negatively affect the regenerative capability of skeletal muscle myofibers and thus facilitate sarcopenia. Skeletal muscle has the ability to regenerate after injury. The main players of muscle regeneration are muscle-specific stem cells, known as satellite cells, which reside between the sarcolemma and basal lamina of myofibers. Satellite cells normally stay quiescent and have a low metabolic rate but initiate the myogenic program in response to muscle injury . ActivatGlucose is an essential energy substrate and an anabolic precursor for various mammalian cells. In anaerobic glycolysis, two ATP molecules are generated from one glucose molecule, and the central metabolite pyruvate is used as a substrate in the mitochondrial tricarboxylic acid (TCA) cycle through oxidative phosphorylation. In addition to its catabolic role, glucose is used for the synthesis of nucleotides through the pentose phosphate pathway, which is essential to produce ribose for DNA synthesis during cell division. Proliferating cells, such as cancer cells, prefer glucose as fuel to support their rapid proliferation . For theHigh-glucose media have also widely been used for culturing muscle cells . HoweverAdult C57BL/6 male mice (8–12 weeks) were used in this study. Mice were fed normal chow and water under standard lighting conditions (12-h:12-h light–dark cycle).2, and media were changed every day, beginning on the third of culture. Myogenic differentiation was induced using the differentiation medium (high-glucose DMEM supplemented with 5% horse serum and 1% penicillin–streptomycin) at 37°C under an atmosphere of 5% CO2. For floating culture, isolated myofibers were cultured in the plating medium  without incubation of Accutase (Extensor digitorum longus (EDL) and soleus muscles were isolated and digested using type I collagenase, as described previously . CollectAccutase .For the cell proliferation assays, 20 myofibers were cultured in each well (24-well plate) in duplicate, and stained with DAPI or Ki67, a marker of cell proliferation. To detect EdU incorporation, the Click-iT EdU Imaging Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. On day-6 of culture, the cells were treated with 10 μM EdU for 6 h, and fixed in 4%PFA for 15 min.TM Plus TUNEL Assay for in situ Apoptosis Detection, Alexa FluorTM 594 dye (Thermo Fisher Scientific) according to the manufacturer’s instructions.To analyze the apoptosis of cultured satellite cells, the TUNEL assay was performed using the Click-iTFor evaluation of myoblast differentiation, cells cultured in low-glucose growth medium for 6-days were passaged in 24-well plates to the same density and cultured for 3-days in differentiation medium. Myosin heavy chain (MHC), a contractile protein expressed in differentiated muscle cells, was stained. The fusion index, the number of nuclei inside myotubes as a percentage of the total number of nuclei, was calculated and compared between glucose concentrations.Glucose oxidase (GOD) and catalase were dissolved in phosphate-buffered saline (PBS) at 100 and 3,700 U/mL, respectively. The enzyme solution was mixed with Biosurfine-AWP-MRH (Tokyo Gosei Kogyo Co.) at a ratio of 1:10 (v/v) and pasted on a glass slide (Matsunami). Each side of the glass slide was exposed to UV light for 30 min to immobilize the enzymes on the slide. FBS was incubated with the glass slide by gently shaking in a 50-mL tube for 3 weeks at 6°C. The glucose concentration was measured using the glucose (HK) assay kit (Sigma Aldrich), according to the manufacturer’s instructions. Glucose-depleted FBS was filtered through a 0.22 μm polyvinylidene fluoride (PVDF) membrane before use for cell culture.Immunocytochemistry of cultured myoblasts and myotubes was performed as described previously . SamplesImmunostaining myofiber-associated satellite cells was performed as described previously . BrieflyTotal protein extracts were obtained from homogenized tissues and cultured cells, and lysed with RIPA or lysis buffer . Proteinp < 0.05.Data are expressed as the mean ± standard error of the mean (SEM). Two-sided unpaired t-tests were used to compare data between the two groups. N in the figure legends indicates the number of mice used in the experiments. The level of significance was set to In general, high-glucose DMEM is the standard medium for culturing primary satellite cells , 2012. TTo examine the effect of glucose concentration on satellite cell proliferation, we determined cell growth curves in high- and low-glucose media. We cultured satellite cells obtained from 20 myofibers in each 24-well plate for 3, 4, 5, and 6 days before counting cells visualized by DAPI staining . As showWe also examined whether glucose regulated cell proliferation in a dose-dependent manner. Satellite cells derived from 20 myofibers were cultured for 6 days in media with different glucose concentrations, and the ratio of Ki67-positive cells to total cell number was quantified. Cell proliferation was enhanced in media with a glucose concentration of 8 mM or less . To miniCell number is potentially affected by cell viability and cell death. Therefore, we evaluated the apoptosis of satellite cells in high- and low-glucose media for 6 days using a TUNEL assay. The percentage of apoptotic cells was lower in the low-glucose medium than in the high-glucose medium, indicating that glucose restriction ameliorated cell damage and maintained cell viability . We analWe also performed experiments to examine whether low-glucose medium maintained the proliferative activity of the satellite cell-derived myoblasts. When high-glucose medium was used, cell proliferation was inhibited in around 1 week. However, we observed that the cells cultured in the low-glucose medium could be passaged, and they maintained their proliferative activity for more than 2 weeks . The pasWe examined whether glucose regulates myoblast differentiation. Myoblasts grown in low-glucose medium were passaged and cultured in differentiation medium (5% horse serum in DMEM) at different glucose concentrations for 3 days. The myotubes were fixed, and fusion indices were calculated by staining the MHC. Myoblasts were normally differentiated even when cultured in low-glucose medium, and fusion indexes were similar regardless of glucose concentration .We determined the myogenic status of the satellite cells using myogenic markers. Pax7 is an important transcription factor for maintaining the stemness of satellite cells, and is a recognized self-renewed cell marker . MyoD isPrimary culture of satellite cells from single myofibers was performed using EDL muscle because the myofibers can be isolated with a low risk of contamination by non-muscle cells or connective tissues . EDL musWhen satellite cells were cultured in a standard high-glucose medium, fibroblasts preferentially grew, as shown above . Mononuc2O2 (1). Since H2O2 is harmful to cells, catalase was used to degrade it to H2O and CO2 (2).As indicated before, the glucose-free DMEM we used in this study contained a small amount of glucose (2 mM) that originated from the FBS component. To test whether satellite cells can survive under even lower glucose conditions, we degraded the glucose in FBS using the enzymes GOD and catalase. GOD breaks down glucose into gluconic acid and HTo remove the enzymes after the reactions, we immobilized them on a glass slide using a UV curable polymer. FBS was incubated with the enzymes immobilized on the glass slide . After eUsing the glucose-depleted medium (termed as very-low-glucose medium), we prepared three different media containing the following glucose concentrations: 0.1, 2.0, and 19.0 mM by adding glucose powder. Satellite cells from 20 myofibers were cultured in each medium for 6 days using 24-well plates. The cell numbers were evaluated by microscopy. Surprisingly, the satellite cells proliferated normally in the very-low-glucose medium , and theTo explore the molecular mechanism of low glucose-promoted cell proliferation, we studied AMP-activated protein kinase (AMPK), a serine/threonine protein kinase that plays a role in several signaling pathways by sensing the intracellular energy state. Because low glucose potentially decreases energy level of the cells, AMPK is expected to be involved in acting as a mediator of energy state-induced cell proliferation regulation . To examSince glucose is a major energy source for cell proliferation, many cell types, such as cancer cells, benefit from using a sufficient amount of glucose . Our finin vitro experiments suggested that satellite cell proliferation was inhibited at glucose concentrations above 8 mM under adherent culture conditions; however, this effect was not observed in suspension culture. Diabetes mellitus is recognized as a risk factor for age-related muscle atrophy (sarcopenia)  , whereascopenia) . Recent copenia) . In addicopenia) , glucoseMetabolic disorders, such as obesity and type two diabetes, are associated with reduced AMPK activity in skeletal muscles . TherefoAlthough glucose restriction increased the number of cells in every population expressed Pax7, MyoD, and myogenin, the relative percentage of reserve cells, which were identified as Pax7+/MyoD-cells, was higher in low glucose medium. This means that lowering glucose concentration enhanced self-renewal of cultured satellite cells and delayed the differentiation, leading the maintenance of cell proliferation capacity. We also observed that Pax7 expression level was higher under the low glucose condition compared to high glucose . BecausePrimary cultures of satellite cells have widely been used to study the myogenesis, metabolism, myokine secretion, and contractile capacity of muscle cells . HoweverIn summary, we showed here, that satellite cell proliferation is promoted by glucose restriction under adherent culture conditions. This finding suggests that a supra-physiological concentration of glucose directly inhibits the regeneration of skeletal muscles and facilitates sarcopenia in diabetes mellitus. More research is required to further elucidate the mechanism by which glucose restriction regulates satellite cell proliferation; identification and investigation of molecules that act as glucose sensors may be useful for this purpose.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Experimental Animal Care and Use Committee of Tokyo Metropolitan University.YF, YK, MA, and YMi performed and analyzed the experiments. YF, YMa, and NF designed the experiments and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Low-grade epilepsy-associated neuroepithelial tumors (LEATs) are a frequent etiology in pediatric patients with epilepsy undergoing surgery. Objective: To identify differences in clinical and post-surgical follow-up between patients with focal meningeal involvement (MI) and those without MI within our cohort of pediatric patients with LEATs. Methods: We retrospectively reviewed all pediatric patients (<18 y) who underwent epilepsy surgery between 2011 and 2017 at our hospital. Cohort inclusion required histological diagnosis of LEATs and post-surgical follow-up of ≥2 y. We subsequently stratified patients according to presence of neuroradiological MI. Results: We identified 37 patients: five with MI and 32 without. Half of patients (19) were drug sensitive at surgery; similar between groups. The group with MI differed mainly for age of epilepsy-onset (0.6 vs. 7.0 y) but not for epilepsy duration (0.9 vs. 1.5 y). Post-surgery radiological follow-up  did not indicate disease progression. Seizure outcome was excellent in both groups, with 34 patients overall being both drug- and seizure-free. Conclusions: Our study identified a new subgroup of LEATs with focal MI and excellent post-surgical outcome. Moreover, this highlights the effectiveness of early surgery in pediatric LEATs. Low-grade epilepsy-associated neuroepithelial tumors (LEATs) are an increasingly recognized cause of focal epilepsy, particularly in young adults and children . The hisDrug-resistant epilepsy is often the major, if not exclusive, neurological symptom of LEATs ,7, so acLesionectomy for neocortical epilepsy or anterior temporal lobectomy (ATL) for mesio-temporal epilepsies are highly effective, standard procedures for LEATs ,10; betwMeningeal involvement (MI) may result from dissemination of cancer cells to both the leptomeninges (pia and arachnoid) and cerebrospinal fluid (CSF) compartments . MI has The aim of this retrospective analysis of our cohort of pediatric patients with LEATs was to identify differences in clinical and post-surgical follow-up between subgroups of patients with MI and those without MI.This single-center study was approved by the local ethics committee. The local ethics committee waived the written informed consent for collection of these data from retrospective review of records. At our institution, we follow about 1200 patients with epilepsy each year. Between 2011 and 2017, 158 patients underwent epilepsy surgery; search of the hospital database for pediatric patients (<18 y) who underwent epilepsy surgery during the same period found 131 patients. We compiled a database on clinical, neuroradiological, and neuropathological data from these 131 patients and filtered according to fulfillment of all study inclusion criteria:Clinical and EEG datasets pre- and postoperative were complete;Follow-up of at least of 2 y; Preoperative and postoperative magnetic resonance imaging (MRI) available for review;LEATs confirmed by postoperative histopathology.Patients with an age at surgery higher than 18 y and patients with non-tumoral etiology of epilepsy were excluded from the analysis.Within our whole cohort of 131 patients who underwent epilepsy surgery before 18 years old, the most common etiologies were (a) LEATs in 37/131 patients (28%), (b) FCD type I and type II in 35/131 patients (27%), and (c) hippocampal sclerosis in 10/131 (8%). The study cohort was 37 patients. We subsequently stratified patients according to presence or absence of neuroradiological MI that had been confirmed by histopathology. MRI were acquired on a 3T scanner  and MI was considered positive if meningeal thickening and/or enhancement was observed in post-contrast 3D-T1-weighted sequences (magnetization-prepared rapid acquisition with gradient echo [MPRAGE] or T1-SPACE) .All children underwent routine presurgical evaluation. This included full history and neurological examination, video-electroencephalographic recording, and brain MRI. Seizures were classified according to the ILAE Position Paper for Classification and Terminology . PotentiThe goal of surgery was the removal of the lesion identified on MRI. The surgical procedure included the complete resection of the tumor and adjacent ineloquent tissue up to the next pial border . For lesHistopathologic diagnosis relied on microscopic inspection of surgical brain samples and followed the current 2016 WHO classification  and ILAEAll operated patients were re-evaluated by general examination in the outpatient clinic by the neurosurgeon 2 weeks after surgery. Further follow-up clinical evaluations by multidisciplinary clinicians occurred at 3, 6, and 12 months after surgery and then every 12 months for at least 5 y. Postoperative brain MRI were performed immediately after surgery to exclude potential residual tumor, and subsequently at 6–10-month intervals. The resection was considered complete if the brain lesion was no longer evident from comparison between the preoperative and immediately postsurgical brain MRI. Seizure outcome was recorded using the Engel classification . In seizThirty-seven patients met the study inclusion criteria, which required histological diagnosis of LEATs, either isolated (28 patients) or in association with FCD type IIIb (9 patients). Part of this clinical series was previously included within aggregated data of two previous reports ,23. DemoAt surgery, 18 patients  were drug resistant according to the current ILAE definition . Long-teAt the time of this review, the median post-surgical follow-up was 4.0 y (interquartile range (IQR) 2.8–5.0 y), with 34 of 37 patients being both drug- and seizure-free, one patient was seizure-free with ongoing drugs (Engel Id), and two patients still experienced seizures (both of these were drug resistant at surgery). Five patients were re-operated due to incomplete resection and seizure persistence; in three of these five, the previous operation was done in another hospital . No patiFive patients were included in Group 1 see . The medThe median age at surgery was 2.0 y. One patient had immediate seizure recurrence after lesionectomy; seizure-freedom was achieved following a second anterior temporal lobectomy. All other patients underwent an anterior temporal lobectomy with resection of the deeper mesial temporal structures, including uncus, amygdala, and hippocampus. For two patients, post-surgical brain MRI revealed the persistence of less than 10% of the lesion.The median follow-up was 4.1 y (IQR 3.0–4.2 y). At the last radiological follow-up, no patient had modifications of the MI or evidence of diffuse leptomeningeal dissemination . MicroscThirty-two patients were included in Group 2 see . The medThe median age at surgery was 9.0 y. Anterior temporal lobectomy was performed in nine patients; the other 23 patients had lesionectomy. Postsurgical brain MRI revealed persistence of less than 10% of operated lesion in five patients; the thalamic part of the lesion was not resected in two patients; and one patient had other lesions (neurofibromatosis) that were not resected.The median follow-up was 4.0 y (IQR 3.0–5.0 y). Microscopic examination revealed FCD IIIb in eight patients with FCD type I associated to LEATs ; the tumor was isolated in the other 24 patients . Seizure outcome was classified as Engel class Ia in 30 patients.Age at seizure onset was the main difference between the groups, being before the first year of life in the main for patients with MI, while generally being above school age (but still within the first decade) for patients without MI. Concordant with the earlier onset of epilepsy, surgery too was at a significantly earlier age for patients with MI; however, duration of epilepsy was similar between the two groups .The tumor was located exclusively in the temporo-mesial structures for the five patients with MI, while nearly half (14/32) of patients without MI had an extratemporal location for LEATs. The two groups were similar with respect to drug resistance, proportion of complete surgical resection, and seizure outcome .We have described a single center cohort of pediatric patients who were operated for resection of LEATs between 2011 and 2017. Procedurally, all patients underwent the same presurgical clinical, electrophysiological, and brain MRI assessments. Our cohort was limited to patients with follow-up for at least 2 y. Post-surgery, 32 of 37 patients were both drug- and seizure-free, which further supports surgery as an effective treatment for LEATs in the pediatric population . MoreoveWithin our cohort with LEATs, we have described a small homogenous group of five patients (Group 1) with focal MI visible on MRI and confirmed by a neuropathologist. In these patients, brain MRI indicated mesio-temporal LEAT localization and gadolinium enhancement of only the adjacent meninges. Enhancement did not progress in the biannual follow-up MRIs, and we saw no sign of diffuse leptomeningeal spread. The clinical significance of focal MI in patients with LEATs has not yet been described in previous published articles and still remains unclear .Case reports have been sporadic, but some have suggested a secondary non-localized leptomeningeal spread with LEATs ,15; howeCompared with the 32 patients without MI (Group 2), the main clinical and radiological differences of Group 1 are an earlier age at seizure onset (within 14 months of birth) and the exclusive localization of lesion within the mesial structures of the temporal lobe. Possibility of an early development of epilepsy in LEATs was already highlighted with other case series , but thoOur patient groups were similar in both having a relatively short duration of epilepsy and a high prevalence of pharmaco-sensitivity prior to surgery, suggesting that both groups were evaluated and undertook surgery relatively early in the development of their conditions. In the previously published series, most patients had surgery only after a more prolonged epilepsy duration and after diagnosis of drug-resistant epilepsy . Our stuThe decision to proceed early with surgery with our patients, even with drug-sensitive epilepsy, considered both the obvious anatomo-electro-clinical association and the absence of functional constraints. The decision for operating specifically prior to drug-resistance was taken mainly due to (a) LEATs-associated epilepsy is characterized by a high prevalence of drug-resistance ,27; (b) All our patients who were seizure-free were also drug-free, and early reduction of drugs was not associated with worse seizure outcome when compared with previously reported patients in whom withdrawal of drug was slower . SuccessClearly, our two groups comprise radically different numbers, and this heavily affects our confidence in identifying differences, so larger studies would better address our points. Further limitations are related to the retrospective nature of this study. We also do not report any of the recently recognized new low-grade tumoral entities, such as polymorphous low-grade neuroepithelial tumor of the young (PLNTY) and multinodular and vacuolating neuronal tumor (MVNT), which have been described after the end of our review period ,30.All patients had an overall excellent postsurgical outcome in our cohort of pediatric patients who underwent surgery for epilepsy associated with LEATs. Presence of local MI was associated with an early onset of epilepsy but did not change the favorable long-term oncological and epileptological outcomes. Future long-term studies are needed to better clarify the prognosis of patients with LEATs associated with focal MI.