Case report: Two cases of Poirier-Bienvenu neurodevelopmental syndrome and review of literature

The Poirier-Bienvenu neurodevelopmental syndrome (POBINDS) is a rare disease caused by mutations in the CSNK2B gene, which is characterized by intellectual disability and early-onset epilepsy. Mosaicism has not been previously reported in CSNK2B gene. POBINDS is autosomal dominant and almost all reported cases were de novo variants. Here, we report two patients were diagnosed with POBINDS. Using Whole Exome Sequencing (WES), we detected two novel CSNK2B variants in the two unrelated individuals: c.634_635del (p.Lys212AspfsTer33) and c.142C > T (p.Gln48Ter) respectively. Both of them showed mild developmental delay with early-onset and clustered seizures. The patient with c.634_635del(p.Lys212AspfsTer33) variant was mutant mosaicism, and the proportion of alleles in peripheral blood DNA was 28%. Further, the literature of patients with a de novo mutation of the CSNK2B gene was reviewed, particularly seizure semiology and genotype-phenotype correlations

Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita (Hamilton) and its in vitro functional analysis

Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-γ genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-γ coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-γ genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-γ) as an adjuvant

Kill one or kill the many: Interplay between mitophagy and apoptosis

Mitochondria are key players of cellular metabolism, Ca2+ homeostasis, and apoptosis. The functionality of mitochondria is tightly regulated, and dysfunctional mitochondria are removed via mitophagy, a specialized form of autophagy that is compromised in hereditary forms of Parkinson's disease. Through mitophagy, cells are able to cope with mitochondrial stress until the damage becomes too great, which leads to the activation of proapoptotic BCL-2 family proteins located on the outer mitochondrial membrane. Active pro-apoptotic BCL-2 proteins facilitate the release of cytochrome c from the mitochondrial intermembrane space (IMS) into the cytosol, committing the cell to apoptosis by activating a cascade of cysteinyl-aspartate specific proteases (caspases). We are only beginning to understand how the choice between mitophagy and the activation of caspases is determined on the mitochondrial surface. Intriguingly in neurons, caspase activation also plays a non-apoptotic role in synaptic plasticity. Here we review the current knowledge on the interplay between mitophagy and caspase activation with a special focus on the central nervous system

Kill one or kill the many: interplay between mitophagy and apoptosis

Mitochondria are key players of cellular metabolism, Ca2+ homeostasis, and apoptosis. The functionality of mitochondria is tightly regulated, and dysfunctional mitochondria are removed via mitophagy, a specialized form of autophagy that is compromised in hereditary forms of Parkinson's disease. Through mitophagy, cells are able to cope with mitochondrial stress until the damage becomes too great, which leads to the activation of proapoptotic BCL-2 family proteins located on the outer mitochondrial membrane. Active pro-apoptotic BCL-2 proteins facilitate the release of cytochrome c from the mitochondrial intermembrane space (IMS) into the cytosol, committing the cell to apoptosis by activating a cascade of cysteinyl-aspartate specific proteases (caspases). We are only beginning to understand how the choice between mitophagy and the activation of caspases is determined on the mitochondrial surface. Intriguingly in neurons, caspase activation also plays a non-apoptotic role in synaptic plasticity. Here we review the current knowledge on the interplay between mitophagy and caspase activation with a special focus on the central nervous system

PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy

PPTC7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass and metabolic capacity with elevated hepatic triglyceride accumulation. Pptc7 knockout animals exhibit increased expression of the mitophagy receptors BNIP3 and NIX, and Pptc

Probing the protein homeostasis mechanisms in long-lived naked mole-rats

The naked mole-rat (NMR) is a fascinating animal which has unique biological features including eusociality, strict subterranean inhabitation and poikilothermy. It is the longest-living rodent, showing negligible senescence over the majority of its lifespan and high resistance to diseases such as cancer and neurodegeneration. This animal is therefore a compelling system for understanding ageing and age-related diseases. Recent evidence suggests that protein homeostasis (proteostasis) mechanisms may play a vital role in mediating the resistance to multiple forms of stress and diseases and, subsequently, contribute to the exceptional longevity of the NMR. However, this view has been predominately based on protein-level and/or cell viability analyses, and our knowledge is still limited about the modulation of proteotoxic stress responses at the transcriptional level due to a lack of reliable and validated molecular tools. This thesis sets out to develop, optimise and apply new methods to investigate two important and complex proteostatic mechanisms, namely the unfolded protein response (UPR) in the endoplasmic reticulum (ER) and macroautophagy (autophagy), in the NMR. Using these methodologies, the effects of pharmacologically induced in vitro stress and the effects of disease-related neurotoxic protein species on the UPR and autophagy in NMR fibroblasts were investigated. In Chapter 3, RNA-based methods, including an Xbp1 splicing assay and quantitative PCR (RT-qPCR) assays were successfully established to probe the activation and outputs of the UPR in a NMR kidney fibroblast cell line in response to tunicamycin (TU) and thapsigargin (TG)-induced ER stress. In Chapter 4, differences between the UPR of NMR kidney fibroblasts and mouse homologues were identified, where a notably higher threshold of pharmacologically induced UPR activation was observed in the NMR under conditions of mild ER stress. In Chapter 5, LC3B turnover and transcriptional changes of autophagy markers under rapamycin (RA) and chloroquine (CQ)-treated conditions were monitored in an NMR skin fibroblast cell line, where the sensitivity of the NMR skin fibroblasts to CQ, when compared to NMR kidney fibroblasts and mouse NIH3T3 embryonic cells, seemed to be partly attributed to the downregulation of TFEB, a master transcription factor of autophagy. In Chapter 6, the effects of amyloid-beta (Aβ) and α-synuclein oligomers, which are believed to be the major pathogenic species of Alzheimer’s and Parkinson’s diseases, respectively, on the UPR and/or autophagy were investigated in NMR and mouse cells using a combination of molecular and cellular tools. Although no significant changes of UPR markers were observed under Aβ oligomer-treated conditions, the chronic toxicity of wild-type α-synuclein oligomers seemed to be associated with downregulation of genes encoding ER chaperones and autophagy proteins. In Chapter 7, we demonstrate the utility of rational design to create a protein-specific binding probe for the NMR LC3B protein by introducing a peptide derived from a LC3-interacting region (LIR) motif into the inter-repeat loop of a consensus-designed tetratricopeptide repeat protein (CTPR). The results provide proof-of-concept validation of using CTPR-based probes to detect proteins in emerging animal models. Having established a set of reliable methods to investigate the molecular details of the UPR and autophagy in the NMR, we have demonstrated unique features of the NMR, at the transcriptional level, when different forms of in vitro stress are employed. Exploiting these assays to measure the UPR and autophagy, as well as other proteostastic mechanisms, in the NMR under more disease-relevant conditions, may ultimately shed light on therapeutic developments to combat age-related neurodegenerative diseases

The Role of TFEB and TFE3 in Mediating Mitochondrial and Lysosomal Adaptations in Skeletal Muscle

Skeletal muscle adapts to external stimuli to meet metabolic and energetic needs imposed on it. As a highly metabolic tissue, mitochondria are the energetic cores of the cell and are central to the adaptive nature of muscle. Essential to the maintenance of mitochondria is the process of mitophagy, a selective form of autophagy through which damaged mitochondria are removed and degraded via the lysosome. Lysosomes and autophagy machinery are regulated by transcription factors, TFEB and TFE3, that are responsive to cellular stresses including exercise, disuse and starvation. Our work aimed to address the role of TFEB and TFE3 in mediating the adaptability of mitochondria in response to exercise and disuse. To understand the role of TFEB and TFE3 in mediating the effects of exercise, we employed an in vitro model and silenced the expression of TFEB and TFE3. While the absence of TFEB or TFE3 alone impacted the mitophagic response to a single bout of contractile activity, mitochondrial and lysosomal function improved with repeated bouts. These data support the notion that exercise stimulates multifaceted and often redundant signaling pathways to promote adaptations. However, the absence of TFEB and TFE3 together abolished functional mitochondrial and lysosomal adaptations to contractile activity, indicating that both TFEB and TFE3 together are required for adaptations. We also sought to evaluate the role of TFE3 in atrophic conditions using denervation of the sciatic nerve as a model of disuse in both males and females. Basally, females exhibited increased lysosomal content, higher mitophagy flux and improved mitochondrial function. In response to denervation however, females appeared to preferentially preserve mitochondrial content at the expense of function by reducing mitophagy flux. Curiously, the absence of TFE3 in vivo preserved muscle mass in males and mitochondrial content in both sexes following denervation but this in turn increased mitochondrial dysfunction similar to wildtype females. The significance of this work is that we provide further evidence of how lysosomes and mitochondrial turnover mediate mitochondrial adaptations to both positive and negative stimuli. Our data also highlight the importance of investigating the effect of biological sex, revealing distinct mitochondrial and lysosomal phenotypes in males and females

Functional and Skeletal Muscle Impairments In Progressive Diabetic CKD

1-in-3 persons with type 2 diabetes (T2DM) develop chronic kidney disease (CKD), which is characterized by progressive renal dysfunction leading to end-stage renal disease. In response to elevated blood glucose and systemic inflammation of diabetes, a process of active thickening of the renal glomerular basement membrane ensues with concomitant damage to the structural supports (podocytes) of the kidneyճ filtration barrier. This results in impaired renal filtration. The metabolic sequelea of T2DM and CKD also, synergistically, alter skeletal muscleճ degradative pathways, satellite cell function (muscle reparative cells), and mitochondrial health (muscle energetic machinery) -- resulting in muscle breakdown, poor muscle quality, and exercise intolerance, and immobility that exacerbates CKD. The temporal nature and extent of these changes in CKD, however, remains unknown. With mandates from the Center for Disease Control (CDC) urging avenues of treatment that impede the progression of CKD, it is critical now, more than ever, to gain a better understanding of the factors that contribute to disease progression. This will inform more effective targeted interventions. We therefore aim to determine how renal dysfunction dictates the activity of muscle degredative pathways, the status of muscle reparative cells, and the energetic production of muscle, to ultimately influence muscle quality, performance and physical mobility. This will be determined across stages of CKD. In chapter 1, we examine how CKD progression in T2DM, impacts muscle performance and physical function. Our results suggest that muscle performance of the lower extremity, particularly the quadriceps, and physical function decline in-parallel with progression of CKD in T2DM, with these declines becoming clinically evident in stage 3. Moreover, we find that CKD-stage, and renal filtration/function (eGFR) are both significant predictors of overall physical function, with increasing CKD stage/worsening kidney filtration predicting worse functional mobility. In chapter 2, we examine the CKD-stage specific functional status of skeletal muscle mitochondrial ATP production, and electron transport chain kinetics, as these are critical cellular processes to fuel muscle cross-bridge cycling, contraction and movement. We find that intrinsic skeletal muscle mitochondrial electron transport chain function is reduced with progression of CKD, with significant reductions in ATP-production capacity emerging as early as stage 3 CKD. Moreover, these changes may derive from transcriptome-level alterations in gene networks governing muscle mitochondrial health and function. In Chapter 3, we examine muscle regenerative and maintenance capacity in relationship to CKD progression. We find the muscle-resident satellite cell pool to decline significantly with CKD progression, and exhibit impaired myogenic capacity with altered gene activation patterns, that relate strongly to findings of poor muscle quality with progressive CKD stage. Using transcriptomics, we report significant dowregulation in gene networks that influence muscle SC behavior and myogenesis. Overall, our data suggests that the progression of diabetes-induced chronic kidney disease, is paralleled by impairments in skeletal muscle ATP-producing capacity, and these energetic deficits are accompanied by CKD-associated reductions in muscle SC abundance, and reparative function. Both changes perhaps stem from alterations in gene pathway expression that is imparted by the altered uremic environment. These impairments may promote the development of poor muscle quality and performance that ultimately impairs functional capacity, even in middle-stage CKD

RETREG3/FAM134C phosphorylation by CSNK2 regulates reticulophagy during starvation

Starvation is the most potent physiological inducer of autophagy, the catabolic process which degrades unessential cytosolic components to sustain cellular homeostasis and survival. During starvation, the mechanisms of autophagy activation have been extensively investigated; however, less is known about how substrate selection occurs. In this punctum, we summarize our recent findings that delineate a novel signaling pathway that promotes selective autophagic removal of parts of the endoplasmic reticulum (reticulophagy) during starvation. We demonstrate that the inhibition of MTORC1 results in the activation of the reticulophagy receptor RETREG3/FAM134C by preventing its phosphorylation by CSNK2/CK2. In vivo, RETREG3 depletion impairs MTORC1-dependent regulation of lipid metabolism in liver. Last, we describe a novel approach to study selective autophagy in vivo, which might be exploited to identify novel physiological roles of autophagy