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.

Intermittent fasting plus early time-restricted eating versus calorie restriction and standard care in adults at risk of type 2 diabetes: a randomized controlled trial.

Intermittent fasting appears an equivalent alternative to calorie restriction (CR) to improve health in humans. However, few trials have considered applying meal timing during the 'fasting' day, which may be a limitation. We developed a novel intermittent fasting plus early time-restricted eating (iTRE) approach. Adults (N = 209, 58 ± 10 years, 34.8 ± 4.7 kg m-2) at increased risk of developing type 2 diabetes were randomized to one of three groups (2:2:1): iTRE (30% energy requirements between 0800 and 1200 hours and followed by a 20-h fasting period on three nonconsecutive days per week, and ad libitum eating on other days); CR (70% of energy requirements daily, without time prescription); or standard care (weight loss booklet). This open-label, parallel group, three-arm randomized controlled trial provided nutritional support to participants in the iTRE and CR arms for 6 months, with an additional 12-month follow-up. The primary outcome was change in glucose area under the curve in response to a mixed-meal tolerance test at month 6 in iTRE versus CR. Glucose tolerance was improved to a greater extent in iTRE compared with CR (-10.10 (95% confidence interval -14.08, -6.11) versus -3.57 (95% confidence interval -7.72, 0.57) mg dl-1 min-1; P = 0.03) at month 6, but these differences were lost at month 18. Adverse events were transient and generally mild. Reports of fatigue were higher in iTRE versus CR and standard care, whereas reports of constipation and headache were higher in iTRE and CR versus standard care. In conclusion, incorporating advice for meal timing with prolonged fasting led to greater improvements in postprandial glucose metabolism in adults at increased risk of developing type 2 diabetes. ClinicalTrials.gov identifier NCT03689608 .

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.

PICALM regulates cathepsin D processing and lysosomal function.

Degradation and clearance of cellular waste in the autophagic and endo-lysosomal systems is important for normal physiology and prevention of common late-onset diseases such as Alzheimer's disease (AD). Phosphatidylinostol-binding clathrin assembly protein (PICALM) is a robust AD risk factor gene and encodes an endosomal protein clathrin-binding cytosolic protein, reduction of which is known to exacerbate tauopathy. Although PICALM is known to regulate initiation of autophagy, its role in maturation of lysosomal enzymes required for proteolysis has not been studied. We sought to determine the importance of PICALM for cellular degradative function by disrupting exon 1 of PICALM using CRISPR/Cas9 in HeLa cells. PICALM disruption increased numbers of early endosomes. Proteomic analysis of endosome-enriched samples showed that disrupting exon 1 of PICALM increased the abundance of lysosomal enzymes in these organelles, and western blotting revealed disruption to processing and maturation of the lysosomal protease, cathepsin D, and a deficit in autophagy. This study shows PICALM is important for the correct maturation of lysosomal enzymes and efficient proteolytic function in the lysosome.

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.

Lysosomal Dysregulation in the Murine AppNL-G-F/NL-G-F Model of Alzheimer's Disease.

Lysosomal network dysfunction is a prominent feature of Alzheimer's disease (AD). Although transgenic mouse models of AD are known to model some aspects of lysosomal network dysfunction, the lysosomal network has not yet been examined in the knock-in AppNL-G-F/NL-G-F mouse. We aimed to determine whether AppNL-G-F/NL-G-F mice exhibit disruptions to the lysosomal network in the brain. Lysosome-associated membrane protein 1 (LAMP1) and cathepsins B, L and D accumulated at amyloid beta plaques in the AppNL-G-F/NL-G-F mice, as occurs in human Alzheimer's patients. The accumulation of these lysosomal proteins occurred early in the development of neuropathology, presenting at the earliest and smallest amyloid beta plaques observed. AppNL-G-F/NL-G-F mice also exhibited elevated activity of ß-hexosaminidase and cathepsins D/E and elevated levels of selected lysosomal network proteins, namely LAMP1, cathepsin D and microtubule-associated protein light chain 3 (LC3-II) in the cerebral cortex, as determined by western blot. Elevation of cathepsin D did not change the extent of co-localisation between cathepsin D and LAMP1 in the AppNL-G-F/NL-G-F mice. These findings demonstrate that perturbations of the lysosomal network occur in the AppNL-G-F/NL-G-F mouse model, further validating its use an animal model of pre-symptomatic AD.

Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-Density Gradient.

The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or fluorophore-tagged lysosomal membrane protein. A simpler approach uses differential density of subcellular organelles, which are characteristic to a particular type of organelle. Separation of organelles along a density-gradient enables fractionation to enrich for specific organelles (such as lysosomes) in their native state. This protocol outlines an optimized method for enriching lysosomes from HeLa cells with a continuous density-gradient that contains Percoll. Gentle cell lysis and extraction conditions yield dense-fractions that are enriched with functional and intact lysosomes, which can be assayed in downstream analyses. This method is quick (conducted in less than 2 h after harvesting cells), and can be easily scaled and optimized for other cell types.

Lysosomal LAMP1 immunoreactivity exists in both diffuse and neuritic amyloid plaques in the human hippocampus.

Lysosomal vesicles around neuritic plaques are thought to drive Alzheimer's disease by providing ideal microenvironments for generation of amyloid-ß. Although lysosomal vesicles are present at every amyloid plaque in mouse models of Alzheimer's disease, the number of amyloid plaques that contain lysosomal vesicles in the human brain remains unknown. This study aimed to quantify lysosomal vesicles at amyloid plaques in the human hippocampus. Lysosome-associated membrane protein 1 (LAMP1)-positive vesicles accumulated in both diffuse (Aß42-positive/AT8-negative) and neuritic (Aß42-positive/AT8-positive) plaques in all regions were analysed. In contrast to mouse models of Alzheimer's disease, however, not all amyloid plaques accumulated LAMP1-positive lysosomal vesicles. Even at neuritic plaques, LAMP1 immunoreactivity was more abundant than phospho-tau (AT8). Further, lysosomal vesicles colocalised weakly with phospho-tau such that accumulation of lysosomal vesicles and phospho-tau appeared to be spatially distinct events that occurred within dystrophic neurites. This quantitative study shows that diffuse plaques, as well as neuritic plaques, contain LAMP1 immunoreactivity in the human hippocampus.

Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease.

Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state.