Altered autophagic flux in GNE mutant cells of Indian origin: Potential drug target for GNE myopathy.

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of ß-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.

Antimicrobial peptide-based strategies to overcome antimicrobial resistance.

Antibiotic resistance has emerged as a global threat, rendering the existing conventional treatment strategies ineffective. In view of this, antimicrobial peptides (AMPs) have proven to be potent alternative therapeutic interventions with a wide range of applications in clinical health. AMPs are small peptides produced naturally as a part of the innate immune responses against a broad range of bacterial, fungal and viral pathogens. AMPs present a myriad of advantages over traditional antibiotics, including their ability to target multiple sites, reduced susceptibility to resistance development, and high efficacy at low doses. These peptides have demonstrated notable potential in inhibiting microbes resistant to traditional antibiotics, including the notorious ESKAPE pathogens, recognized as the primary culprits behind nosocomial infections. AMPs, with their multifaceted benefits, emerge as promising candidates in the ongoing efforts to combat the escalating challenges posed by antibiotic resistance. This in-depth review provides a detailed discussion on AMPs, encompassing their classification, mechanism of action, and diverse clinical applications. Focus has been laid on combating newly emerging drug-resistant organisms, emphasizing the significance of AMPs in mitigating this pressing challenge. The review also illuminates potential future strategies that may be implemented to improve AMP efficacy, such as structural modifications and using AMPs in combination with antibiotics and matrix-inhibiting compounds.

Potential small effector molecules restoring cellular defects due to sialic acid biosynthetic enzyme deficiency: Pathological relevance to GNE myopathy.

GNEM (GNE Myopathy) is a rare neuromuscular disease caused due to biallelic mutations in sialic acid biosynthetic GNE enzyme (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine Kinase). Recently direct or indirect role of GNE in other cellular functions have been elucidated. Hyposialylation of IGF-1R leads to apoptosis due to mitochondrial dysfunction while hyposialylation of ß1 integrin receptor leads to altered F-actin assembly, disrupted cytoskeletal organization and slow cell migration. Other cellular defects in presence of GNE mutation include altered ER redox state and chaperone expression such as HSP70 or PrdxIV. Currently, there is no cure to treat GNEM. Possible therapeutic trials focus on supplementation with sialic acid, ManNAc, sialyllactose and gene therapy that slows the disease progression. In the present study, we analyzed the effect of small molecules like BGP-15 (HSP70 modulator), IGF-1 (IGF-1R ligand) and CGA (cofilin activator) on cellular phenotypes of GNE heterozygous knock out L6 rat skeletal muscle cell line (SKM­GNEHz). Treatment with BGP-15 improved GNE epimerase activity by 40 % and reduced ER stress by 45 % for SKM­GNEHz. Treatment with IGF-1 improved epimerase activity by 37.5 %, F-actin assembly by 100 %, cell migration upto 36 % (36 h) and atrophy by 0.44-fold for SKM­GNEHz. Treatment with CGA recovered epimerase activity by 49 %, F-actin assembly by 132 % and cell migration upto 41 % (24 h) in SKM­GNEHz. Our study shows that treatment with these small effector molecules reduces the detrimental phenotype observed in SKM­GNEHz, thereby, providing insights into potential therapeutic targets for GNEM.

Understanding pathophysiology of GNE myopathy and current progress towards drug development.

GNE myopathy is a rare genetic neuromuscular disease that is caused due to mutations in the GNE gene responsible for sialic acid biosynthesis. Foot drop is the most common initial symptom observed in GNE myopathy patients. There is slow progressive muscle weakness in the lower and upper extremities while the quadriceps muscles are usually spared. The exact pathophysiology of the disease is unknown. Besides sialic acid biosynthesis, recent studies suggest either direct or indirect involvement of GNE in other cellular functions such as protein aggregation, apoptosis, ER stress, cell migration, HSP70 chaperone activity, autophagy, muscle atrophy, and myogenesis. Both animal and in vitro cell-based model systems are generated to elucidate the mechanism of GNE myopathy and evaluate the efficacy of therapies. The many therapeutic avenues explored include supplementation with sialic acid derivatives or precursors and gene therapy. Recent studies suggest other therapeutic options such as modulators of HSP70 chaperone (BGP-15), cofilin activator (CGA), and ligands like IGF-1 that may help to rescue cellular defects due to GNE dysfunction. This review provides an overview of the pathophysiology associated with GNE function in the cell and promising therapeutic leads to be explored for future drug development.

Effect of GNE Mutations on Cytoskeletal Network Proteins: Potential Gateway to Understand Pathomechanism of GNEM.

GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide. In Indian subcontinent, several GNE mutations have been recently identified with unknown functional correlation. Alternate role of GNE in various cellular processes such as cell adhesion, migration, apoptosis, protein aggregation, and cytoskeletal organization have been proposed in recent studies. We aim to understand and compare the effect of various GNE mutations from Indian origin on regulation of the cytoskeletal network. In particular, F-actin dynamics was determined quantitatively by determining F/G-actin ratios in immunoblots for specific proteins. The extent of F-actin polymerization was visualized by immunostaining with Phalloidin using confocal microscopy. The proteins regulating F-actin dynamics such as RhoA, cofilin, Arp2, and alpha-actinin were studied in various GNE mutants. The altered level of cytoskeletal organization network proteins affected cell migration of GNE mutant proteins as measured by wound healing assay. The functional comparison of GNE mutations will help in better understanding of the genotypic severity of the disease in the Indian population. Our study offers a potential for identification of therapeutic molecules regulating actin dynamics in GNE specific mutations.

Role of HSP70 chaperone in protein aggregate phenomenon of GNE mutant cells: Therapeutic lead for GNE Myopathy.

Limited treatment options and research in understanding the pathomechanisms of rare diseases has raised concerns about their therapeutic development. One such poorly understood ultra-rare neuromuscular disorder is GNE Myopathy (GNEM) which is caused due to mutation in key sialic acid biosynthetic enzyme, GNE. Treatment with sialic acid or its derivatives/precursors slows the disease progression, but curative strategies need to be explored further. Pathologically, muscle biopsy samples of GNEM patients reveal rimmed vacuole formation due to aggregation of ß-amyloid, Tau, presenilin proteins with unknown mechanism. The present study aims to understand the mechanism of protein aggregate formation in GNE mutant cells to decipher role of chaperones in disease phenotype. The pathologically relevant GNE mutations expressed as recombinant proteins in HEK cells was used as a model system for GNEM to estimate extent of protein aggregation. We identified HSP70, a chaperone, as binding partner of GNE. Downregulation of HSP70 with altered BAG3, JNK, BAX expression levels was observed in GNE mutant cells. The cell apoptosis was observed in GNE mutation specific manner. An activator of HSP70 chaperone, BGP-15, rescued the phenotypic defects due to GNE mutation, thereby, reducing protein aggregation significantly. The results were further validated in rat skeletal muscle cell lines carrying single Gne allele. Our study suggests that HSP70 activators can be a promising therapeutic target in the treatment of ultra-rare GNE Myopathy disease.