Child Neurology: Progressive Cerebellar Atrophy and Retinal Dystrophy: Clues to an Ultrarare ACO2-Related Neurometabolic Diagnosis.

Pathogenic biallelic variants in ACO2, which encodes the enzyme mitochondrial aconitase, are associated with the very rare diagnosis of ACO2-related infantile cerebellar retinal degeneration (OMIM 614559). We describe the diagnostic odyssey of a 4-year-old female patient with profound global developmental delays, microcephaly, severe hypotonia, retinal dystrophy, seizures, and progressive cerebellar atrophy. Whole-exome sequencing revealed 2 variants in ACO2; c.2105_2106delAG (p.Gln702ArgfsX9), a likely pathogenic variant, and c.988C>T (p.Pro330Ser) which was classified as a variant of uncertain significance (VUS). While the VUS was confirmed to be maternally inherited, the phase of the other variant could not be confirmed due to lack of a paternal sample. Functional biochemical studies were performed on a research basis to clarify the interpretation of the VUS, which enabled clinical confirmation of the diagnosis of ACO2-related infantile cerebellar retinal degeneration for our patient.

Effect of hyperglycemia on tbx5a and nppa gene expression and its correlation to structural and functional changes in developing zebrafish heart.

The objective of the current study is to analyze the effects of gestational diabetes on structural and functional changes in correlation with these two essential regulators of developing hearts in vivo using zebrafish embryos. We employed fertilized zebrafish embryos exposed to a hyperglycemic condition of 25 mM glucose for 96 h postfertilization. The embryos were subjected to various structural and functional analyses in a time-course manner. The data showed that exposure to high glucose significantly affected the embryo's size, heart length, heart rate, and looping of the heart compared to the control. Further, we observed an increased incidence of ventricular standstill and valvular regurgitation with a marked reduction of peripheral blood flow in the high glucose-exposed group compared to the control. In addition, the histological data showed that the high-glucose exposure markedly reduced the thickness of the wall and the number of cardiomyocytes in both atrium and ventricles. We also observed striking alterations in the pericardium like edema, increase in diameter with thinning of the wall compared to the control group. Interestingly, the expression of tbx5a and nppa was increased in the early development and found to be repressed in the later stage of development in the hyperglycemic group compared to the control. In conclusion, the developing heart is more susceptible to hyperglycemia in the womb, thereby showing various developmental defects possibly by altering the expression of crucial gene regulators such as tbx5a and nppa.

Inhibition of mitochondrial LonP1 protease by allosteric blockade of ATP binding and hydrolysis via CDDO and its derivatives.

The mitochondrial protein LonP1 is an ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Biallelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its -methyl and -imidazole derivatives reversibly inhibit LonP1 by a noncompetitive mechanism, blocking ATP-hydrolysis and thus proteolysis. By contrast, we found that CDDO-anhydride inhibits the LonP1 ATPase competitively. Docking of CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the ATP-binding site. The binding site of CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (CODAS) syndrome, which ablated inhibition. CDDO failed to inhibit the ATPase activity of the purified 26S proteasome, which like LonP1 belongs to the AAA+ superfamily of ATPases Associated with diverse cellular Activities, suggesting that CDDO shows selectivity within this family of ATPases. Furthermore, we show that noncytotoxic concentrations of CDDO derivatives in cultured cells inhibited LonP1, but not the 26S proteasome. Taken together, these findings provide insights for future development of LonP1-specific inhibitors with chemotherapeutic potential.

Understanding diabetes-induced cardiomyopathy from the perspective of renin angiotensin aldosterone system.

Experimental and clinical evidence suggests that diabetic subjects are predisposed to a distinct cardiovascular dysfunction, known as diabetic cardiomyopathy (DCM), which could be an autonomous disease independent of concomitant micro and macrovascular disorders. DCM is one of the prominent causes of global morbidity and mortality and is on a rising trend with the increase in the prevalence of diabetes mellitus (DM). DCM is characterized by an early left ventricle diastolic dysfunction associated with the slow progression of cardiomyocyte hypertrophy leading to heart failure, which still has no effective therapy. Although the well-known "Renin Angiotensin Aldosterone System (RAAS)" inhibition is considered a gold-standard treatment in heart failure, its role in DCM is still unclear. At the cellular level of DCM, RAAS induces various secondary mechanisms, adding complications to poor prognosis and treatment of DCM. This review highlights the importance of RAAS signaling and its major secondary mechanisms involving inflammation, oxidative stress, mitochondrial dysfunction, and autophagy, their role in establishing DCM. In addition, studies lacking in the specific area of DCM are also highlighted. Therefore, understanding the complex role of RAAS in DCM may lead to the identification of better prognosis and therapeutic strategies in treating DCM.

Roles of host mitochondria in the development of COVID-19 pathology: Could mitochondria be a potential therapeutic target?

The recent emergence of severe acute respiratory syndrome-Corona Virus 2 (SARS-CoV-2) in late 2019 and its spread worldwide caused an acute pandemic of Coronavirus disease 19 (COVID-19). Since then, COVID-19 has been under intense scrutiny as its outbreak led to significant changes in healthcare, social activities, and economic settings worldwide. Although angiotensin-converting enzyme-2 (ACE-2) receptor is shown to be the primary port of SARS-CoV-2 entry in cells, the mechanisms behind the establishment and pathologies of COVID-19 are poorly understood. As recent studies have shown that host mitochondria play an essential role in virus-mediated innate immune response, pathologies, and infection, in this review, we will discuss in detail the entry and progression of SARS-CoV-2 and how mitochondria could play roles in COVID-19 disease. We will also review the potential interactions between SARS-CoV-2 and mitochondria and discuss possible treatments, including whether mitochondria as a potential therapeutic target in COVID-19. Understanding SARS-CoV-2 and mitochondrial interactions mediated virus establishment, inflammation, and other consequences may provide a unique mechanism and conceptual advancement in finding a novel treatment for COVID-19.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy.

[Figure: see text].

Proteomic analysis of mitochondrial biogenesis in cardiomyocytes differentiated from human induced pluripotent stem cells.

Mitochondria play key roles in the differentiation and maturation of human cardiomyocytes (CMs). As human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold potential in the treatment of heart diseases, we sought to identify key mitochondrial pathways and regulators, which may provide targets for improving cardiac differentiation and maturation. Proteomic analysis was performed on enriched mitochondrial protein extracts isolated from hiPSC-CMs differentiated from dermal fibroblasts (dFCM) and cardiac fibroblasts (cFCM) at time points between 12 and 115 days of differentiation, and from adult and neonatal mouse hearts. Mitochondrial proteins with a twofold change at time points up to 120 days relative to 12 days were subjected to ingenuity pathway analysis (IPA). The highest upregulation was in metabolic pathways for fatty acid oxidation (FAO), the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and branched chain amino acid (BCAA) degradation. The top upstream regulators predicted to be activated were peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1-α), the insulin receptor (IR), and the retinoblastoma protein (Rb1) transcriptional repressor. IPA and immunoblotting showed upregulation of the mitochondrial LonP1 protease-a regulator of mitochondrial proteostasis, energetics, and metabolism. LonP1 knockdown increased FAO in neonatal rat ventricular cardiomyocytes (nRVMs). Our results support the notion that LonP1 upregulation negatively regulates FAO in cardiomyocytes to calibrate the flux between glucose and fatty acid oxidation. We discuss potential mechanisms by which IR, Rb1, and LonP1 regulate the metabolic shift from glycolysis to OXPHOS and FAO. These newly identified factors and pathways may help in optimizing the maturation of iPSC-CMs.

Cell stress management by the mitochondrial LonP1 protease - Insights into mitigating developmental, oncogenic and cardiac stress.

Mitochondrial LonP1 is an essential stress response protease that mediates mitochondrial proteostasis, metabolism and bioenergetics. Homozygous and compound heterozygous variants in the LONP1 gene encoding the LonP1 protease have recently been shown to cause a diverse spectrum of human pathologies, ranging from classical mitochondrial disease phenotypes, profound neurologic impairment and multi-organ dysfunctions, some of which are uncommon to mitochondrial disorders. In this review, we focus primarily on human LonP1 and discuss findings, which demonstrate its multidimensional roles in maintaining mitochondrial proteostasis and adapting cells to metabolic flux and stress during normal physiology and disease processes. We also discuss emerging roles of LonP1 in responding to developmental, oncogenic and cardiac stress.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

In-silico design and synthesis of N9-substituted ß-Carbolines as PLK-1 inhibitors and their in-vitro/in-vivo tumor suppressing evaluation.

A new series of ß-Carboline/Schiff bases was designed, synthesized, characterised and biologically evaluated as inhibitors of PLK-1. The synthesized compounds exhibited strong to moderate cytotoxic activities against NCI-60 panel cell assay. Compound SB-2 was the most potent, particularly against colon with GI50 of 3-45 µM on NCI-60 panel cell lines. SB-2 selectively inhibited PLK-1 at 15 µM on KinomeScan screening. It also showed a dose-dependent cell cycle arrest at S/G2 phase on HCT-116 and induced apoptosis by the activation of procaspase-3 and cleaved PARP. Further, the antitumor studies on DLA and EAC model revealed that SB-2, at 100 mg/kg/bd.wt significantly increased their average lifespan. Further, a decrease in the body weight of the tumor-bearing mice was also observed when compared to the tumor controlled mice. SB-2 thus shows good potential as antitumor agent.

Mitochondrial LonP1 protects cardiomyocytes from ischemia/reperfusion injury in vivo.

RATIONALE: LonP1 is an essential mitochondrial protease, which is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. However, the importance of LonP1 during cardiac stress is largely unknown. OBJECTIVE: To determine the functions of LonP1 during ischemia/reperfusion (I/R) injury in vivo, and hypoxia-reoxygenation (H/R) stress in vitro. METHODS AND RESULTS: LonP1 was induced 2-fold in wild-type mice during cardiac ischemic preconditioning (IPC), which protected the heart against ischemia-reperfusion (I/R) injury. In contrast, haploinsufficiency of LonP1 (LONP1+/-) abrogated IPC-mediated cardioprotection. Furthermore, LONP1+/- mice showed significantly increased infarct size after I/R injury, whereas mice with 3-4 fold cardiac-specific overexpression of LonP1 (LonTg) had substantially smaller infarct size and reduced apoptosis compared to wild-type controls. To investigate the mechanisms underlying cardioprotection, LonTg mice were subjected to ischemia (45 min) followed by short intervals of reperfusion (10, 30, 120 min). During early reperfusion, the left ventricles of LonTg mice showed substantially reduced oxidative protein damage, maintained mitochondrial redox homeostasis, and showed a marked downregulation of both Complex I protein level and activity in contrast to NTg mice. Conversely, when LonP1 was knocked down in isolated neonatal rat ventricular myocytes (NRVMs), an up-regulation of Complex I subunits and electron transport chain (ETC) activities was observed, which was associated with increased superoxide production and reduced respiratory efficiency. The knockdown of LonP1 in NRVMs caused a striking dysmorphology of the mitochondrial inner membrane, mitochondrial hyperpolarization and increased hypoxia-reoxygenation (H/R)-activated apoptosis. Whereas, LonP1 overexpression blocked H/R-induced cell death. CONCLUSIONS: LonP1 is an endogenous mediator of cardioprotection. Our findings show that upregulation of LonP1 mitigates cardiac injury by preventing oxidative damage of proteins and lipids, preserving mitochondrial redox balance and reprogramming bioenergetics by reducing Complex I content and activity. Mechanisms that promote the upregulation of LonP1 could be beneficial in protecting the myocardium from cardiac stress and limiting I/R injury.

Bi-allelic mutations of LONP1 encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Acetylation and phosphorylation of human TFAM regulate TFAM-DNA interactions via contrasting mechanisms.

Mitochondrial transcription factor A (TFAM) is essential for the maintenance, expression and transmission of mitochondrial DNA (mtDNA). However, mechanisms for the post-translational regulation of TFAM are poorly understood. Here, we show that TFAM is lysine acetylated within its high-mobility-group box 1, a domain that can also be serine phosphorylated. Using bulk and single-molecule methods, we demonstrate that site-specific phosphoserine and acetyl-lysine mimics of human TFAM regulate its interaction with non-specific DNA through distinct kinetic pathways. We show that higher protein concentrations of both TFAM mimics are required to compact DNA to a similar extent as the wild-type. Compaction is thought to be crucial for regulating mtDNA segregation and expression. Moreover, we reveal that the reduced DNA binding affinity of the acetyl-lysine mimic arises from a lower on-rate, whereas the phosphoserine mimic displays both a decreased on-rate and an increased off-rate. Strikingly, the increased off-rate of the phosphoserine mimic is coupled to a significantly faster diffusion of TFAM on DNA. These findings indicate that acetylation and phosphorylation of TFAM can fine-tune TFAM-DNA binding affinity, to permit the discrete regulation of mtDNA dynamics. Furthermore, our results suggest that phosphorylation could additionally regulate transcription by altering the ability of TFAM to locate promoter sites.

Effect of densely ionizing radiation on cardiomyocyte differentiation from human-induced pluripotent stem cells.

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.

HSP60 Takes a Hit: Inhibition of Mitochondrial Protein Folding.

In this issue of Cell Chemical Biology,Wiechmann et al. (2017) identify mitochondrial chaperonin HSP60 as a direct target of myrtucommulone (MC), a nonprenylated acylphloroglucinol that is well known for its apoptotic activity in cancer cells. The authors propose MC as a chemical probe to study HSP60 biology and a potential chemotherapeutic agent in treating cancer and other HSP60-associated diseases.

CODAS syndrome is associated with mutations of LONP1, encoding mitochondrial AAA+ Lon protease.

CODAS syndrome is a multi-system developmental disorder characterized by cerebral, ocular, dental, auricular, and skeletal anomalies. Using whole-exome and Sanger sequencing, we identified four LONP1 mutations inherited as homozygous or compound-heterozygous combinations among ten individuals with CODAS syndrome. The individuals come from three different ancestral backgrounds (Amish-Swiss from United States, n = 8; Mennonite-German from Canada, n = 1; mixed European from Canada, n = 1). LONP1 encodes Lon protease, a homohexameric enzyme that mediates protein quality control, respiratory-complex assembly, gene expression, and stress responses in mitochondria. All four pathogenic amino acid substitutions cluster within the AAA(+) domain at residues near the ATP-binding pocket. In biochemical assays, pathogenic Lon proteins show substrate-specific defects in ATP-dependent proteolysis. When expressed recombinantly in cells, all altered Lon proteins localize to mitochondria. The Old Order Amish Lon variant (LONP1 c.2161C>G[p.Arg721Gly]) homo-oligomerizes poorly in vitro. Lymphoblastoid cell lines generated from affected children have (1) swollen mitochondria with electron-dense inclusions and abnormal inner-membrane morphology; (2) aggregated MT-CO2, the mtDNA-encoded subunit II of cytochrome c oxidase; and (3) reduced spare respiratory capacity, leading to impaired mitochondrial proteostasis and function. CODAS syndrome is a distinct, autosomal-recessive, developmental disorder associated with dysfunction of the mitochondrial Lon protease.

Phosphorylation of human TFAM in mitochondria impairs DNA binding and promotes degradation by the AAA+ Lon protease.

Human mitochondrial transcription factor A (TFAM) is a high-mobility group (HMG) protein at the nexus of mitochondrial DNA (mtDNA) replication, transcription, and inheritance. Little is known about the mechanisms underlying its posttranslational regulation. Here, we demonstrate that TFAM is phosphorylated within its HMG box 1 (HMG1) by cAMP-dependent protein kinase in mitochondria. HMG1 phosphorylation impairs the ability of TFAM to bind DNA and to activate transcription. We show that only DNA-free TFAM is degraded by the Lon protease, which is inhibited by the anticancer drug bortezomib. In cells with normal mtDNA levels, HMG1-phosphorylated TFAM is degraded by Lon. However, in cells with severe mtDNA deficits, nonphosphorylated TFAM is also degraded, as it is DNA free. Depleting Lon in these cells increases levels of TFAM and upregulates mtDNA content, albeit transiently. Phosphorylation and proteolysis thus provide mechanisms for rapid fine-tuning of TFAM function and abundance in mitochondria, which are crucial for maintaining and expressing mtDNA.

Segregation of sperm subpopulations in normozoospermic infertile men.

Sperm function is essential for fertilization and embryogenesis yet semen contain a heterogeneous population of sperm. This study was designed to evaluate two different sperm populations separated by the density gradient method. Semen from 25 idiopathic normozoospermic infertile men was processed by double density gradient centrifugation and evaluated for sperm present in the 50% (upper) layer and the 90% (lower) layer for reactive oxygen species (ROS), sperm chromatin integrity, and morphology. The population of sperm in the 90% layer showed significantly lower ROS levels (22.90 (0.92, 85.32) vs. 382.03 (158.30, 1409.51) and lower DNA fragmentation index (DFI) (24.26 (22.54, 25.50) vs. 29.93 (28.48, 31.25) and higher number of sperm with normal morphology (55 (45.0, 60.0) vs. 32.5 (20, 40) compared to sperm in the 50% layer. However, in the original raw semen, sperm DFI (27.02 (26.19, 27.76)) and percentage high DNA stainability (% HDS) (3.1 (2.40, 3.78)) cells were significantly higher compared to the 90% layer population. Density gradient separation of the sperm subpopulation from the original semen favors the selection of sperm with genome integrity, low levels of ROS, and normal morphology. Therefore presence of pathological sperm in the semen may disrupt the function of normal spermatozoa, and hence the selection of the normal sperm subpopulation may be a better candidate for assisted conception. Further studies are required to evaluate the gradient separated sperm population in assisted reproductive techniques (ART).