Rare Variants in Autophagy and Non-Autophagy Genes in Late-Onset Pompe Disease: Suggestions of Their Disease-Modifying Role in Two Italian Families

Pompe disease is an autosomal recessive disorder caused by a deficiency in the enzyme acid alpha-glucosidase. The late-onset form of Pompe disease (LOPD) is characterized by a slowly progressing proximal muscle weakness, often involving respiratory muscles. In LOPD, the levels of GAA enzyme activity and the severity of the clinical pictures may be highly variable among individuals, even in those who harbour the same combination of GAA mutations. The result is an unpredictable genotype–phenotype correlation. The purpose of this study was to identify the genetic factors responsible for the progression, severity and drug response in LOPD. We report here on a detailed clinical, morphological and genetic study, including a whole exome sequencing (WES) analysis of 11 adult LOPD siblings belonging to two Italian families carrying compound heterozygous GAA mutations. We disclosed a heterogeneous pattern of myopathic impairment, associated, among others, with cardiac defects, intracranial vessels abnormality, osteoporosis, vitamin D deficiency, obesity and adverse response to enzyme replacement therapy (ERT). We identified deleterious variants in the genes involved in autophagy, immunity and bone metabolism, which contributed to the severity of the clinical symptoms observed in the LOPD patients. This study emphasizes the multisystem nature of LOPD and highlights the polygenic nature of the complex phenotype disclosed in these patients.     

Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies

Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation.     

Systematic Structure–Activity Study on Potential Chaperone Lead Compounds for Acid α‐Glucosidase

Acid α‐glucosidase (GAA) is a lysosomal enzyme and a pharmacological target for Pompe disease, an inherited lysosomal storage disorder (LSD). An emerging treatment for LSDs is the use of pharmacological chaperones, small molecules that enhance total cellular activity of the target lysosomal protein. We have systematically studied thirteen inhibitors, which provide good lead compounds for the development of GAA chaperones. We have verified binding on GAA at low and neutral pH, mapping the range of pH during transport to lysosomes. These ligands inhibit GAA competitively and reversibly, and a few of the compounds show higher molecular stabilisation capacity than would be expected from their binding affinity. These molecules also increase lysosomal localisation of GAA variants in cells. In order to understand the specific molecular mechanism of the interactions, we docked the compounds to a homology model of the human GAA. Three factors contribute to the tightness of binding. Firstly, well‐positioned hydroxy groups are essential to orient the ligand and make the binding specific. Secondly, the open nature of the GAA active site allows both large and small ligands to bind. The third and most important binding determinant is the positive charge on the ligand, which is neutralised by Asp 518 or Asp 616 on GAA. Our study creates a firm basis for the design of drugs to treat Pompe disease, as it provides a comparable study of the ligand properties. Our analysis suggests a useful drug design framework for specific pharmacological chaperones for human GAA.     

Identification of Oxidative Stress Related Proteins as Biomarkers for Lung Cancer and Chronic Obstructive Pulmonary Disease in Bronchoalveolar Lavage

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. Cigarette smoke causes oxidative stress and an inflammatory response in lung cells, which in turn may be involved in COPD and lung cancer development. The aim of this study was to identify differential proteomic profiles related to oxidative stress response that were potentially involved in these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC, and control (neither COPD nor LC). Proteins were separated into spots by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 16 oxidative stress regulatory proteins were differentially expressed in BAL samples from LC and/or COPD patients as compared with the control group. A distinct proteomic reactive oxygen species (ROS) protein signature emerged that characterized lung cancer and COPD. In conclusion, our findings highlight the role of the oxidative stress response proteins in the pathogenic pathways of both diseases, and provide new candidate biomarkers and predictive tools for LC and COPD diagnosis.     

Structural Basis of Membrane Trafficking by Rab Family Small G Protein

The Ras-superfamily of small G proteins is a family of GTP hydrolases that is regulated by GTP/GDP binding states. One member of the Ras-superfamily, Rab, is involved in the regulation of vesicle trafficking, which is critical to endocytosis, biosynthesis, secretion, cell differentiation and cell growth. The active form of the Rab proteins, which contains GTP, can recruit specific binding partners, such as sorting adaptors, tethering factors, kinases, phosphatases and motor proteins, thereby influencing vesicle formation, transport, and tethering. Many Rab proteins share the same interacting partners and perform unique roles in specific locations. Because functional loss of the Rab pathways has been implicated in a variety of diseases, the Rab GTPase family has been extensively investigated. In this review, we summarize Rab GTPase- mediated membrane trafficking while focusing on the structures of Rab protein and Rab-effector complexes. This review provides detailed information that helps explain how the Rab GTPase family is involved in membrane trafficking.     

Alteration of Dynein Function Affects α-Synuclein Degradation via the Autophagosome-Lysosome Pathway

Growing evidence suggests that dynein dysfunction may be implicated in the pathogenesis of neurodegeneration. It plays a central role in aggresome formation, the delivery of autophagosome to lysosome for fusion and degradation, which is a pro-survival mechanism essential for the bulk degradation of misfolded proteins and damaged organells. Previous studies reported that dynein dysfuntion was associated with aberrant aggregation of α-synuclein, which is a major component of inclusion bodies in Parkinson’s disease (PD). However, it remains unclear what roles dynein plays in α-synuclein degradation. Our study demonstrated a decrease of dynein expression in neurotoxin-induced PD models in vitro and in vivo, accompanied by an increase of α-synuclein protein level. Dynein down-regulation induced by siRNA resulted in a prolonged half-life of α-synuclein and its over-accumulation in A53T overexpressing PC12 cells. Dynein knockdown also prompted the increase of microtubule-associated protein 1 light chain 3 (LC3-II) and sequestosome 1 (SQSTM1, p62) expression, and the accumulation of autophagic vacuoles. Moreover, dynein suppression impaired the autophagosome fusion with lysosome. In summary, our findings indicate that dynein is critical for the clearance of aberrant α-synuclein via autophagosome-lysosome pathway.     

The Roles of Monomeric GTP-Binding Proteins in Macroautophagy in Saccharomyces cerevisiae

Autophagy is a cellular degradation process that sequesters components into a double-membrane structure called the autophagosome, which then fuses with the lysosome or vacuole for hydrolysis and recycling of building blocks. Bulk phase autophagy, also known as macroautophagy, controlled by specific Atg proteins, can be triggered by a variety of stresses, including starvation. Because autophagy relies extensively on membrane traffic to form the membranous structures, factors that control membrane traffic are essential for autophagy. Among these factors, the monomeric GTP-binding proteins that cycle between active and inactive conformations form an important group. In this review, we summarize the functions of the monomeric GTP-binding proteins in autophagy, especially with reference to experiments in Saccharomyces cerevisiae.     

The Critical Role of Membrane Cholesterol in Salmonella-Induced Autophagy in Intestinal Epithelial Cells

It was previously observed that plasma membrane cholesterol plays a critical role in the Salmonella-induced phosphatidylinositol 3-kinase-dependent (PI3K)-dependent anti-inflammatory response in intestinal epithelial cells (IECs). The PI3K/Akt pathway is associated with autophagy which has emerged as a critical mechanism of host defense against several intracellular bacterial pathogens. Plasma membrane contributes directly to the formation of early Atg16L1-positive autophagosome precursors. Therefore, this study aimed to investigate the role of plasma membrane cholesterol on the Salmonella-induced autophagy in IECs. By using methyl-beta-cyclodextrin (MBCD), it was demonstrated that disruption of membrane cholesterol by MBCD enhanced NOD2 and Atg16L1 proteins expression in membrane, and autophagic LC3II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells, which was counteracted by Atg16L1 siRNA. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) siRNA enhanced the Salmonella-induced activation of Akt in Caco-2 cells. However, inhibitors of Akt or extracellular signal-regulated kinases (ERK) had no significant effect on Salmonella-induced autophagy Beclin 1 or LC3 proteins expression. In conclusion, our study suggests that cholesterol accumulation in the plasma membrane at the entry site of Salmonella results in the formation of Salmonella-containing vacuole (SCV) and decreased autophagy. Our results offer mechanistic insights on the critical role of membrane cholesterol in the pathogenesis of Salmonella infection in intestinal epithelial cells and the therapeutic potential of its antagonists.     

Distinct Mechanisms for Processing Autophagy Protein LC3-PE by RavZ and ATG4B

Autophagy is a conserved catabolic process involved in the elimination of proteins, organelles and pathogens in eukaryotic cells. Lipidated LC3 proteins that are conjugated to phosphatidylethanolamine (PE) play a key role in autophagosome biogenesis. Endogenous ATG4-mediated deconjugation of LC3-PE is required for LC3 recycling. However, the Legionella effector RavZ irreversibly deconjugates LC3-PE to inhibit autophagy. It is not clear how ATG4 and RavZ process LC3-PE with distinct modes. Herein, a series of semisynthetic LC3-PE proteins containing C-terminal mutations or insertions were used to investigate the relationship of the C-terminal structure of LC3-PE with ATG4/RavZ-mediated deconjugation. Using a combination of molecular docking and biochemical assays, we found that Gln116, Phe119 and Gly120 of LC3-PE are required for cleavage by both RavZ and ATG4B, whereas Glu117(LC3) is specific to cleavage by RavZ. The molecular ruler mechanism exists in the active site of ATG4B, but not in RavZ. Met63 and Gln64 at the active site of RavZ are involved in accommodating LC3 C-terminal motif. Our findings show that the distinct binding modes of the LC3 C-terminal motif (116–120) with ATG4 and RavZ might determine the specificity of cleavage site.     

Revising the Role of Cortical Cytoskeleton during Secretion: Actin and Myosin XI Function in Vesicle Tethering

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.     

Characterization of New Proteomic Biomarker Candidates in Mucopolysaccharidosis Type IVA

Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Skeletal dysplasia and the related clinical features of MPS IVA are caused by disruption of the cartilage and its extracellular matrix, leading to a growth imbalance. Enzyme replacement therapy (ERT) with recombinant human GALNS has yielded positive results in activity of daily living and endurance tests. However, no data have demonstrated improvements in bone lesions and bone grow thin MPS IVA after ERT, and there is no correlation between therapeutic efficacy and urine levels of keratan sulfate, which accumulates in MPS IVA patients. Using qualitative and quantitative proteomics approaches, we analyzed leukocyte samples from healthy controls (n = 6) and from untreated (n = 5) and ERT-treated (n = 8, sampled before and after treatment) MPS IVA patients to identify potential biomarkers of disease. Out of 690 proteins identified in leukocytes, we selected a group of proteins that were dysregulated in MPS IVA patients with ERT. From these, we identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: lactotransferrin, coronin 1A, neutral alpha-glucosidase AB, and vitronectin. Further studies of cartilage and bone alterations in MPS IVA will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.     

Structure‐Specific Recognition of Friedreich's Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)_n repeats form non‐B‐DNA structures, including triple helix H‐DNA and higher‐order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non‐B‐DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)_n binding by triplex‐stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex‐DNA‐cleaving BQQ‐1,10‐phenanthroline (BQQ‐OP) compounds. We also examined the ability of these compounds to act as structural probes for H‐DNA formation within higher‐order structures at pathological frataxin sequences in plasmids. DNA‐complex‐formation analyses with a gel‐mobility‐shift assay and sequence‐specific probing of H‐DNA‐forming (GAA)_n sequences by single‐strand oligonucleotides and triplex‐directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)_n repeats under physiologically relevant conditions.     

Different miRNA Profiles in Plasma Derived Small and Large Extracellular Vesicles from Patients with Neurodegenerative Diseases

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.     

Identification of Potential Muscle Biomarkers in McArdle Disease: Insights from Muscle Proteome Analysis

Glycogen storage disease type V (GSDV, McArdle disease) is a rare genetic myopathy caused by deficiency of the muscle isoform of glycogen phosphorylase (PYGM). This results in a block in the use of muscle glycogen as an energetic substrate, with subsequent exercise intolerance. The pathobiology of GSDV is still not fully understood, especially with regard to some features such as persistent muscle damage (i.e., even without prior exercise). We aimed at identifying potential muscle protein biomarkers of GSDV by analyzing the muscle proteome and the molecular networks associated with muscle dysfunction in these patients. Muscle biopsies from eight patients and eight healthy controls showing none of the features of McArdle disease, such as frequent contractures and persistent muscle damage, were studied by quantitative protein expression using isobaric tags for relative and absolute quantitation (iTRAQ) followed by artificial neuronal networks (ANNs) and topology analysis. Protein candidate validation was performed by Western blot. Several proteins predominantly involved in the process of muscle contraction and/or calcium homeostasis, such as myosin, sarcoplasmic/endoplasmic reticulum calcium ATPase 1, tropomyosin alpha-1 chain, troponin isoforms, and alpha-actinin-3, showed significantly lower expression levels in the muscle of GSDV patients. These proteins could be potential biomarkers of the persistent muscle damage in the absence of prior exertion reported in GSDV patients. Further studies are needed to elucidate the molecular mechanisms by which PYGM controls the expression of these proteins.     

Biomarkers in Glycogen Storage Diseases: An Update

Glycogen storage diseases (GSDs) are a group of 19 hereditary diseases caused by a lack of one or more enzymes involved in the synthesis or degradation of glycogen and are characterized by deposits or abnormal types of glycogen in tissues. Their frequency is very low and they are considered rare diseases. Except for X-linked type IX, the different types are inherited in an autosomal recessive pattern. In this study we reviewed the literature from 1977 to 2020 concerning GSDs, biomarkers, and metabolic imbalances in the symptoms of some GSDs. Most of the reported studies were performed with very few patients. Classification of emerging biomarkers between different types of diseases (hepatics GSDs, McArdle and PDs and other possible biomarkers) was done for better understanding. Calprotectin for hepatics GSDs and urinary glucose tetrasaccharide for Pompe disease have been approved for clinical use, and most of the markers mentioned in this review only need clinical validation, as a final step for their routine use. Most of the possible biomarkers are implied in hepatocellular adenomas, cardiomyopathies, in malfunction of skeletal muscle, in growth retardation, neutropenia, osteopenia and bowel inflammation. However, a few markers have lost interest due to a great variability of results, which is the case of biotinidase, actin alpha 2, smooth muscle, aorta and fibroblast growth factor receptor 4. This is the first review published on emerging biomarkers with a potential application to GSDs.     

Structural Features of a Full-Length Ubiquitin Ligase Responsible for the Formation of Patches at the Plasma Membrane

Plant U-box armadillo repeat (PUB-ARM) ubiquitin (Ub) ligases have important functions in plant defense through the ubiquitination of target proteins. Defense against pathogens involves vesicle trafficking and the formation of extracellular vesicles. The PUB-ARM protein SENESCENCE ASSOCIATED UBIQUITIN E3 LIGASE1 (SAUL1) can form patches at the plasma membrane related to tethering multi-vesicular bodies (MVBs) to the plasma membrane. We uncovered the structure of a full-length plant ubiquitin ligase and the structural requirements of SAUL1, which are crucial for its function in patch formation. We resolved the structure of SAUL1 monomers by small-angle X-ray scattering (SAXS). The SAUL1 model showed that SAUL1 consists of two domains: a domain containing the N-terminal U-box and armadillo (ARM) repeats and the C-terminal ARM repeat domain, which includes a positively charged groove. We showed that all C-terminal ARM repeats are essential for patch formation and that this function requires arginine residue at position 736. By applying SAXS to polydisperse SAUL1 systems, the oligomerization of SAUL1 is detectable, with SAUL1 tetramers being the most prominent oligomers at higher concentrations. The oligomerization domain consists of the N-terminal U-box and some N-terminal ARM repeats. Deleting the U-box resulted in the promotion of the SAUL1 tethering function. Our findings indicate that structural changes in SAUL1 may be fundamental to its function in forming patches at the plasma membrane.     

Possibilities and Limits of Application of Electrical Resistance Tomography in Hydrodynamics of Bubble Columns

Knowing the hydrodynamic regime in which is working a bubble column is of great importance because the regime affects strongly the mass transfer between the phases. To this end, we examine the potentialities of an Electrical Resistance Tomography (ERT) device. We analyse cross correlation of electrode pair measurements, of neighbouring pixels and power spectra of averaged pixels within a single plane as well as cross correlations of averaged pixels between two planes, without finding a clear signature of the churn turbulent flow. Variable gas flow rate inputs are used to determine the time resolution of the ERT.     

Complex Transposon Insertion as a Novel Cause of Pompe Disease

Pompe disease (OMIM#232300) is an autosomal recessive lysosomal storage disorder caused by mutations in the GAA gene. According to public mutation databases, more than 679 pathogenic variants have been described in GAA, none of which are associated with mobile genetic elements. In this article, we report a novel molecular genetic cause of Pompe disease, which could be hardly detected using routine molecular genetic analysis. Whole genome sequencing followed by comprehensive functional analysis allowed us to discover and characterize a complex mobile genetic element insertion deep in the intron 15 of the GAA gene in a patient with infantile onset Pompe disease.     

Angiotensin-(1-7) Prevents Lipopolysaccharide-Induced Autophagy via the Mas Receptor in Skeletal Muscle

Skeletal muscle atrophy, which occurs in lipopolysaccharide (LPS)-induced sepsis, causes a severe muscle function reduction. The increased autophagy contributes to sepsis-induced skeletal muscle atrophy in a model of LPS injection, increasing LC3II/LC3I ratio, autophagy flux, and autophagosomes. Angiotensin-(1-7) (Ang-(1-7)) has anti-atrophic effects via the Mas receptor in skeletal muscle. However, the impact of Ang-(1-7) on LPS-induced autophagy is unknown. In this study, we determined the effect of Ang-(1-7) on sepsis-induced muscle autophagy. C57BL6 wild-type (WT) mice and mice lacking the Mas receptor (KO Mas) were injected with LPS together with the systemic administration of Ang-(1-7) to determine autophagy in skeletal muscle. We also evaluated autophagy and p38 and c-Jun N-terminal kinase (JNK)activation. Our results show that Ang-(1-7) prevents LPS-induced autophagy in the diaphragm, tibialis anterior, and gastrocnemius of WT mice, which is demonstrated by a decrease in the LC3II/LC3I ratio and mRNA levels of lc3b and ctsl. This effect was lost in KO Mas mice, suggesting the role of the Mas receptor. The results in C2C12 cells show that Ang-(1-7) reduces several LPS-dependent effects, such as autophagy (LC3II/LC3I ratio, autophagic flux, and autophagosomes), activation of p38 and JNK, B-cell lymphoma-2 (BCL2) phosphorylation, and disassembly of the Beclin1/BCL2 complex. In conclusion, Ang-(1-7)/Mas receptor reduces LPS-induced autophagy in skeletal muscle. In vitro assays indicate that Ang-(1-7) prevents LPS-induced autophagy and modifies the MAPK signaling and the disassembly of a complex involved at the beginning of autophagy.