Ion Channels and Pumps in Autophagy: A Reciprocal Relationship

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP3Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps—including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases—in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases.

HPMA-Based Polymer Conjugates for Repurposed Drug Mebendazole and Other Imidazole-Based Therapeutics

Recently, the antitumor potential of benzimidazole anthelmintics, such as mebendazole and its analogues, have been reported to have minimal side effects, in addition to their well-known anti-parasitic abilities. However, their administration is strongly limited owing to their extremely poor solubility, which highly depletes their overall bioavailability. This study describes the design, synthesis, and physico-chemical properties of polymer-mebendazole nanomedicines for drug repurposing in cancer therapy. The conjugation of mebendazole to water-soluble and biocompatible polymer carrier was carried out via biodegradable bond, relying on the hydrolytic action of lysosomal hydrolases for mebendazole release inside the tumor cells. Five low-molecular-weight mebendazole derivatives, differing in their inner structure, and two polymer conjugates differing in their linker structure, were synthesized. The overall synthetic strategy was designed to enable the modification and polymer conjugation of most benzimidazole-based anthelmintics, such as albendazole, fenbendazole or albendazole, besides the mebendazole. Furthermore, the described methodology may be suitable for conjugation of other biologically active compounds with a heterocyclic N-H group in their molecules.

Accumulation of saposin in dystrophic neurites is linked to impaired lysosomal functions in Alzheimer’s disease brains

Neuritic plaques in Alzheimer’s disease (AD) brains refer to β-amyloid (Aβ) plaques surrounded by dystrophic neurites (DNs), activated microglia and reactive astrocytes. Most recently, we showed that DNs form sequentially in three layers during plaque growth. Although lysosomal proteins such as LAMP1 are found in DNs, it is not clear how many and how early lysosomal proteins are involved in forming neuritic plaques. To answer this unmet question, we examined APP knock-in (APPNL-G-F), 5xFAD and APP/PS1ΔE9 mouse brains and found that the lysosomal activator proteins saposins (SAPs) and LAMP1 were accumulated to surround Aβ plaques at the earliest stage, namely the 1st layer of DNs. Noticeably, lysosomal hydrolases were not detectable in these early DNs, suggesting that DNs at this early stage likely enrich dysfunctional lysosomes. In old AD mouse brains and in the later stage of human AD brains, SAP-C+-DNs and LAMP1+-DNs were gradually reduced in concomitant with the growth of amyloid plaques. Remarkably, the observed LAMP1 immunoreactivity near plaques in aged AD mouse and human brains were actually associated with disease-associated microglia rather than neuronal sources, likely reflecting more severely impaired lysosomal functions in neurons. Western blot analyses showed increased levels of SAP-C in AD mouse brains, and Aβ oligomers induced elevated levels of SAP-C in cellular assays. The elevated protein levels of SAP-C in AD mouse brains during plaque growth potentially contributed lysosomal membrane leakage and loss of hydrolases. Together, our study indicates that lysosomal functions are impaired by being entrapped in DNs early during plaque growth, and this may viciously facilitate growth of amyloid plaques.

Glucose-dependent miR-125b is a negative regulator of β-cell function

Impaired pancreatic β-cell function and insulin secretion are hallmarks of type 2 diabetes. MicroRNAs are short non-coding RNAs that silence gene expression, vital for the development and function of endocrine cells. MiR-125b is a highly conserved miRNA abundant in β-cells, though its role in these cells remains unclear. Here, we show that miR-125b expression in human islets correlates with body mass index (BMI) of the donors and is regulated by glucose in an AMP-activated protein kinase-dependent manner in both mice and humans. Using and unbiased high-throughput approach, we identify dozens of direct gene targets, including the transporter of lysosomal hydrolases M6pr and the mitochondrial fission regulator Mtfp1. Whereas inactivation of miR-125b in human β-cells led to shorter mitochondria and improved glucose stimulated insulin secretion, mice over-expressing mir-125b selectively in β-cells displayed defective insulin secretion and marked glucose intolerance. Moreover, the β-cells of these transgenic animals showed strongly reduced insulin content and secretion and contained enlarged lysosomal structures. Thus, miR125b provides a glucose-controlled regulator of organelle dynamics that negatively regulates insulin secretion in β-cells.

Levels of marker lysosomal hydrolases in women with coronary heart disease depending on age and sex hormone level

Aim of the study was to evaluate the serum concentrations of three marker lysosomal hydrolases (cathepsin D, acid phosphatase (AP) and acid DNase (aDNAase)) in women with coronary heart disease (CHD) depending on the level of follicle-­stimulating hormone (FSH), testosterone (T), age and find if those parameters associated with anthropometric parameters, glycemia, insulinemia and HOMA-IR index, biomarkers of atherosclerosis. The study included 285 women aged 35–65 years (median age was 54.4 years (25% and 75% percentiles — 43.2 and 61.3 years, respectively) who had had myocardial infarction no earlier than 30 days before the examination. Patients were divided into the following age groups: 35–55 and 56–65 years (first and second age groups, respectively), and into groups according to the levels of sex hormones: FSH ≥ and <30 mIU/mL and testosterone ≥ and <3 nmol/L. Results of comparative and correlation analyzes demonstrates that in women 35–65 years old with FSH ≥30 mIU/mL, the levels of cathepsin D are higher (p<0.05) than in patients with FSH <30 mIU/mL, and in women 35–55 years old, the content of AP was also higher (p=0.025). Associations of a high level of androgen with lysosomal hyperenzymemia were demonstrated only in the second age group, where at a level of T ≥3 nmol/L, higher values of all three lysosomal enzymes were recorded. Multivariate analysis in both age groups is confirmed direct impact of periand postmenopausal periods on the levels of lysosomal enzymemia and, accordingly, a negative effect on the state of lysosomal membranes. Thus, FSH levels directly determined the concentrations of AP and cardiotropic cathepsin D. The levels of aDNAase in women with CHD of 56–65 years of age were positively correlated with indicators that determine insulin-­glucose homeostasis: glycemia (p<0.001), HOMA-IR index (p<0.001). Such associations of three marker lysosomal enzymes demonstrate the primary contribution of FSH ≥30 mIU/mL to an increase in the concentration of lysosomal hydrolases in women with CHD35–65 years old and the correlation of aDNAase with the processes triggered by insulin resistance.

Organelle tethering, pore formation and SNARE compensation in the late endocytic pathway

To provide insights into the kiss-and-run and full fusion events resulting in endocytic delivery to lysosomes, we investigated conditions causing increased tethering and pore formation between late endocytic organelles in HeLa cells. Knockout of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) VAMP7 and VAMP8 showed, by electron microscopy, the accumulation of tethered LAMP (lysosome associated membrane protein)-carrier vesicles around multivesicular bodies, as well as the appearance of ‘hourglass’ profiles of late endocytic organelles attached by filamentous tethers, but did not prevent endocytic delivery to lysosomal hydrolases. Subsequent depletion of the SNARE YKT6 reduced this delivery, consistent with it compensating for the absence of VAMP7 and VAMP8. We also investigated filamentous tethering between multivesicular bodies and enlarged endolysosomes following depletion of CHMP6 (charged multi-vesicular body protein 6) and provide the first evidence that pore formation commences at the edge of tether arrays, with pore expansion required for full membrane fusion.

Autophagy in Viral Development and Progression of Cancer

Autophagy is a complex degradative process by which eukaryotic cells capture cytoplasmic components for subsequent degradation through lysosomal hydrolases. Although this catabolic process can be triggered by a great variety of stimuli, action in cells varies according to cellular context. Autophagy has been previously linked to disease development modulation, including cancer. Autophagy helps suppress cancer cell advancement in tumor transformation early stages, while promoting proliferation and metastasis in advanced settings. Oncoviruses are a particular type of virus that directly contribute to cell transformation and tumor development. Extensive molecular studies have revealed complex ways in which autophagy can suppress or improve oncovirus fitness while still regulating viral replication and determining host cell fate. This review includes recent advances in autophagic cellular function and emphasizes its antagonistic role in cancer cells.

TFEB Gene Promoter Variants Effect on Gene Expression in Acute Myocardial Infarction

Autophagy is involved in many physiological processes. Transcription factor EB (TFEB) is a master regulator of autophagy and coordinates the expression of autophagic proteins, lysosomal hydrolases, and lysosomal membrane proteins. Though autophagy has been implicated in several human diseases, little is known regarding TFEB gene expression and regulation in the process. Since dysfunctional autophagy plays critical roles in acute myocardial infarction (AMI), dysregulated TFEB gene expression may be associated with AMI by regulating autophagy. In this study, the TFEB gene promoter was genetically and functionally analyzed in AMI patients (n = 352) and ethnic-matched controls (n = 337). A total of fifteen regulatory variants of the TFEB gene, including eight single-nucleotide polymorphisms (SNPs), were identified in this population. Among these, six regulatory variants [g.41737274T&gt;C (rs533895008), g.41737144A&gt;G, g.41736987C &gt; T (rs760293138), g.41736806C &gt; T (rs748537297), g.41736635T &gt; C (rs975050638), and g.41736544C &gt; T] were only identified in AMI patients. These regulatory variants significantly altered the transcriptional activity of the TFEB gene promoter. Further electrophoretic mobility shift assay revealed that three of the variants evidently affected the binding of transcription factors. Therefore, this study identified novel TFEB gene regulatory variants which affect the gene expression. These TFEB gene regulatory variants may contribute to AMI development as a rare risk factor.

Lysosomal Exocytosis: The Extracellular Role of an Intracellular Organelle

Lysosomes are acidic cell compartments containing a large set of hydrolytic enzymes. These lysosomal hydrolases degrade proteins, lipids, polysaccharides, and nucleic acids into their constituents. Materials to be degraded can reach lysosomes either from inside the cell, by autophagy, or from outside the cell, by different forms of endocytosis. In addition to their degradative functions, lysosomes are also able to extracellularly release their contents by lysosomal exocytosis. These organelles move from the perinuclear region along microtubules towards the proximity of the plasma membrane, then the lysosomal and plasma membrane fuse together via a Ca2+-dependent process. The fusion of the lysosomal membrane with plasma membrane plays an important role in plasma membrane repair, while the secretion of lysosomal content is relevant for the remodelling of extracellular matrix and release of functional substrates. Lysosomal storage disorders (LSDs) and age-related neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases, share as a pathological feature the accumulation of undigested material within organelles of the endolysosomal system. Recent studies suggest that lysosomal exocytosis stimulation may have beneficial effects on the accumulation of these unprocessed aggregates, leading to their extracellular elimination. However, many details of the molecular machinery required for lysosomal exocytosis are only beginning to be unravelled. Here, we are going to review the current literature on molecular mechanisms and biological functions underlying lysosomal exocytosis, to shed light on the potential of lysosomal exocytosis stimulation as a therapeutic approach.

The multifaceted functions of ATG16L1 in autophagy and related processes

ABSTRACTAutophagy requires the formation of membrane vesicles, known as autophagosomes, that engulf cellular cargoes and subsequently recruit lysosomal hydrolases for the degradation of their contents. A number of autophagy-related proteins act to mediate the de novo biogenesis of autophagosomes and vesicular trafficking events that are required for autophagy. Of these proteins, ATG16L1 is a key player that has important functions at various stages of autophagy. Numerous recent studies have begun to unravel novel activities of ATG16L1, including interactions with proteins and lipids, and how these mediate its role during autophagy and autophagy-related processes. Various domains have been identified within ATG16L1 that mediate its functions in recognising single and double membranes and activating subsequent autophagy-related enzymatic activities required for the recruitment of lysosomes. These recent findings, as well as the historical discovery of ATG16L1, pathological relevance, unresolved questions and contradictory observations, will be discussed here.