In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Alzheimer's disease (AD) is a genetically complex and heterogeneous disorder. To date four genes have been established to either cause early-onset autosomal-dominant AD (APP, PSEN1, and PSEN21–4) or to increase susceptibility for late-onset AD (APOE5). However, the heritability of late-onset AD is as high as 80%,6 and much of the phenotypic variance remains unexplained to date. We performed a genome-wide association (GWA) analysis using 484,522 single-nucleotide polymorphisms (SNPs) on a large (1,376 samples from 410 families) sample of AD families of self-reported European descent. We identified five SNPs showing either significant or marginally significant genome-wide association with a multivariate phenotype combining affection status and onset age. One of these signals (p = 5.7 × 10−14) was elicited by SNP rs4420638 and probably reflects APOE-ɛ4, which maps 11 kb proximal (r2 = 0.78). The other four signals were tested in three additional independent AD family samples composed of nearly 2700 individuals from almost 900 families. Two of these SNPs showed significant association in the replication samples (combined p values 0.007 and 0.00002). The SNP (rs11159647, on chromosome 14q31) with the strongest association signal also showed evidence of association with the same allele in GWA data generated in an independent sample of ∼1,400 AD cases and controls (p = 0.04). Although the precise identity of the underlying locus(i) remains elusive, our study provides compelling evidence for the existence of at least one previously undescribed AD gene that, like APOE-ɛ4, primarily acts as a modifier of onset age.

Genome-wide Association Analysis Reveals Putative Alzheimer's Disease Susceptibility Loci in Addition to APOE

Bisphenol A and human chronic diseases: current evidences, possible mechanisms, and future perspectives.

The regulation of mitochondrial dynamics by the GTPase OPA1, which is located at the inner mitochondrial membrane, is crucial for adapting mitochondrial function and preserving cellular health. OPA1 governs the delicate balance between fusion and fission in the dynamic mitochondrial network. A disturbance of this balance, often observed under stress and pathologic conditions, causes mitochondrial fragmentation and can ultimately result in cell death. As discussed in this Commentary, these morphological changes are regulated by proteolytic processing of OPA1 by the inner-membrane peptidases YME1L (also known as YME1L1) and OMA1. Long, membrane-bound forms of OPA1 are required for mitochondrial fusion, but their processing to short, soluble forms limits fusion and can facilitate mitochondrial fission. Excessive OPA1 processing by the stress-activated protease OMA1 promotes mitochondrial fragmentation and, if persistent, triggers cell death and tissue degeneration in vivo The prevention of OMA1-mediated OPA1 processing and mitochondrial fragmentation might thus offer exciting therapeutic potential for human diseases associated with mitochondrial dysfunction.

/pdf/opa1-processing-in-cell-death-and-disease-the-long-and-short-qun5ns0o6r.pdf

OPA1 processing in cell death and disease – the long and short of it

The search for therapeutic agents that bind specifically to precursor protein conformations and inhibit amyloid assembly is an important challenge. Identifying such inhibitors is difficult because many protein precursors of aggregation are partially folded or intrinsically disordered, which rules out structure-based design. Furthermore, inhibitors can act by a variety of mechanisms, including specific or nonspecific binding, as well as colloidal inhibition. Here we report a high-throughput method based on ion mobility spectrometry-mass spectrometry (IMS-MS) that is capable of rapidly detecting small molecules that bind to amyloid precursors, identifying the interacting protein species and defining the mode of inhibition. Using this method we have classified a variety of small molecules that are potential inhibitors of human islet amyloid polypeptide (hIAPP) aggregation or amyloid-beta 1-40 aggregation as specific, nonspecific, colloidal or non-interacting. We also demonstrate the ability of IMS-MS to screen for inhibitory small molecules in a 96-well plate format and use this to discover a new inhibitor of hIAPP amyloid assembly.

/pdf/screening-and-classifying-small-molecule-inhibitors-of-4ixa2wgpax.pdf

Screening and classifying small-molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry

Protein misfolding and aggregation are associated with more than twenty diseases, such as neurodegenerative diseases and metabolic diseases. The amyloid oligomers and fibrils may induce cell membrane disruption and lead to cell apoptosis. A great number of studies have focused on discovery of amyloid inhibitors which may prevent or treat amyloidosis diseases. Polyphenols have been extensively studied as a class of amyloid inhibitors, with several polyphenols under clinical trials as anti-neurodegenerative drugs. As oxidative intermediates of natural polyphenols, quinones widely exist in medicinal plants or food. In this study, we used insulin as an amyloid model to test the anti-amyloid effects of four simple quinones and four natural anthraquinone derivatives from rhubarb, a traditional herbal medicine used for treating Alzheimer's disease. Our results demonstrated that all eight quinones show inhibitory effects to different extent on insulin oligomerization, especially for 1,4-benzoquinone and 1,4-naphthoquinone. Significantly attenuated oligomerization, reduced amount of amyloid fibrils and reduced hemolysis levels were found after quinones treatments, indicating quinones may inhibit insulin from forming toxic oligomeric species. The results suggest a potential action of native anthraquinone derivatives in preventing protein misfolding diseases, the quinone skeleton may thus be further explored for designing effective anti-amyloidosis compounds.

/pdf/effects-of-several-quinones-on-insulin-aggregation-3ouqvyrlgf.pdf

Effects of several quinones on insulin aggregation

Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide

Bisphenol A (BPA) is a chemical compound widely used in manufacturing plastic products. Recent epidemiological studies suggest BPA exposure is positively associated with the incidence of type 2 diabetes mellitus (T2DM), however the mechanisms underlying this link remain unclear. Human islet amyloid polypeptide (hIAPP) is a hormone synthesized and secreted by the pancreatic β-cells. Misfolding of hIAPP into toxic oligomers and mature fibrils can disrupt cell membrane and lead to β-cell death, which is regarded as one of the causative factors of T2DM. To test whether there are any connections between BPA exposure and hIAPP misfolding, we investigated the effects of BPA on hIAPP aggregation using thioflavin-T based fluorescence, transmission electronic microscopy, circular dichroism, dynamic light scattering, size-exclusion chromatography,fluorescence-dye leakage assay in an artificial micelle system and the generation of reactive oxygen species in INS-1 cells. We demonstrated that BPA not only dose-dependently promotes the aggregation of hIAPP and enhances the membrane disruption effects of hIAPP, but also promotes the extent of hIAPP aggregation related oxidative stress. Taken together, our results suggest that BPA exposure increased T2DM risk may involve the exacerbated toxic aggregation of hIAPP.

/pdf/bisphenol-a-accelerates-toxic-amyloid-formation-of-human-24r5camglu.pdf

Bisphenol A Accelerates Toxic Amyloid Formation of Human Islet Amyloid Polypeptide: A Possible Link between Bisphenol A Exposure and Type 2 Diabetes

Autophagy plays critical and complex roles in many human diseases, including diabetes and its complications. However, the role of autophagy in the development of diabetic retinopathy remains uncertain. Core histone modifications have been reported involved in the development of diabetic retinopathy, but little is known about the histone variants. Here, we observed increased autophagy and histone HIST1H1C/H1.2, an important variant of the linker histone H1, in the retinas of type 1 diabetic rodents. Overexpression of histone HIST1H1C upregulates SIRT1 and HDAC1 to maintain the deacetylation status of H4K16, leads to upregulation of ATG proteins, then promotes autophagy in cultured retinal cell line. Histone HIST1H1C overexpression also promotes inflammation and cell toxicity in vitro. Knockdown of histone HIST1H1C reduces both the basal and stresses (including high glucose)-induced autophagy, and inhibits high glucose induced inflammation and cell toxicity. Importantly, AAV-mediated histone HIST1H1C overexpression in the retinas leads to increased autophagy, inflammation, glial activation and neuron loss, similar to the pathological changes identified in the early stage of diabetic retinopathy. Furthermore, knockdown of histone Hist1h1c by siRNA in the retinas of diabetic mice significantly attenuated the diabetes-induced autophagy, inflammation, glial activation and neuron loss. These results indicate that histone HIST1H1C may offer a novel therapeutic target for preventing diabetic retinopathy.

https://www.tandfonline.com/doi/pdf/10.1080/15548627.2017.1293768

Yue Sun

Papers

Effects of several quinones on insulin aggregation

Silibinin inhibits the toxic aggregation of human islet amyloid polypeptide

Bisphenol A Accelerates Toxic Amyloid Formation of Human Islet Amyloid Polypeptide: A Possible Link between Bisphenol A Exposure and Type 2 Diabetes

Histone HIST1H1C/H1.2 regulates autophagy in the development of diabetic retinopathy

Proanthocyanidins are the major anti-diabetic components of cinnamon water extract