The factorial structure and psychometric properties of the Committed Action Questionnaire (CAQ-8) in a Portuguese clinical sample with eating disorders.

The Committed Action Questionnaire (CAQ-8) is an instrument developed to measure committed action, an adaptive psychological process. The main goal in the current study was to confirm the factorial structure of the Portuguese version of the CAQ-8 in a transdiagnostic clinical sample of participants diagnosed with an eating disorder (ED). Participants were 102 female outpatients (Mage = 28.1, SD = 10.6; MBMI = 20.0, SD = 5.5) recruited from a clinical setting specialized in the treatment of ED. Confirmatory factor analysis (CFA) was used to confirm the CAQ-8's factorial structure. Both first- and second-order models revealed adequate goodness-of-fit indices (e.g. χ2 /df = 1.545, p = .06; SRMR = 0.049; RMSEA = 0.073; CFI/TLI > 0.95). A moderation model revealed that the conditional effect of weight, shape and eating concerns on experiential avoidance was significantly moderated by increased levels of committed action, F(3, 97) = 23.79, p < .001, accounting for 42% of the final variance. The present study supports the usefulness of the CAQ-8 as a measure of levels of committed action with patients diagnosed with an ED.

The impact of COVID-19 lockdown on disordered eating behaviors: the mediation role of psychological distress.

PURPOSE: This study aimed to explore the early associations between the experienced psychosocial impact of the COVID-19 pandemic crisis during lockdown, depressive symptomatology, anxiety/stress levels, and disordered eating behaviors in adults during a first COVID-19 lockdown period. METHODS: This was a community-based cross-sectional study assessing 254 Portuguese adults (82.7% women; 35.82 ± 11.82 years) 1 week after the end of the first mandatory COVID-19 lockdown in Portugal. An online survey was conducted to evaluate psychological distress, disordered eating, and psychosocial impact of the COVID-19 pandemic. Pearson correlations and Structural Equation Modeling were performed. RESULTS: Participants reported the presence of meal skipping (52.8%), grazing eating behavior (80.9%), overeating (81.0%), loss of control over eating (47.2%), and binge eating episodes (39.2%) during lockdown. Structural equation modeling analyses, controlling for age and sex, indicated that there was a significant indirect effect of the experienced psychosocial impact of COVID-19 pandemic on disordered eating behaviors mediated through psychological distress. CONCLUSION: The psychosocial impact of the COVID-19 pandemic crisis may lead to disordered eating, and this relation may occur through the elevation of psychological distress. These findings can be used to inform interventions, to enhance mental health and manage disordered eating during similar future situations. Level of evidence V: cross-sectional descriptive study.

A preliminary study on the psychosocial impact of COVID-19 lockdown in post-bariatric surgery women: the importance of eating behavior, health care access, and social support.

This study aims to characterize the psychosocial impact of COVID-19 lockdown for post-bariatric surgery (≥ 36 months) women and its association with disordered eating and psychological distress. The medium to long-time follow up is a period of increased susceptibility for poorer weight outcomes which might be triggered by the lockdown. Twenty-four participants responded to an online questionnaire and a telephone interview. About half (n = 14; 58.3%) reported perceived weight gain during the lockdown, 13 (54.1%) limited access to social support, and 12 (50%) limited access to medical care. Co-habiting with a higher number of persons during lockdown was associated with fewer difficulties in dealing with emotionally activating situations, less fear of gaining weight, less fear of losing control over eating, and less disordered eating. The global perceived psychosocial impact of lockdown was significantly correlated with difficulties in dealing with emotionally activating situations and stress symptoms. Results highlight the need to monitor post-bariatric patients, facilitate health care access, and promote social support during the lockdown period. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12144-021-01529-6.

Eating Disorder Examination - Questionnaire short forms: A comparison.

OBJECTIVE: The Eating Disorder Examination - Questionnaire (EDE-Q) is a widely used self-report measure of eating-disordered behaviors and attitudes. Recent studies utilizing confirmatory factor analyses (CFA) have proposed alternative and shorter forms. The aim of this study was to compare the full-length version of the EDE-Q and several proposed short forms (7-item, 8-item, and 18-item) in terms of their psychometric properties, including concurrent, convergent and discriminant validity, factor structure, and sensitivity to change. METHODS: Participants from two eating-disorder clinical samples (N = 175 and 38) and from a nonclinical sample (N = 3,413) completed a battery of measures, including the Portuguese version of the EDE-Q. Analyses compared psychometric properties of the available short forms of the EDE-Q among each other. RESULTS: All forms of the EDE-Q showed good internal consistency values, correlated highly among each other (r > .90) and with different measures of eating psychopathology (r > .80). All EDE-Q forms were able to distinguish between cases and controls with moderate-to-high accuracy and were sensitive to change. CFA failed to support the proposed factor structure for all the EDE-Q forms, except for the 7-item form. DISCUSSION: The present study provides empirical background for choosing between different forms of the EDE-Q. Findings indicate that for nonclinical and for clinical research, including studies of treatment change and outcome, the short forms of the EDE-Q can be used. A shorter version is a viable alternative when less time-consuming alternatives are needed, such to quickly screen for eating-disorder psychopathology or to perform session-by-session treatment monitoring.

The utility of DSM-5 indicators of loss of control eating for the bariatric surgery population.

OBJECTIVES: This study investigated the utility of DSM-5 indicators of loss of control (LOC) eating in adult bariatric surgery patients who presented with binge-eating episodes. METHODS: Participants (all women) were 40 preoperative and 28 postoperative bariatric surgery patients reporting objective binge eating (OBE), 46 preoperative and 52 postoperative with subjective binge-eating (SBE), 53 bulimia nervosa (BN) controls, and 34 binge-eating disorder (BED) controls. Face-to-face Eating Disorder Examination interviews and questionnaires were administered. ANOVA, T-test, χ 2 , and regressions compared the groups in terms of LOC indicators endorsed and to explain disordered eating psychopathology. RESULTS: The indicator most commonly reported by bariatric patients with OBE was "feeling disgusted" (90% and 75% of pre- and postoperative groups), and the least endorsed was "eating alone" (40 and 28.6%). These indicators were reported by >84.9% of the BN and BED. Bariatric patients (pre- or post-surgery) with OBE only reported a higher number of indicators than patients with SBE only (t(150) = 2.34, p = .021). A higher number of indicators reported were associated with increased eating-related psychopathology (F(1,134) = 31.06, p < .001), but only for the post-surgery patients. CONCLUSIONS: The LOC indicators proposed by DSM-5 need to be refined or revised for the bariatric population. Highlights Bariatric patients endorse fewer LOC indicators than BN or BED during a binge-eating episode. Some of the DSM-5 LOC indicators may not be suited to assess episodes of loss of control eating among bariatric patients. The Higher the number of LOC indicators reported, the higher the eating-related psychopathology.

Validation of the Portuguese version of the Clinical Impairment Assessment (CIA) in eating disorders' patients.

PURPOSE: The purpose of the study was to assess the psychometric properties of the Portuguese version of the Clinical Impairment Assessment (CIA) in eating disorders (ED) patients. METHOD: The CIA is a 16-item brief self-reported instrument developed to assess psychosocial impairment secondary to EDs. The CIA was administered to a clinical sample of 237 women with EDs and a college sample of 196 women. The clinical sample completed the Eating Disorders Examination Questionnaire, the Beck Depression Inventory and the Outcome-45 Questionnaire. Reliability, confirmatory factor analysis, validity, and clinically significant change were calculated. RESULTS: Confirmatory factor analysis validated the original 3-factor structure showing an adequate model fit. CIA showed good psychometric properties with high internal consistency, good convergent validity with the EDE-Q, the OQ-45, and the BDI. For divergent validity, participants CIA scores in the clinical sample were significantly higher than in the non-clinical sample. ROC curve analysis provided a cutoff of 15. For known-groups validity participants' scoring above CIA cutoff reported significantly higher CIA scores. In addition, non-underweight participants and participants reporting the presence of dysfunctional ED behaviors had significantly higher CIA scores. Finally, for clinically significant change, a reliable change index of 5 points was obtained to consider a reliable change in the CIA global score. CONCLUSIONS: Our findings support the validity and clinical utility of the CIA as a good self-report measure to be used in both clinical and research settings. LEVEL OF EVIDENCE: Level V. Cross-sectional descriptive study.

Perceived social support before and after bariatric surgery: association with depression, problematic eating behaviors, and weight outcomes.

PURPOSE: Engaging in a healthy lifestyle after bariatric surgery is essential to optimize and sustain weight loss in the long term. There is promising evidence that social support of patients who undergo bariatric surgery plays an important role in promoting a better quality of life and adherence to the required behavioral changes and medical appointments. This study sought to investigate: (a) if post-operative patients experience different levels of perceived social support compared to pre-operative patients; (b) correlations between perceived social support, depression, disordered eating, and weight outcomes; (c) if social support is a moderator between psychological distress, and disordered eating behavior and weight outcomes. METHODS: A group of 65 patients assessed pre-surgery and another group of 65 patients assessed post-surgery (M = 26.12; SD 7.97 months since surgery) responded to a set of self-report measures assessing social support, eating disorder psychopathology, disordered eating, and depression. RESULTS: Greater social support was associated with lower depression, emotional eating, weight and shape concerns, and greater weight loss in pre- and post-surgery groups. Social support was found to be a moderator between different psychological/weight variables but only for the post-surgery group: the relation between depression and eating disorder psychopathology or weight loss was significant for patients scoring medium to high level is social support; the relation between grazing and weight regain was significant for patients scoring medium to low levels of social support. CONCLUSIONS: The associations found between perceived social support and depression, disordered eating and weight outcomes highlight the importance of considering and working with the social support network of patients undergoing bariatric surgery to optimize treatment outcomes. Level of Evidence  Level III: case-control study.

Eating Disorder-15 (ED-15): Factor structure, psychometric properties, and clinical validation.

OBJECTIVE: The purpose of the current study was to explore the factor structure and psychometric properties of the Portuguese version of the Eating Disorder-15 (ED-15), as well as to establish cutoff scores and normative data for the Portuguese version. METHODS: Participants from a nonclinical sample (N = 860) and an eating disorders clinical sample (N = 260) were invited to complete a set of questionnaires, including the Portuguese version of the ED-15. RESULTS: The first-order two-factor structure originally proposed by the ED-15 authors was endorsed through a confirmatory factor analysis (χ2 /df = 2.610; standardized root-mean-square residual = 0.0325; root-mean-square error of approximation = 0.079; Tucker-Lewis index/goodness-of-fit index/incremental fit index > 0.95). Items revealed adequate construct validity (λ = .54-.90; R2  = .29-.81). The ED-15 revealed excellent internal consistency (α = .91) and temporal stability (intraclass correlation coefficient = .92; 95% CI [.84-.95]). Normative data for the ED-15 were provided. The ED-15 demonstrated acceptable concurrent and convergent validity. Receiver operating characteristic analysis revealed that the ED-15 total score accurately discriminates between participants with and without an eating disorder (area under de curve = .80; SE = .017; p ≤ .001; 95% CI [.766-.834]). A cutoff score for clinical significance and a reliable change index were computed. CONCLUSIONS: The Portuguese version of the ED-15 is a reliable and valid measure of eating psychopathology and symptoms, whenever a brief measure is needed, as in session-by-session assessment of therapy progress and outcome.

Prevalence of eating disorders and picking/nibbling in elderly women.

OBJECTIVE: The aim of this study was to examine the point prevalence of eating disorders and picking/nibbling in elderly women. METHODS: This was a two-stage epidemiological study that assessed 342 women aged 65-94 years old. In Stage 1, the following screening measures were used to identify possible cases: the Mini-Mental State Examination, to screen and exclude patients with cognitive impairment; Weight Concerns Scale; SCOFF (Sick, Control, One, Fat, Food) Questionnaire; Eating Disorder Examination Questionnaire-dietary restraint subscale; and three questions to screen for picking/nibbling and night eating syndrome. Women selected for Stage 2 (n = 118) were interviewed using the diagnostic items of the Eating Disorder Examination. RESULTS: According to the DSM-5, the prevalence of all eating disorders was 3.25% (1.83-5.7, 95% C.I.). Prevalence of binge-eating disorder was 1.68% (0.82-3.82, 95% C.I.), of other specified feeding or eating disorders was 1.48% (0.63-3.42, 95% C.I.), and of bulimia nervosa 0.3% (.05-1.7, 95% C.I.)]. Binge-eating episodes were reported by 5.62% of women. No cases of anorexia nervosa or night eating syndrome were identified. The prevalence of picking/nibbling was 18.9%. Picking/nibbling was associated with increased body mass index (t(322) = -3.28, p < .001) and binge-eating episodes (χ2 (1) = 5.65, p < .017). DISCUSSION: Prevalence rates of eating disorders on elderly Portuguese women were comparable to those found on young women. Our data support the literature that suggests that binge-eating disorder is particularly prevalent in older adults. Picking/nibbling was the most prevalent eating behavior and we provide further evidence for its association with weight and disordered eating.

Repetitive eating questionnaire [Rep(eat)-Q]: Enlightening the concept of grazing and psychometric properties in a Portuguese sample.

BACKGROUND/OBJECTIVE: Grazing has been associated with poor weight loss or weight regain in obese patients undergoing bariatric surgery, but research remains scarce and complicated by the use of different non-validated measures. The aim of this paper is to describe the validation of the Rep(eat)-Q, a self-report measure developed to assess grazing, and investigates its relationship with BMI and psychopathology. SUBJECTS/METHODS: 1223 university students and community participants (non-clinical; Study A) and 154 pre-bariatric and 84 post-bariatric patients (Study B) completed a set of self-report measures, including the Rep(eat)-Q (worded in Portuguese), to assess disordered eating, depression, anxiety, stress and impulsivity. Exploratory and confirmatory factor analyses tested the factor structure; internal consistency construct, convergent and divergent validity were also tested. RESULTS: The Rep(eat)-Q scales showed good internal consistency (α ≥ 0.849) and temporal stability (rsp = 0.824, p < 0.000). Factor analyses generated two subscales: compulsive grazing and repetitive eating. Significant correlations (p < 0.05) were found between the Rep(eat)-Q and BMI in the non-clinical population and weight loss and weight regain in the bariatric sample. Generally, the correlations with psychological distress were weak (rsp < 0.4). Strong and significant (rsp≥0.4; p's < 0.05) correlations were found between compulsive grazing and eating disorder psychopathology. Repetitive eating subscale was inversely correlated with cognitive restraint (rsp -0.321, p < 0.05) and directly correlated with uncontrolled eating and emotional eating (rsp = 0.754; rsp = 0.691; p < 0.05). DISCUSSION/CONCLUSION: The Rep(eat)-Q is a valid measure to assess grazing in non-clinical and in bariatric surgery populations. Grazing can be conceptualized on the spectrum of disordered eating behavior, and appears associated with loss of control over eating. Considering the link between grazing and weight outcomes, the Rep(eat)-Q represents a necessary strategy for the systematic screening of grazing.

Early response as a predictor of success in guided self-help treatment for bulimic disorders.

The aims of this study were to investigate the number of sessions and time required for a clinical meaningful symptomatic change with a guided self-help treatment and to assess the predictive value of early response and other potential predictors of end-of-treatment clinical status. Participants were 42 patients with a diagnosis of bulimia nervosa or ED not otherwise specified. Survival analyses (Kaplan-Meier) were performed to estimate the median time required to attain a 51% reduction in bulimic symptoms. Logistic regression was used to assess predictors of symptom remission. Results showed that the median time to achieve a 51% reduction in binge and purge frequencies was 3.68 and 3.77, respectively. This change occurred at session 3 for 50% of the participants. Early response was the most significant predictor of binge eating remission. No pretreatment predictors of time to achieve early response were found. These results have implications for allocating treatment resources in a stepped-care intervention model.

Eating disorder examination questionnaire: psychometric properties and norms for the Portuguese population.

OBJECTIVE: The first aim of the current study was to establish general population norms for the Portuguese version of the Eating Disorder Examination Questionnaire (EDE-Q) in a large community sample of female adolescents and young women, as well, for a diverse Eating Disorder (ED) clinical sample, and for women with obesity without an ED. A second aim of the study was to assess the discriminant validity of the EDE-Q and providing cut-off scores for the total scale and subscales. METHOD: A sample of female adolescents and young women (N = 4091) from the general population, 416 women who met diagnostic criteria for an ED and 138 women seeking obesity treatment completed the EDE-Q. RESULTS: Norms for the EDE-Q global subscale were provided. Within the community sample, norms were provided for both high school and college samples. Receiver operating characteristic analysis showed that the EDE-Q total score accurately discriminate between participants with and without an ED. Current norm contributes to the clinical utility of the EDE-Q, providing both a cut-off score and reliable change index. Results showed that the EDE-Q is a reliable instrument, but the theorized four subscales structure was not supported by an explorative factor analysis. CONCLUSION: Results will help both researchers and clinicians interpreting the EDE-Q scores and to establish comparison with data produced in different countries.

Neurotoxic Astrocytes Directly Converted from Sporadic and Familial ALS Patient Fibroblasts Reveal Signature Diversities and miR-146a Theragnostic Potential in Specific Subtypes.

A lack of stratification methods in patients with amyotrophic lateral sclerosis (ALS) is likely implicated in therapeutic failures. Regional diversities and pathophysiological abnormalities in astrocytes from mice with SOD1 mutations (mSOD1-ALS) can now be explored in human patients using somatic cell reprogramming. Here, fibroblasts from four sporadic (sALS) and three mSOD1-ALS patients were transdifferentiated into induced astrocytes (iAstrocytes). ALS iAstrocytes were neurotoxic toward HB9-GFP mouse motor neurons (MNs) and exhibited subtype stratification through GFAP, CX43, Ki-67, miR-155 and miR-146a expression levels. Up- (two cases) and down-regulated (three cases) miR-146a values in iAstrocytes were recapitulated in their secretome, either free or as cargo in small extracellular vesicles (sEVs). We previously showed that the neuroprotective phenotype of depleted miR-146 mSOD1 cortical astrocytes was reverted by its mimic. Thus, we tested such modulation in the most miR-146a-depleted patient-iAstrocytes (one sALS and one mSOD1-ALS). The miR-146a mimic in ALS iAstrocytes counteracted their reactive/inflammatory profile and restored miR-146a levels in sEVs. A reduction in lysosomal activity and enhanced synaptic/axonal transport-related genes in NSC-34 MNs occurred after co-culture with miR-146a-modulated iAstrocytes. In summary, the regulation of miR-146a in depleted ALS astrocytes may be key in reestablishing their normal function and in restoring MN lysosomal/synaptic dynamic plasticity in disease sub-groups.

Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin.

Microglia are the main players of the brain immune response. They act as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglial phagocytic phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings revealed that conditioned media derived from UCB-treated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to curtail microglial over activation. Conditioned medium from UCB-challenged neurons, although down-regulating tumor necrosis factor-α and interleukin-1ß promoted the release of interleukin-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared with UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons intensified the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia were able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise.

Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation.

Microglia constitute the brain's immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1ß, and IL-6, upregulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target.

N-methyl-aspartate receptor and neuronal nitric oxide synthase activation mediate bilirubin-induced neurotoxicity.

Hyperbilirubinemia may lead to neurotoxicity and neuronal death. Although the mechanisms of nerve cell damage by unconjugated bilirubin (UCB) appear to involve a disruption of the redox status and excitotoxicity, the contribution of nitric oxide (NO·) and of N-methyl-D-aspartate (NMDA) glutamate receptors is unclear. We investigated the role of NO· and NMDA glutamate receptors in the pathways of nerve cell demise by UCB. Neurons were incubated with 100 micromol/L UCB, in the presence of 100 micromol/L human serum albumin for 4 h at 37ºC, alone or in combination with N-ω-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of neuronal nitric oxide synthase [nNOS]), hemoglobin (an NO· scavenger) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (an NMDA-receptor antagonist). Exposure to UCB led to increased expression of nNOS and production of both NO· and cyclic guanosine 3',5'-monophosphate (cGMP), along with protein oxidation and depletion of glutathione. These events concurred for cell dysfunction and death and were counteracted by L-NAME. Moreover, the UCB-induced loss of neuronal viability was abolished by hemoglobin, whereas the activation of nNOS and production of both NO· and cGMP were counteracted by MK-801, resulting in significant protection from cell dysfunction and death. These results reinforce the involvement of oxidative stress by showing that nerve cell damage by UCB is mediated by NO· and therefore is counteracted by NO· inhibitors or scavengers. Our findings strongly suggest that the activation of nNOS and neurotoxicity occur through the engagement of NMDA receptors. These data reveal a role for overstimulation of glutamate receptors in mediating oxidative damage by UCB.

Targeting gliomas with triazene-based hybrids: Structure-activity relationship, mechanistic study and stability.

Herein we report novel hybrid compounds based on valproic acid and DNA-alkylating triazene moieties, 1, with therapeutic potential for glioblastoma multiforme chemotherapy. We identified hybrid compounds 1d and 1e to be remarkably more potent against glioma and more efficient in decreasing invasive cell properties than temozolomide and endowed with chemical and plasma stability. In contrast to temozolomide, which undergoes hydrolysis to release an alkylating metabolite, the valproate hybrids showed a low potential to alkylate DNA. Key physicochemical properties align for optimal CNS penetration, highlighting the potential of these effective triazene based-hybrids for enhanced anticancer chemotherapy.

Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs). Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase 1 (mSOD1) from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs) that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN)-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A) and extended the study into their derived exosomes (mSOD1 exosomes). Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells [either transfected with either wild-type (wt) human SOD1 or mutant SOD1(G93A)] showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP-2, and MMP-9 activation, as well as upregulation of IL-1ß, TNF-α, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number of senescent-like positive N9 cells. Data suggest that miR-124 is translocated from the mSOD1 MNs to exosomes, which determine early and late phenotypic alterations in the recipient N9-microglial cells. In conclusion, modulation of the inflammatory-associated miR-124, in mSOD1 NSC-34 MNs, with potential benefits in the cargo of their exosomes may reveal a promising therapeutic strategy in halting microglia activation and associated effects in MN degeneration.

Key Aging-Associated Alterations in Primary Microglia Response to Beta-Amyloid Stimulation.

Alzheimer's disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-ß (Aß) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive Aß production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM Aß1-42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after Aß treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated ß-galactosidase (SA-ß-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by Aß in 2 DIV microglia. Additionally, Aß downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after Aß treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with Aß, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Dipeptidyl Vinyl Sulfone as a Novel Chemical Tool to Inhibit HMGB1/NLRP3-Inflammasome and Inflamma-miRs in Aß-Mediated Microglial Inflammation.

Rapid microglial activation and associated inflammatory pathways contribute to immune-defense and tissue repair in the central nervous system (CNS). However, persistent activation of these cells will ultimately result in vast production of pro-inflammatory mediators and other neurotoxic factors, which may induce neuronal damage and contribute to chronic neurodegenerative diseases, as Alzheimer's disease (AD). Therefore, small molecules with immunomodulatory effects on microglia may be considered as potential tools to counteract their proinflammatory phenotype and neuroimmune dysregulation in such disorders. Indeed, reducing amyloid-ß (Aß)-induced microglia activation is believed to be effective in treating AD. In this study, we investigated whether dipeptidyl vinyl sulfone (VS) was able to attenuate Aß-mediated inflammatory response using a mouse microglial (N9) cell line and a solution containing a mixture of Aß aggregates. We show that low levels of VS are able to prevent cell death while reducing microglia phagocytosis upon Aß treatment. VS also suppressed Aß-induced expression of inflammatory mediators in microglia, such as matrix metalloproteinase (MMP)-2 and MMP-9, as well as high-mobility group box protein-1 (HMGB1), nod-like receptor protein 3 (NLRP3)-inflammasome, and interleukin (IL)-1ß. Interestingly, increased expression of the two critical inflammation-related microRNAs (miR)-155 and miR-146a in microglia upon Aß treatment was also prevented by VS coincubation. Taken together, VS emerges as a potential new therapeutic strategy worthy of further investigation in improved cellular and animal models of AD.