Study protocol for two stepped-wedge interventional trials evaluating the effects of holistic information technology-based patient-oriented management in older multimorbid patients with cancer: The GERONTE trials.

INTRODUCTION: Current hospital-based care pathways are generally single-disease centred. As a result, coexisting morbidities are often suboptimally evaluated and managed, a deficiency becoming increasingly apparent among older patients who exhibit heterogeneity in health status, functional abilities, frailty, and other geriatric impairments. To address this issue, our study aims to assess a newly developed patient-centred care pathway for older patients with multimorbidity and cancer. The new care pathway was based on currently available evidence and co-designed by end-users including health care professionals, patients, and informal caregivers. Within this care pathway, all healthcare professionals involved in the care of older patients with multimorbidity and cancer will form a Health Professional Consortium (HPC). The role of the HPC will be to centralise oncologic and non-oncologic treatment recommendations in accordance with the patient's priorities. Moreover, an Advanced Practice Nurse will act as case-manager by being the primary point of contact for the patient, thus improving coordination between specialists, and by organising and leading the consortium. Patient monitoring and the HPC collaboration will be facilitated by digital communication tools designed specifically for this purpose, with the added benefit of being customisable for each patient. MATERIALS AND METHODS: The GERONTE study is a prospective international, multicentric study consisting of two stepped-wedge trials performed at 16 clinical sites across three European countries. Each trial will include 720 patients aged 70 years and over with a new or progressive cancer (breast, lung, colorectal, prostate) and at least one moderate or severe multimorbidity. The patients in the intervention group will receive the new care pathway whereas patients in the control group will receive usual oncologic care. DISCUSSION: GERONTE will evaluate whether this kind of holistic, patient-oriented healthcare management can improve quality of life (primary outcome) and other valuable endpoints in older patients with multimorbidity and cancer. An ancillary study will assess in depth the socio-economic impact of the intervention and deliver concrete implementation guidelines for the GERONTE intervention care pathway. TRIAL REGISTRATION: FRONE: NCT05720910 TWOBE: NCT05423808.

Emotional Blunting in Depression in the PREDDICT Clinical Trial: Inflammation-Stratified Augmentation of Vortioxetine With Celecoxib.

BACKGROUND: Emotional symptoms are recognized as a key feature in individuals with major depressive disorder. Previously, emotional blunting has been described both as a side effect of antidepressant treatment and as a symptom of depression. Little is known about the change of emotional blunting during antidepressant treatment. METHODS: The PREDDICT trial is a randomized, placebo-controlled, 6-week trial on the augmentation of vortioxetine with the anti-inflammatory agent celecoxib or placebo. Presently we report on exploratory secondary outcomes of changes in emotional blunting in depression assessed with the Oxford Depression Questionnaire (ODQ) total score and subscores from baseline to 8-week, 3-month, and 6-month follow-up assessments. RESULTS: In the whole group, there was a significant improvement in the ODQ total score and all subscores after 8 weeks. After stratification of participants into the treatment groups, the ODQ total score as well as subscores related to emotional blunting as a symptom of depression (reduction in positive emotions, not caring) improved between baseline and all follow-up time points in both treatment groups. Changes in subscores considered as a side effect of antidepressants (general reduction in emotions, emotional detachment) were inconclusive in both treatment groups. Overall, the placebo-augmented group showed slightly better results in changes of emotional blunting scores than the celecoxib group as did those with elevated inflammation at screening, regardless of treatment group. CONCLUSIONS: This analysis suggests favorable effects of vortioxetine on emotional blunting in both short- and long-term course. The beneficial impact of vortioxetine on emotional blunting was weaker in celecoxib-augmented patients compared with placebo, possibly due to pharmacokinetic interactions. Clinical Trials Registration: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12617000527369p. Registered on 11 April 2017, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617000527369p.

Efficacy and safety of a 4-week course of repeated subcutaneous ketamine injections for treatment-resistant depression (KADS study): randomised double-blind active-controlled trial.

BACKGROUND: Prior trials suggest that intravenous racemic ketamine is a highly effective for treatment-resistant depression (TRD), but phase 3 trials of racemic ketamine are needed. AIMS: To assess the acute efficacy and safety of a 4-week course of subcutaneous racemic ketamine in participants with TRD. Trial registration: ACTRN12616001096448 at www.anzctr.org.au. METHOD: This phase 3, double-blind, randomised, active-controlled multicentre trial was conducted at seven mood disorders centres in Australia and New Zealand. Participants received twice-weekly subcutaneous racemic ketamine or midazolam for 4 weeks. Initially, the trial tested fixed-dose ketamine 0.5 mg/kg versus midazolam 0.025 mg/kg (cohort 1). Dosing was revised, after a Data Safety Monitoring Board recommendation, to flexible-dose ketamine 0.5-0.9 mg/kg or midazolam 0.025-0.045 mg/kg, with response-guided dosing increments (cohort 2). The primary outcome was remission (Montgomery-Åsberg Rating Scale for Depression score ≤10) at the end of week 4. RESULTS: The final analysis (those who received at least one treatment) comprised 68 in cohort 1 (fixed-dose), 106 in cohort 2 (flexible-dose). Ketamine was more efficacious than midazolam in cohort 2 (remission rate 19.6% v. 2.0%; OR = 12.1, 95% CI 2.1-69.2, P = 0.005), but not different in cohort 1 (remission rate 6.3% v. 8.8%; OR = 1.3, 95% CI 0.2-8.2, P = 0.76). Ketamine was well tolerated. Acute adverse effects (psychotomimetic, blood pressure increases) resolved within 2 h. CONCLUSIONS: Adequately dosed subcutaneous racemic ketamine was efficacious and safe in treating TRD over a 4-week treatment period. The subcutaneous route is practical and feasible.

Cognitive improvement in patients with major depressive disorder after personalised multi domain training in the CERT-D study.

The CERT-D program offers a new treatment approach addressing disturbed cognitive and psychosocial functioning in major depressive disorder (MDD). The current analysis of a randomised controlled trial (RCT) comprises two objectives: Firstly, evaluating the program's efficacy of a personalised versus standard treatment and secondly, assessing the treatment's persistence longitudinally. Participants (N = 112) were randomised into a personalised or standard treatment group. Both groups received 8 weeks of cognitive training, followed by a three-month follow-up without additional training. The type of personalised training was determined by pre-treatment impairments in the domains of cognition, emotion-processing and social-cognition. Standard training addressed all three domains equivalent. Performance in these domains was assessed repeatedly during RCT and follow-up. Treatment comparisons during the RCT-period showed benefits of personalised versus standard treatment in certain aspects of social-cognition. Conversely, no benefits in the remaining domains were found, contradicting a general advantage of personalisation. Exploratory follow-up analysis on persistence of the program's effects indicated sustained intervention outcomes across the entire sample. A subsequent comparison of clinical outcomes between personalised versus standard treatment over a three-month follow-up period showed similar results. First evidence suggests that existing therapies for MDD could benefit from an adjunct administration of the CERT-D program.

Exploratory Analysis of the Effects of Celecoxib on Cognitive Function in Vortioxetine-Treated Patients With Major Depressive Disorder in the PREDDICT Study: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Objective: Major depressive disorder (MDD) remains difficult to treat, with many patients resistant to existing treatments or experiencing relapse. Cognitive dysfunction is associated with more severe clinical outcomes. Vortioxetine has shown efficacy in remediating depression-associated cognitive impairment. Anti-inflammatory augmentation of antidepressants is a new strategy in treating depression and has not previously been assessed for effects on cognition in depression.Methods: Exploratory analyses were performed on secondary outcome cognitive data from the PREDDICT parallel-group, randomized, double-blind, placebo-controlled trial at the University of Adelaide (Australia). Participants (N = 119) with MDD (validated with Mini-International Neuropsychiatric Interview for DSM-IV) were treated with vortioxetine and celecoxib or vortioxetine and placebo for 6 weeks between December 2017 and April 2020. Measures included objective cognition composite scores (Choice Reaction Time, N-Back, Digit Symbol Substitution Test, Trail Making Task Part B), subjective cognition scores (Perceived Deficits Questionnaire), and global cognition composite scores (combined objective and subjective scores) derived from the THINC integrated tool (THINC-it). High-sensitivity C-reactive protein (hsCRP) measured at baseline and week 6 was tested for a predictive relationship with cognitive outcomes.Results: Cognition composite scores demonstrated improvement by week 6 in both treatment groups. However, there was no significant interaction between change over time and treatment group. HsCRP did not have a significant relationship with any tested cognition measures.Conclusions: Both treatment groups showed a reduction in depression-associated cognitive impairment. No superior clinical effect was reported for the add-on celecoxib group. HsCRP was modulated by neither vortioxetine nor add-on celecoxib.Trial Registration: ANZCTR identifier: ACTRN12617000527369.

Inflammation-stratified augmentation of vortioxetine with celecoxib: Results from a double-blind, randomized, placebo-controlled trial in major depressive disorder.

Low-grade inflammation is considered as a pathophysiological mechanism in a subtype of patients with major depressive disorder (MDD). Anti-inflammatory drugs have shown efficacy in treating MDD. However, it remains unclear how to identify suitable patients for anti-inflammatory treatment of depression. This study investigates the predictive value of pre-treatment high-sensitivity C-Reactive Protein (hsCRP) stratification on the outcome of celecoxib augmentation of vortioxetine. The PREDDICT study was conducted as a randomized, double-blind, placebo-controlled 6-week trial on augmentation of vortioxetine with celecoxib between December 2017 and April 2020 at the University of Adelaide (Australia). The present analysis focusses on the question of whether the pre-treatment hsCRP measurement and stratification of patients to depression with inflammation (hsCRP >3 mg/L) or without inflammation (hsCRP ≤3 mg/L) has an impact on the outcome of anti-inflammatory treatment with celecoxib. A total of n = 119 mostly treatment-resistant MDD patients with moderate to severe symptomatology were recruited in the trial. There was no effect of treatment group (celecoxib or placebo), pre-treatment hsCRP strata (with/without inflammation), or interaction between the two terms on treatment outcome. The results of the current analysis do not support the hypothesis that pre-treatment hsCRP level is predictive for response to anti-inflammatory treatment with celecoxib in MDD patients. Further research is needed to identify appropriate biomarkers for the prediction of anti-inflammatory treatment outcome in depression. CLINICAL TRIALS REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12617000527369p. Registered on 11 April 2017, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617000527369p.

Basal autophagic flux measured in blood correlates positively with age in adults at increased risk of type 2 diabetes.

Preclinical data show that autophagy delays age-related disease. It has been postulated that age-related disease is-at least in part-caused by an age-related decline in autophagy. However, autophagic flux has never been measured in humans across a spectrum of aging in a physiologically relevant context. To address this critical gap in knowledge, the objective of this cross-sectional observational study was to measure basal autophagic flux in whole blood taken from people at elevated risk of developing type 2 diabetes and correlate it with chronological age. During this study, 119 people were recruited and five people were excluded during sample analysis such that 114 people were included in the final analysis. Basal autophagic flux measured in blood and correlations with parameters such as age, body weight, fat mass, AUSDRISK score, blood pressure, glycated hemoglobin HbA1c, blood glucose and insulin, blood lipids, high-sensitivity C-reactive protein, plasma protein carbonylation, and plasma ß-hexosaminidase activity were analysed. Despite general consensus in the literature that autophagy decreases with age, we found that basal autophagic flux increased with age in this human cohort. This is the first study to report measurement of basal autophagic flux in a human cohort and its correlation with age. This increase in basal autophagy could represent a stress response to age-related damage. These data are significant not only for their novelty but also because they will inform future clinical studies and show that measurement of basal autophagic flux in a human cohort is feasible.

Human monocyte-derived microglia-like cell models: A review of the benefits, limitations and recommendations.

In the last few decades, mounting evidence has highlighted that microglia have crucial roles in both health and disease. This has led to a growing interest in studying human microglia in disease-relevant models. However, current models present limitations that can make them unsuitable for moderate throughput studies in human cohorts. Primary human microglia are ethically and technically difficult to obtain and only allow low throughput studies; immortalized cell lines have been shown to differ too greatly from primary human microglia; and induced pluripotent stem cell-derived microglia, although physiologically relevant in most contexts, have limited potential for use in large cohorts of people or for personalised drug screening. In this review, we discuss monocyte-derived microglia-like (MDMi) cells, a model that has been developed and increasingly used in the last decade, using human monocytes isolated from blood samples. We describe the variety of protocols that have been used to develop MDMi cell models and highlight a need for standardization across protocols. We then summarize data that validate MDMi cells as an appropriate model to study human microglia in health and disease. We also present the benefits and limitations of using this approach to study human microglia compared with other microglial models, when used in combination with the relevant downstream applications and with cross-validation of findings in other systems. Finally, we summarize the paradigms in which MDMi models have been used to advance research on microglia in immune-related disease. This review is an important reference for scientists who seek to establish MDMi cells as a microglial model for the advancement of our understanding of microglia in human health and disease.

The Break-Fast study protocol: a single arm pre-post study to measure the effect of a protein-rich breakfast on autophagic flux in fasting healthy individuals.

BACKGROUND: Autophagy is a cellular process that cleanses cells and is particularly important during ageing. Autophagy has been extensively studied in vitro and in animal models and is known to be sensitive to nutrition. However, human data are limited because autophagic flux (autophagic degradative activity) has been challenging to measure in humans. This protocol paper describes the Break-Fast study, in which autophagic flux will be measured using a recently developed blood test, before and after ingestion of whey protein. This aims to determine whether an acute nutritional intervention can change autophagy in humans. METHODS: A minimum of forty healthy participants (both male and female) aged 20-50 years, BMI 18.5-29.9 kg/m2 will be recruited into this single arm pre-post study. Participants will visit the clinic after an overnight fast for a first blood collection after which they will consume a whey protein-rich drink. A second blood collection will be performed 60 minutes after consumption of the drink. The primary outcome is the change in autophagic flux at 60 minutes post drink. Secondary outcomes include changes in blood glucose, autophagy-related proteins and mRNA, plasma hormones (e.g. insulin, C-peptide, adiponectin, GLP-1, GIP, ghrelin), cytokines, amino acids and lipids, protein synthesis, and correlation between molecular cell damage and autophagic flux. DISCUSSION: This study will provide information about whether autophagy responds to nutrients in humans, and if nutritional strategies could be used to treat or prevent autophagy-related diseases such as Alzheimer's disease or cancer. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), anzctr.org.au ACTRN12621001029886. Registered on 5 August 2021.

Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of ß-hexosaminidase) result in accumulation of amyloid-ß (Aß) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for ß-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between ß-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of ß-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

The mTOR-lysosome axis at the centre of ageing.

Age-related diseases represent some of the largest unmet clinical needs of our time. While treatment of specific disease-related signs has had some success (for example, the effect of statin drugs on slowing progression of atherosclerosis), slowing biological ageing itself represents a target that could significantly increase health span and reduce the prevalence of multiple age-related diseases. Mechanistic target of rapamycin complex 1 (mTORC1) is known to control fundamental processes in ageing: inhibiting this signalling complex slows biological ageing, reduces age-related disease pathology and increases lifespan in model organisms. How mTORC1 inhibition achieves this is still subject to ongoing research. However, one mechanism by which mTORC1 inhibition is thought to slow ageing is by activating the autophagy-lysosome pathway. In this review, we examine the special bidirectional relationship between mTORC1 and the lysosome. In cells, mTORC1 is located on lysosomes. From this advantageous position, it directly controls the autophagy-lysosome pathway. However, the lysosome also controls mTORC1 activity in numerous ways, creating a special two-way relationship. We then explore specific examples of how inhibition of mTORC1 and activation of the autophagy-lysosome pathway slow the molecular hallmarks of ageing. This body of literature demonstrates that the autophagy-lysosome pathway represents an excellent target for treatments that seek to slow biological ageing and increase health span in humans.

No evidence for clinical efficacy of adjunctive celecoxib with vortioxetine in the treatment of depression: A 6-week double-blind placebo controlled randomized trial.

Given the role of low-grade inflammation in the pathophysiology of major depressive disorder (MDD), anti-inflammatory strategies may improve treatment outcomes in some patients. However, it is controversial whether they can be used as adjunctive treatments and whether pre-treatment levels of inflammation can predict treatment outcomes. This study was conducted to measure the efficacy of anti-inflammatory augmentation of antidepressant treatment in MDD patients; and to investigate whether treatment response was dependent on baseline inflammation levels. This parallel-group randomised, double-blind, placebo-controlled trial was conducted at the University of Adelaide (Australia). Participants with MDD were randomised to receive vortioxetine with celecoxib or vortioxetine with placebo for six weeks, and baseline blood high sensitivity C reactive protein levels were measured. Primary outcome was change in depressive symptoms (Montgomery-Åsberg Depression Rating Scale) and secondary outcomes included change in cognition (THINC-integrated tool - Codebreaker task) and functioning (Functioning Assessment Short Test) over 6 weeks. There was no evidence of superior efficacy of celecoxib augmentation over placebo on depressive symptom severity, response and remission rates, cognition and psychosocial functioning. There was also no evidence that pre-treatment inflammation levels modified the effect of celecoxib augmentation versus placebo. This observed lack of efficacy of celecoxib add-on does not support the use of celecoxib augmentation of antidepressants in the treatment of MDD in a cohort that mostly comprises treatment-resistant individuals. Additionally, C-reactive protein may not be suitable to predict treatment selection and response in MDD. The study was registered on the Australian New Zealand Clinical Trials Registry: ACTRN12617000527369 (www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617000527369p).

Inhibiting mTOR activity using AZD2014 increases autophagy in the mouse cerebral cortex.

Autophagy is a catabolic process that collects and degrades damaged or unwanted cellular materials such as protein aggregates. Defective brain autophagy has been linked to diseases such as Alzheimer's disease. Autophagy is regulated by the protein kinase mTOR (mechanistic target of rapamycin). Although already demonstrated in vitro, it remains contentious whether inhibiting mTOR can enhance autophagy in the brain. To address this, mice were intraperitoneally injected with the mTOR inhibitor AZD2014 for seven days. mTOR complex 1 (mTORC1) activity was decreased in liver and brain. Autophagic activity was increased by AZD2014 in both organs, as measured by immunoblotting for LC3 (microtubule-associated proteins-1A/1B light chain 3B) and measurement of autophagic flux in the cerebral cortex of transgenic mice expressing the EGFP-mRFP-LC3B transgene. mTOR activity was shown to correlate with changes in LC3. Thus, we show it is possible to promote autophagy in the brain using AZD2014, which will be valuable in tackling conditions associated with defective autophagy, especially neurodegeneration.

Psychological training to improve psychosocial function in patients with major depressive disorder: A randomised clinical trial.

Cognitive and emotional remediation training for depression (CERT-D): a randomised controlled trial to improve cognitive, emotional and functional outcomes in depression The aim of the current study was to evaluate an experimental treatment designed to improve psychosocial function in patients with Major Depressive Disorder (MDD) by reinforcing cognitive, emotional, and social-cognitive abilities. Participants (N = 112) with current or lifetime MDD were recruited to participate in a randomised, blinded, controlled trial. Exclusion criteria included diagnosis of a substance abuse disorder, bipolar disorder organic, eating disorders, or illness which affect cognitive function. The treatment involved repeated cognitive training designed to improve cognitive, emotional, and social-cognitive abilities. In training sessions, the principles of cognitive training were applied across cognitive, emotional, and social domains, with participants completing repeated mental exercises. Exercises included critically analysing interpretations of social interactions (e.g., body language), exploring emotional reactions to stimuli, and completing game-like cognitive training tasks. Training sessions placed great emphasis on the application of trained cognitive, emotional, and social cognitive skills to psychosocial outcomes. Outcomes demonstrated significant improvement in psychosocial function, symptom severity, self-reported cognition, and social-cognition. Our findings demonstrate the efficacy of multi-domain cognitive training to improve psychosocial functioning in individuals with MDD. We suggest that the present treatment could be deployed at a lower cost and with minimal training in comparison to established psychological therapies.

Gut Microbiome Regulation of Autophagic Flux and Neurodegenerative Disease Risks.

The gut microbiome-brain axis exerts considerable influence on the development and regulation of the central nervous system. Numerous pathways have been identified by which the gut microbiome communicates with the brain, falling largely into the two broad categories of neuronal innervation and immune-mediated mechanisms. We describe an additional route by which intestinal microbiology could mediate modifiable risk for neuropathology and neurodegeneration in particular. Autophagy, a ubiquitous cellular process involved in the prevention of cell damage and maintenance of effective cellular function, acts to clear and recycle cellular debris. In doing so, autophagy prevents the accumulation of toxic proteins and the development of neuroinflammation, both common features of dementia. Levels of autophagy are influenced by a range of extrinsic exposures, including nutrient deprivation, infection, and hypoxia. These relationships between exposures and rates of autophagy are likely to be mediated, as least in part, by the gut microbiome. For example, the suppression of histone acetylation by microbiome-derived short-chain fatty acids appears to be a major contributor to upregulation of autophagic function. We discuss the potential contribution of the microbiome-autophagy axis to neurological health and examine the potential of exploiting this link to predict and prevent neurodegenerative diseases.

Clinical Switching Strategies of Various Antidepressants to Vortioxetine in the PREDDICT Trial.

BACKGROUND: Partial response to antidepressant medication as well as relapse and treatment resistance are common in major depressive disorder (MDD). Therefore, for most patients with MDD, there will be a need to consider changing antidepressant medication at some stage during the course of the illness. The PREDDICT study investigates the efficacy of augmenting vortioxetine with celecoxib. METHODS: We describe the method used in the PREDDICT study to change participants, who were already taking antidepressant medication at the time of the screening visit, to vortioxetine. We used a cross-titration to change study participants to vortioxetine. RESULTS: Of a total of 122 study participants who were randomized to receive vortioxetine plus celecoxib or vortioxetine plus placebo at the study baseline visit, 82 were taking antidepressant medication (other than vortioxetine) prior to randomization. These medications were selective serotonin reuptake inhibitors, serotonin noradrenaline reuptake inhibitors, tricyclic antidepressants, mirtazapine, or agomelatine. Eighty of these 82 participants completed the changeover to vortioxetine as well as the study baseline visit. We found side effects were generally mild during this changeover period. In addition, there was a reduction in mean total Montgomery-Åsberg Depression Rating Scale score of 2.5 (SD 6.0) from study baseline to week 2 and a further reduction in mean total Montgomery-Åsberg Depression Rating Scale of 2.5 (SD 5.9) from week 2 to week 4. CONCLUSION: Changing other antidepressants to vortioxetine can be done safely and was generally well-tolerated. However, there are some antidepressant classes, in particular monoamine oxidase inhibitors that require a washout period, which were not represented in this study. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR); ID number 12617000527369p; http://www.anzctr.org.au/ACTRN12617000527369p.aspx.

Measurement of autophagic flux in humans: an optimized method for blood samples.

Autophagic flux is a critical cellular process that is vastly under-appreciated in terms of its importance to human health. Preclinical studies have demonstrated that reductions in autophagic flux cause cancer and exacerbate chronic diseases, including heart disease and the pathological hallmarks of dementia. Autophagic flux can be increased by targeting nutrition-related biochemical signaling. To date, translation of this knowledge has been hampered because there has been no way to directly measure autophagic flux in humans. In this study we detail a method whereby human macroautophagic/autophagic flux can be directly measured from human blood samples. We show that whole blood samples can be treated with the lysosomal inhibitor chloroquine, and peripheral blood mononuclear cells isolated from these samples could be used to measure autophagic machinery protein LC3B-II. Blocking of autophagic flux in cells while still in whole blood represents an important advance because it preserves genetic, nutritional, and signaling parameters inherent to the individual. We show this method was reproducible and defined LC3B-II as the best protein to measure autophagic flux in these cells. Finally, we show that this method is relevant to assess intra-individual variation induced by an intervention by manipulating nutrition signaling with an ex vivo treatment of whole blood that comprised leucine and insulin. Significantly, this method will enable the identification of factors that alter autophagic flux in humans, and better aid their translation in the clinic. With further research, it could also be used as a novel biomarker for risk of age-related chronic disease.Abbreviations: AMPK: AMP-activated protein kinase; ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; CQ: chloroquine; DMSO: dimethyl sulfoxide; DPBS: Dulbecco's phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; KO: knockout; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; PBMCs: peripheral blood mononuclear cells; PMNs: polymorphonuclear cells; RPMI: Roswell Park Memorial Institute; SQSTM1: sequestosome 1; TBST: Tris-buffered saline containing 0.1% (v:v) Tween 20; TEM: transmission electron microscopy.

Comparison of chloroquine-like molecules for lysosomal inhibition and measurement of autophagic flux in the brain.

Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.