α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.     

Mitochondrial Dysfunction in Spinocerebellar Ataxia Type 3 Is Linked to VDAC1 Deubiquitination

Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.     

Molecular Pathogenesis of Pulmonary Fibrosis,with Focus on Pathways Related to TGF-β and the Ubiquitin-Proteasome Pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. During the past decade, novel pathogenic mechanisms of IPF have been elucidated that have shifted the concept of IPF from an inflammatory-driven to an epithelial-driven disease. Dysregulated repair responses induced by recurrent epithelial cell damage and excessive extracellular matrix accumulation result in pulmonary fibrosis. Although there is currently no curative therapy for IPF, two medications, pirfenidone and nintedanib, have been introduced based on understanding the pathogenesis of the disease. In this review, we discuss advances in understanding IPF pathogenesis, highlighting epithelial–mesenchymal transition (EMT), the ubiquitin-proteasome system, and endothelial cells. TGF-β is a central regulator involved in EMT and pulmonary fibrosis. HECT-, RING finger-, and U-box-type E3 ubiquitin ligases regulate TGF-β-Smad pathway-mediated EMT via the ubiquitin-proteasome pathway. p27 degradation mediated by the SCF-type E3 ligase, Skp2, contributes to the progression of pulmonary fibrosis by promotion of either mesenchymal fibroblast proliferation, EMT, or both. In addition to fibroblasts as key effector cells in myofibroblast differentiation and extracellular matrix deposition, endothelial cells also play a role in the processes of IPF. Endothelial cells can transform into myofibroblasts; therefore, endothelial–mesenchymal transition can be another source of myofibroblasts.     

A Commonly Used Biocide 2-N-octyl-4-isothiazolin-3-oneInduces Blood–Brain Barrier Dysfunction via Cellular Thiol Modification and Mitochondrial Damage

Isothiazolinone (IT) biocides are potent antibacterial substances commonly used as preservatives or disinfectants, and 2-n-Octyl-4-isothiazolin-3-one (OIT; octhilinone) is a common IT biocide that is present in leather products, glue, paints, and cleaning products. Although humans are exposed to OIT through personal and industrial use, the potentially deleterious effects of OIT on human health are still unknown. To investigate the effects of OIT on the vascular system, which is continuously exposed to xenobiotics through systemic circulation, we treated brain endothelial cells with OIT. OIT treatment significantly activated caspase-3-mediated apoptosis and reduced the bioenergetic function of mitochondria in a bEnd.3 cell-based in vitro blood–brain barrier (BBB) model. Interestingly, OIT significantly altered the thiol redox status, as evidenced by reduced glutathione levels and protein S-nitrosylation. The endothelial barrier function of bEnd.3 cells was significantly impaired by OIT treatment. OIT affected mitochondrial dynamics through mitophagy and altered mitochondrial morphology in bEnd.3 cells. N-acetyl cysteine significantly reversed the effects of OIT on the metabolic capacity and endothelial function of bEnd.3 cells. Taken together, we demonstrated that the alteration of the thiol redox status and mitochondrial damage contributed to OIT-induced BBB dysfunction, and we hope that our findings will improve our understanding of the potential hazardous health effects of IT biocides.     

Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet–Mediated Amyloid Aggregation in Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein. Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro. Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αII_bβ₃ activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C. Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.     

Rlip76: An Unexplored Player in Neurodegeneration and Alzheimer’s Disease?

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia in older people. AD is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional abnormalities, microRNA deregulation, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aβ) and phosphorylated tau (p-tau). AD occurs in two forms: early onset, familial AD and late-onset, sporadic AD. Causal factors are still unknown for a vast majority of AD patients. Genetic polymorphisms are proposed to contribute to late-onset AD via age-dependent increases in oxidative stress and mitochondrial abnormalities. Recent research from our lab revealed that reduced levels of Rlip76 induce oxidative stress, mitochondrial dysfunction and synaptic damage, leading to molecular and behavioral phenotypes resembling late-onset AD. Rlip76 is a multifunctional 76 kDa protein encoded by the RALBP1 gene, located on chromosome 18. Rlip is a stress-protective ATPase of the mercapturic acid pathway that couples clathrin-dependent endocytosis with the efflux of glutathione–electrophile conjugates. Rlip is evolutionarily highly conserved across species and is ubiquitously expressed in all tissues, including AD-affected brain regions, the cerebral cortex and hippocampus, where highly active neuronal metabolisms render the cells highly susceptible to intracellular oxidative damage. In the current article, we summarize molecular and cellular features of Rlip and how depleted Rlip may exacerbate oxidative stress, mitochondrial dysfunction and synaptic damage in AD. We also discuss the possible role of Rlip in aspects of learning and memory via axonal growth, dendritic remodeling, and receptor regulation. We conclude with a discussion of the potential for the contribution of genetic polymorphisms in Rlip to AD progression and the potential for Rlip-based therapies.     

Pathogenesis of Target Organ Damage in Hypertension: Role of Mitochondrial Oxidative Stress

Hypertension causes target organ damage (TOD) that involves vasculature, heart, brain and kidneys. Complex biochemical, hormonal and hemodynamic mechanisms are involved in the pathogenesis of TOD. Common to all these processes is an increased bioavailability of reactive oxygen species (ROS). Both in vitro and in vivo studies explored the role of mitochondrial oxidative stress as a mechanism involved in the pathogenesis of TOD in hypertension, especially focusing on atherosclerosis, heart disease, renal failure, cerebrovascular disease. Both dysfunction of mitochondrial proteins, such as uncoupling protein-2 (UCP2), superoxide dismutase (SOD) 2, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), calcium channels, and the interaction between mitochondria and other sources of ROS, such as NADPH oxidase, play an important role in the development of endothelial dysfunction, cardiac hypertrophy, renal and cerebral damage in hypertension. Commonly used anti-hypertensive drugs have shown protective effects against mitochondrial-dependent oxidative stress. Notably, few mitochondrial proteins can be considered therapeutic targets on their own. In fact, antioxidant therapies specifically targeted at mitochondria represent promising strategies to reduce mitochondrial dysfunction and related hypertensive TOD. In the present article, we discuss the role of mitochondrial oxidative stress as a contributing factor to hypertensive TOD development. We also provide an overview of mitochondria-based treatment strategies that may reveal useful to prevent TOD and reduce its progression.     

Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca²⁺-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson’s disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.     

Fabry Disease and the Heart: A Comprehensive Review

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by mutations of the GLA gene that result in a deficiency of the enzymatic activity of α-galactosidase A and consequent accumulation of glycosphingolipids in body fluids and lysosomes of the cells throughout the body. GB3 accumulation occurs in virtually all cardiac cells (cardiomyocytes, conduction system cells, fibroblasts, and endothelial and smooth muscle vascular cells), ultimately leading to ventricular hypertrophy and fibrosis, heart failure, valve disease, angina, dysrhythmias, cardiac conduction abnormalities, and sudden death. Despite available therapies and supportive treatment, cardiac involvement carries a major prognostic impact, representing the main cause of death in FD. In the last years, knowledge has substantially evolved on the pathophysiological mechanisms leading to cardiac damage, the natural history of cardiac manifestations, the late-onset phenotypes with predominant cardiac involvement, the early markers of cardiac damage, the role of multimodality cardiac imaging on the diagnosis, management and follow-up of Fabry patients, and the cardiac efficacy of available therapies. Herein, we provide a comprehensive and integrated review on the cardiac involvement of FD, at the pathophysiological, anatomopathological, laboratory, imaging, and clinical levels, as well as on the diagnosis and management of cardiac manifestations, their supportive treatment, and the cardiac efficacy of specific therapies, such as enzyme replacement therapy and migalastat.     

Targetable Pathways for Alleviating Mitochondrial Dysfunction in Neurodegeneration of Metabolic and Non-Metabolic Diseases

Many neurodegenerative and inherited metabolic diseases frequently compromise nervous system function, and mitochondrial dysfunction and oxidative stress have been implicated as key events leading to neurodegeneration. Mitochondria are essential for neuronal function; however, these organelles are major sources of endogenous reactive oxygen species and are vulnerable targets for oxidative stress-induced damage. The brain is very susceptible to oxidative damage due to its high metabolic demand and low antioxidant defence systems, therefore minimal imbalances in the redox state can result in an oxidative environment that favours tissue damage and activates neuroinflammatory processes. Mitochondrial-associated molecular pathways are often compromised in the pathophysiology of neurodegeneration, including the parkin/PINK1, Nrf2, PGC1α, and PPARγ pathways. Impairments to these signalling pathways consequently effect the removal of dysfunctional mitochondria, which has been suggested as contributing to the development of neurodegeneration. Mitochondrial dysfunction prevention has become an attractive therapeutic target, and there are several molecular pathways that can be pharmacologically targeted to remove damaged mitochondria by inducing mitochondrial biogenesis or mitophagy, as well as increasing the antioxidant capacity of the brain, in order to alleviate mitochondrial dysfunction and prevent the development and progression of neurodegeneration in these disorders. Compounds such as natural polyphenolic compounds, bioactive quinones, and Nrf2 activators have been reported in the literature as novel therapeutic candidates capable of targeting defective mitochondrial pathways in order to improve mitochondrial function and reduce the severity of neurodegeneration in these disorders.     

Analysis of miR-9-5p,miR-124-3p,miR-21-5p,miR-138-5p,and miR-1-3p in Glioblastoma Cell Lines and Extracellular Vesicles

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM’s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.     

Parkinson’s Disease Etiology: Insights and Associations with Phosphate Toxicity

The present paper investigated the association of Parkinson’s disease etiology with phosphate toxicity, a pathophysiological condition in which dysregulated phosphate metabolism causes excessive inorganic phosphate sequestration in body tissue that damages organ systems. Excessive phosphate is proposed to reduce Complex I function of the mitochondrial electron transport chain in Parkinson’s disease and is linked to opening of the mitochondrial permeability transition pore, resulting in increased reactive oxygen species, inflammation, DNA damage, mitochondrial membrane depolarization, and ATP depletion causing cell death. Parkinson’s disease is associated with α-synuclein and Lewy body dementia, a secondary tauopathy related to hyperphosphorylation of tau protein, and tauopathy is among several pathophysiological pathways shared between Parkinson’s disease and diabetes. Excessive phosphate is also associated with ectopic calcification, bone mineral disorders, and low levels of serum vitamin D in patients with Parkinson’s disease. Sarcopenia and cancer in Parkinson’s disease patients are also associated with phosphate toxicity. Additionally, Parkinson’s disease benefits are related to low dietary phosphate intake. More studies are needed to investigate the potential mediating role of phosphate toxicity in the etiology of Parkinson’s disease.     

Autophagy Signaling by Neural-Induced Human Adipose Tissue-Derived Stem Cell-Conditioned Medium during Rotenone-Induced Toxicity in SH-SY5Y Cells

Rotenone (ROT) inhibits mitochondrial complex I, leading to reactive oxygen species formation, which causes neurodegeneration and alpha-synuclein (α-syn) aggregation and, consequently, Parkinson’s disease. We previously found that a neurogenic differentiated human adipose tissue-derived stem cell-conditioned medium (NI-hADSC-CM) was protective against ROT-induced toxicity in SH-SY5Y cells. In the present study, ROT significantly decreased the phospho (p)-mTORC1/total (t)-mTOR, p-mTORC2/t-mTOR, and p-/t-ULK1 ratios and the ATG13 level by increasing the DEPTOR level and p-/t-AMPK ratio. Moreover, ROT increased the p-/t-Akt ratio and glycogen synthase kinase-3β (GSK3β) activity by decreasing the p-/t-ERK1/2 ratios and beclin-1 level. ROT also promoted the lipidation of LC3B-I to LC3B-II by inducing autophagosome formation in Triton X-100-soluble and -insoluble cell lysate fractions. Additionally, the levels of ATG3, 5, 7, and 12 were decreased, along with those of lysosomal LAMP1, LAMP2, and TFEB, leading to lysosomal dysfunction. However, NI-hADSC-CM treatment increased the p-mTORC1, p-mTORC2, p-ULK1, p-Akt, p-ERK1/2, ATG13, and beclin-1 levels and decreased the p-AMPK level and GSK3β activity in response to ROT-induced toxicity. Additionally, NI-hADSC-CM restored the LC3B-I level, increased the p62 level, and normalized the ATG and lysosomal protein amounts to control levels. Autophagy array revealed that the secreted proteins in NI-hADSC-CM could be crucial in the neuroprotection. Taken together, our results showed that the neuroprotective effects of NI-hADSC-CM on the autophagy signaling pathways could alleviate the aggregation of α-syn in Parkinson’s disease and other neurodegenerative disorders.     

P62 Links the Autophagy Pathway and the Ubiquitin–Proteasome System in Endothelial Cells during Atherosclerosis

Among autophagy-related molecules, p62/SQSTM1 is an adaptor for identifying and delivering intracellular cargo for degradation. Since ubiquitination is reversible, it has a switch role in autophagy. Ubiquitination is also involved in regulating autophagy in a timely manner. This study aimed to elucidate how p62-mediated autophagy is regulated in human endothelial cells and macrophages under atherosclerotic conditions, focusing on the lysosomal and proteasomal pathways. Co-cultured HUVECs and THP-1 cells were exposed to oxLDL (50 μg/mL) and autophagy was assessed. To downregulate p62, siRNA was administered, and the E3 ligases were inhibited by Heclin or MLN4924 treatment under the condition that cellular inflammatory processes were stimulated by oxLDL simultaneously initiated autophagy. Downregulating p62 induced an alternative degradation system, and the E3 ligases were found to be involved in the progression of atherosclerosis. Collectively, the present study demonstrated that the endothelial lipid accumulation under atherosclerotic conditions was caused by lysosomal dysfunction associated with autophagy.     

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.     

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe–4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron–sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron–sulfur cluster-binding region of BOLA3, but without abolishing [2Fe–2S]²⁺ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron–sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3–GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.