β-carbolines RCC and C5 induce death of Leishmania amazonensis intracellular amastigotes

Background: Cutaneous leishmaniasis is caused by Leishmania spp., and its treatment is limited. The β-carbolines have shown activity against kinetoplastids. Aim: To evaluate the activity and effects of the β-carbolines, N-{2-[(4,6-bis(isopropylamino)-1,3,5-triazin-2-yl)amino]ethyl}-1-(4-methoxyphenyl)-β-carboline-3-carboxamide (RCC) and N-benzyl-1-(4-methoxy)phenyl-9H-beta-carboline-3-carboxamide (C5), against L. amazonensis intracellular amastigotes and to suggest their mechanism of action. Methods: We analyzed the activity and cytotoxicity of β-carbolines and the morphological alterations by electron microscopy. Mitochondrial membrane potential, production nitric oxide, reactive oxygen species, lipidic bodies, autophagic vacuoles and ATP were also evaluated. Results & conclusion: The results showed that RCC and C5 are active against intracellular amastigotes and were able to induce oxidative stress and ultrastructural alterations such as accumulation of lipid bodies and autophagic vacuoles, leading to parasite death.

The Secrets of Alternative Autophagy

For many years, it was thought that ATG5 and ATG7 played a pivotal role in autophagy, and that the knockdown of one of these genes would result in its inhibition. However, cells with ATG5 or ATG7 depletion still generate autophagic vacuoles with mainly trans-Golgi-originated isolation membranes and do not die. This indicates that autophagy can occur via ATG5/ATG7-independent alternative autophagy. Its molecular mechanism differs from that of the canonical pathway, including inter alia the phosphorylation of ULK1, and lack of LC3 modifications. As the alternative autophagy pathway has only recently been described, little is known of its precise role; however, a considerable body of evidence suggests that alternative autophagy participates in mitochondrion removal. This review summarizes the latest progress made in research on alternative autophagy and describes its possible molecular mechanism, roles and methods of detection, and possible modulators. There is a need for further research focused on types of autophagy, as this can elucidate the functioning of various cell types and the pathogenesis of human and animal diseases.

Lysosomal Storage Disorders

Lysosomes are membrane-bound organelles that degrade various macromolecules. Lysosomal storage diseases are a clinically, enzymatically, and genetically heterogeneous group of disorders resulting from intracellular accumulation of substrates. Mechanisms of lysosomal storage disorders include 1) primary deficiency of specific hydrolases; 2) defects in activator proteins required for enzyme-substrate interactions in posttranslational modification of enzymes or in transport of the substrate from lysosomes; and 3) abnormalities of fusion between autophagic vacuoles and lysosomes. Substrate accumulation is slowly progressive, leading to considerable morbidity and mortality.

Detection of intracellular histological abnormalities using cardiac magnetic resonance T1 mapping in patients with Danon disease: a case series

Background Danon disease is an X-linked dominant disorder with defects in the lysosome-associated membrane protein 2 (LAMP2) gene and is characterized histologically by intracellular autophagic vacuoles in skeletal and cardiac muscles. Cardiac magnetic resonance (CMR) T1 mapping potentially allows to differentiate intracellular and extracellular cardiac abnormalities with a combination of native T1 value and extracellular volume (ECV) fraction. Case summary We assessed CMR T1 mapping in two Danon disease patients (a 22-year-old man and his 48-year-old mother), who had a LAMP2 c.864G>A p. Val288Val mutation, and two blood relatives without Danon disease (his 47-year-old maternal aunt and 49-year-old father). The male patient underwent a left ventricular (LV) assist device implantation at 15 months after the image acquisition because he was inotrope dependent (INTERMACS profile 3) and had no noticeable psychological or musculoskeletal symptoms. His mother was in New York Heart Association Class II with mildly reduced LV ejection fraction (46%). The Danon group showed late gadolinium enhancement (LGE) in the anterior and posterolateral LV walls. In the interventricular wall, where evident LGE was not noted, the Danon group had high native T1 value, compared with the T1 value in the non-Danon group, and normal ECV fraction. Cardiac biopsy from the interventricular wall showed intracytoplasmic autophagic vacuoles, which are characteristics of Danon disease. Discussion This characteristic pattern of high native T1 and normal ECV fraction in the areas without LGE, which may reflect the existence of intracytoplasmic autophagic vacuoles, may support the differential diagnosis of Danon disease from other cardiomyopathies.

Danon disease: a case report and literature review

Background Danon disease (DD) is a rare x-linked dominant multisystemic disorder with a clinical triad of severe cardiomyopathy, skeletal myopathy, and mental retardation. It is caused by a defect in the lysosomal-associated membrane protein-2 (LAMP2) gene, which leads to the formation of autophagic vacuoles containing glycogen granule deposits in skeletal and cardiac muscle fibers. So far, more than 50 different mutations in LAMP2 have been identified. Case presentation Here, we report an 18-year-old male patient who was hospitalized for heart failure. Biopsy of the left lateral femoral muscle revealed scattered autophagic vacuoles in the muscle fibers with increased glycogen. Next generation sequencing (NGS) was used to detect gene mutations of the proband sample and a novel frameshift mutation (c.1052delG) has been identified in exon 8 of LAMP2, which leads to truncation of the protein. Conclusion We found a novel frameshift mutation, a hemizygous mutation (c.1052delG) in exon 8 of LAMP2, identified as presenting the hypertrophic cardiomyopathy (HCM) phenotype. Genetic analysis is the gold standard for the diagnosis of DD and is essential to determine appropriate treatment strategies and to confirm the genetic risk of family members.

Electroacupuncture Inhibits Autophagy of Glial Cells in Postherpetic Neuralgia by Increasing the Expression of miR-223-3p

Postherpetic neuralgia (PHN) is a complication of herpes zoster viral infection. Its main manifestations are continuous or intermittent burning-like and electroshock-like pain in the affected nerves. Electroacupuncture (EA) is widely used in clinical treatment and exerts effects in alleviating neuropathic pain. In this study, we investigated the effect and underlying mechanism of EA on PHN. Sprague-Dawley rats were treated with resiniferatoxin (RTX) to establish a PHN model and subjected to EA and/or miR-223-3p overexpression (OV) or interference. Mechanical withdrawal latency was measured as an indication of pain sensitivity. Hematoxylin-eosin staining and transmission electron microscopy were performed to observe glial cell morphology and autophagic vacuoles, respectively. ELISA was performed to detect reactive oxygen species (ROS) production and the levels of tumor necrosis factor- (TNF-) α, inducible nitric oxide synthase (iNOS), interleukin- (IL-) 6, and IL-10. Changes in autophagy and apoptosis-related miRNAs were detected by immunofluorescence and qRT-PCR, respectively. In RTX-treated rats, OV and EA reduced pain sensitivity, decreased the number of eosinophils, and increased that of nerve cells. ROS generation and the levels of TNF-α and iNOS were significantly reduced, while those of IL-6 and IL-10 were increased. OV and EA induced fewer autophagic vacuoles than those in the model group. The expression of autophagy-related protein microtubule-associated protein 1 light chain 3-II, ATG9, and Rab1 was decreased by OV and EA, whereas that of P62 was increased. qRT-PCR revealed that miR-223-3p expression in the model group decreased but was increased by EA. EA inhibits glial cell autophagy in PHN by increasing miR-223-3p expression.

Loss of supervillin causes myopathy with myofibrillar disorganization and autophagic vacuoles

The muscle specific isoform of the supervillin protein (SV2), encoded by the SVIL gene, is a large sarcolemmal myosin II- and F-actin-binding protein. Supervillin (SV2) binds and co-localizes with costameric dystrophin and binds nebulin, potentially attaching the sarcolemma to myofibrillar Z-lines. Despite its important role in muscle cell physiology suggested by various in vitro studies, there are so far no reports of any human disease caused by SVIL mutations. We here report four patients from two unrelated, consanguineous families with a childhood/adolescence onset of a myopathy associated with homozygous loss-of-function mutations in SVIL. Wide neck, anteverted shoulders and prominent trapezius muscles together with variable contractures were characteristic features. All patients showed increased levels of serum creatine kinase but no or minor muscle weakness. Mild cardiac manifestations were observed. Muscle biopsies showed complete loss of large supervillin isoforms in muscle fibres by western blot and immunohistochemical analyses. Light and electron microscopic investigations revealed a structural myopathy with numerous lobulated muscle fibres and considerable myofibrillar alterations with a coarse and irregular intermyofibrillar network. Autophagic vacuoles, as well as frequent and extensive deposits of lipoproteins, including immature lipofuscin, were observed. Several sarcolemma-associated proteins, including dystrophin and sarcoglycans, were partially mis-localized. The results demonstrate the importance of the supervillin (SV2) protein for the structural integrity of muscle fibres in humans and show that recessive loss-of-function mutations in SVIL cause a distinctive and novel myopathy.