AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.

Improving Neuroplasticity through Robotic Verticalization Training in Patients with Minimally Conscious State: A Retrospective Study.

In disorders of consciousness, verticalization is considered an effective type of treatment to improve motor and cognitive recovery. Our purpose is to investigate neurophysiological effects of robotic verticalization training (RVT) in patients with minimally conscious state (MCS). Thirty subjects affected by MCS due to traumatic or vascular brain injury, attending the intensive Neurorehabilitation Unit of the IRCCS Neurolesi (Messina, Italy), were included in this retrospective study. They were equally divided into two groups: the control group (CG) received traditional verticalization with a static bed and the experimental group (EG) received advanced robotic verticalization using the Erigo device. Each patient was evaluated using both clinical scales, including Levels of Cognitive Functioning (LCF) and Functional Independence Measure (FIM), and quantitative EEG pre (T0) and post each treatment (T1). The treatment lasted for eight consecutive weeks, and sessions were held three times a week, in addition to standard neurorehabilitation. In addition to a notable improvement in clinical parameters, such as functional (FIM) (p < 0.01) and cognitive (LCF) (p < 0.01) outcomes, our findings showed a significant modification in alpha and beta bands post-intervention, underscoring the promising effect of the Erigo device to influence neural plasticity and indicating a noteworthy difference between pre-post intervention. This was not observed in the CG. The observed changes in alpha and beta bands underscore the potential of the Erigo device to induce neural plasticity. The device's custom features and programming, tailored to individual patient needs, may contribute to its unique impact on brain responses.

Special Issue "Mitochondrial Respiration in Physiology and Pathology".

Mitochondria are key organelles that regulate several functions essential for maintaining cellular homeostasis [...].

CSF N-Glycomics Using High-Throughput UPLC-ESI Techniques in Alzheimer's Disease.

In this chapter, we will present a high-throughput method applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. This methodology is based on a commercial equipment developed by WATERS™ to speed up N-deglycosylation and N-glycan labeling of glycoproteins of pharmaceutical and biological interest such as monoclonal antibodies. This analytical kit is sold under the trade name of RapiFluor-MS (RFMS). We have slightly modified the methodology, increasing the glycosylation time and using a high-resolution mass analyzer for the analysis of CSF N-glycans, thus obtaining a high-throughput method (up to 96 samples simultaneously), mass accuracy better than 5 ppm, and the ability to separate and identify isomers.

CSF N-Glycomics Using MALDI MS Techniques.

In this chapter, we will present the methodology currently applied in our laboratory for the structural elucidation of the cerebrospinal fluid (CSF) N-glycome. N-glycans are released from denatured carboxymethylated glycoproteins by digestion with peptide-N-glycosidase F (PNGase F) and purified using both C18 Sep-Pak® and porous graphitized carbon (PGC) HyperSep™ Hypercarb™ solid phase extraction (SPE) cartridges. The glycan pool is subsequently permethylated to increase mass spectrometry sensitivity. Molecular assignments are performed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis considering either the protein N-linked glycosylation pathway or MALDI TOF MS/MS data. Each stage has been optimized to obtain high-quality mass spectra in reflector mode with an optimal signal-to-noise ratio up to m/z 4800. This method has been successfully adopted to associate specific N-glycome profiles to the early and the advanced phases of Alzheimer's disease (AD).

Severe kidney dysfunction in sialidosis mice reveals an essential role for neuraminidase 1 in reabsorption.

Sialidosis is an ultra-rare multisystemic lysosomal disease caused by mutations in the neuraminidase 1 (NEU1) gene. The severe type II form of the disease manifests with a prenatal/infantile or juvenile onset, bone abnormalities, severe neuropathology, and visceromegaly. A subset of these patients present with nephrosialidosis, characterized by abrupt onset of fulminant glomerular nephropathy. We studied the pathophysiological mechanism of the disease in 2 NEU1-deficient mouse models, a constitutive Neu1-knockout, Neu1ΔEx3, and a conditional phagocyte-specific knockout, Neu1Cx3cr1ΔEx3. Mice of both strains exhibited terminal urinary retention and severe kidney damage with elevated urinary albumin levels, loss of nephrons, renal fibrosis, presence of storage vacuoles, and dysmorphic mitochondria in the intraglomerular and tubular cells. Glycoprotein sialylation in glomeruli, proximal distal tubules, and distal tubules was drastically increased, including that of an endocytic reabsorption receptor megalin. The pool of megalin bearing O-linked glycans with terminal galactose residues, essential for protein targeting and activity, was reduced to below detection levels. Megalin levels were severely reduced, and the protein was directed to lysosomes instead of the apical membrane. Together, our results demonstrated that desialylation by NEU1 plays a crucial role in processing and cellular trafficking of megalin and that NEU1 deficiency in sialidosis impairs megalin-mediated protein reabsorption.

N-acetylneuraminate pyruvate lyase controls sialylation of muscle glycoproteins essential for muscle regeneration and function.

Deleterious variants in N-acetylneuraminate pyruvate lyase (NPL) cause skeletal myopathy and cardiac edema in humans and zebrafish, but its physiological role remains unknown. We report generation of mouse models of the disease: NplR63C, carrying the human p.Arg63Cys variant, and Npldel116 with a 116-bp exonic deletion. In both strains, NPL deficiency causes drastic increase in free sialic acid levels, reduction of skeletal muscle force and endurance, slower healing and smaller size of newly formed myofibers after cardiotoxin-induced muscle injury, increased glycolysis, partially impaired mitochondrial function, and aberrant sialylation of dystroglycan and mitochondrial LRP130 protein. NPL-catalyzed degradation of sialic acid in the muscle increases after fasting and injury and in human patient and mouse models with genetic muscle dystrophy, demonstrating that NPL is essential for muscle function and regeneration and serves as a general marker of muscle damage. Oral administration of N-acetylmannosamine rescues skeletal myopathy, as well as mitochondrial and structural abnormalities in NplR63C mice, suggesting a potential treatment for human patients.

Human VDAC pseudogenes: an emerging role for VDAC1P8 pseudogene in acute myeloid leukemia.

BACKGROUND: Voltage-dependent anion selective channels (VDACs) are the most abundant mitochondrial outer membrane proteins, encoded in mammals by three genes, VDAC1, 2 and 3, mostly ubiquitously expressed. As 'mitochondrial gatekeepers', VDACs control organelle and cell metabolism and are involved in many diseases. Despite the presence of numerous VDAC pseudogenes in the human genome, their significance and possible role in VDAC protein expression has not yet been considered. RESULTS: We investigated the relevance of processed pseudogenes of human VDAC genes, both in physiological and in pathological contexts. Using high-throughput tools and querying many genomic and transcriptomic databases, we show that some VDAC pseudogenes are transcribed in specific tissues and pathological contexts. The obtained experimental data confirm an association of the VDAC1P8 pseudogene with acute myeloid leukemia (AML). CONCLUSIONS: Our in-silico comparative analysis between the VDAC1 gene and its VDAC1P8 pseudogene, together with experimental data produced in AML cellular models, indicate a specific over-expression of the VDAC1P8 pseudogene in AML, correlated with a downregulation of the parental VDAC1 gene.

Relationship of Inflammatory Parameters and Nutritional Status in Cancer Patients.

BACKGROUND/AIM: Chronic inflammation and cytokine storm can cause uncontrolled events in cancer. Pro-inflammatory molecules released by malignant cells send signals to the brain, liver, and neuroendocrine cells, interfering with appetite and promoting anorexia. Malnutrition in cancer patients is associated with increased treatment toxicity, reduced physical efficiency, and decreased survival. Therefore, early recognition of malnutrition could improve quality of life, treatment compliance, and survival. The aim of the study was to explore the relationship between inflammatory parameters with disease stage and nutritional status in patients with solid cancers. PATIENTS AND METHODS: We screened 77 consecutive patients from 3 clinical Institutions in Sicily, Italy, with solid tumors who were either in follow-up after curative treatment or being treated for metastatic disease using the Mini Nutritional Assessment (MNA) questionnaire. Inflammatory parameters, including interleukin 6 (IL6), C-reactive protein (CRP), ß2-microglobulin, ferritin, and transferrin were evaluated. RESULTS: A statistically significant difference was found in mean values of IL6, CRP, ß2-microglobulin, ferritin, and transferrin between patients without evidence of disease and metastatic patients. Among the metastatic group, there was a significant difference in mean values of these inflammatory parameters between patients with malnutrition and those with normal nutritional status. The difference in average IL6, CRP, ß2-microglobulin, and ferritin between patients at risk of malnutrition and those with normal nutritional status was also significant. However, the difference between patients at risk of malnutrition and those with malnutrition was not significant. CONCLUSION: IL6, CRP, transferrin, ferritin, and ß2-microglobulin are functional inflammatory parameters that indicate risk of malnutrition and support the MNA screening test to identify patients with solid tumors who require nutritional support.

VDAC as a Cellular Hub: Docking Molecules and Interactions.

The voltage-dependent anion channel (VDAC) is the primary regulating pathway of water-soluble metabolites and ions across the mitochondrial outer membrane [...].

ERK1/2-dependent TSPO overactivation associates with the loss of mitophagy and mitochondrial respiration in ALS.

Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mouse model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinson's disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.

VDAC1 Knockout Affects Mitochondrial Oxygen Consumption Triggering a Rearrangement of ETC by Impacting on Complex I Activity.

Voltage-Dependent Anion-selective Channel isoform 1 (VDAC1) is the most abundant isoform of the outer mitochondrial membrane (OMM) porins and the principal gate for ions and metabolites to and from the organelle. VDAC1 is also involved in a number of additional functions, such as the regulation of apoptosis. Although the protein is not directly involved in mitochondrial respiration, its deletion in yeast triggers a complete rewiring of the whole cell metabolism, with the inactivation of the main mitochondrial functions. In this work, we analyzed in detail the impact of VDAC1 knockout on mitochondrial respiration in the near-haploid human cell line HAP1. Results indicate that, despite the presence of other VDAC isoforms in the cell, the inactivation of VDAC1 correlates with a dramatic impairment in oxygen consumption and a re-organization of the relative contributions of the electron transport chain (ETC) enzymes. Precisely, in VDAC1 knockout HAP1 cells, the complex I-linked respiration (N-pathway) is increased by drawing resources from respiratory reserves. Overall, the data reported here strengthen the key role of VDAC1 as a general regulator of mitochondrial metabolism.

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry.

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.

Small Extracellular Vesicles Secreted by Nigrostriatal Astrocytes Rescue Cell Death and Preserve Mitochondrial Function in Parkinson's Disease.

Extracellular vesicles (EVs) are emerging as powerful players in cell-to-cell communication both in healthy and diseased brain. In Parkinson's disease (PD)-characterized by selective dopaminergic neuron death in ventral midbrain (VMB) and degeneration of their terminals in striatum (STR)-astrocytes exert dual harmful/protective functions, with mechanisms not fully elucidated. Here, this study shows that astrocytes from the VMB-, STR-, and VMB/STR-depleted brains release a population of small EVs  in a region-specific manner. Interestingly, VMB-astrocytes secreted the highest rate of EVs, which is further exclusively increased in response to CCL3, a chemokine that promotes robust dopaminergic neuroprotection in different PD models. The neuroprotective potential of nigrostriatal astrocyte-EVs is investigated in differentiated versus undifferentiated SH-SY5Y cells exposed to oxidative stress and mitochondrial toxicity. EVs from both VMB- and STR-astrocytes counteract H2 O2 -induced caspase-3 activation specifically in differentiated cells, with EVs from CCL3-treated astrocytes showing a higher protective effect. High resolution respirometry further reveals that nigrostriatal astrocyte-EVs rescue neuronal mitochondrial complex I function impaired by the neurotoxin MPP+ . Notably, only EVs from VMB-astrocyte fully restore ATP production, again specifically in differentiated SH-SY5Y. These results highlight a regional diversity in the nigrostriatal system for the secretion and activities of astrocyte-EVs, with neuroprotective implications for PD.

α-Synuclein A53T Promotes Mitochondrial Proton Gradient Dissipation and Depletion of the Organelle Respiratory Reserve in a Neuroblastoma Cell Line.

α-synuclein (αSyn) is a small neuronal protein whose accumulation correlates with Parkinson's disease. αSyn A53T mutant impairs mitochondrial functions by affecting substrate import within the organelle, activity of complex I and the maximal respiratory capacity. However, the precise mechanism initiating the bioenergetic dysfunction is not clearly understood yet. By overexpressing αSyn A53T in SH-SY5Y cells, we investigated the specific changes in the mitochondrial respiratory profile using High-Resolution Respirometry. We found that αSyn A53T increases dissipative fluxes across the intermembrane mitochondrial space: this does not compromise the oxygen flows devoted to ATP production while it reduces the bioenergetic excess capacity of mitochondria, providing a possible explanation of the increased cell susceptibility observed in the presence of further stress stimuli.

Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress.

Unraveling the role of VDAC3 within living cells is challenging and still requires a definitive answer. Unlike VDAC1 and VDAC2, the outer mitochondrial membrane porin 3 exhibits unique biophysical features that suggest unknown cellular functions. Electrophysiological studies on VDAC3 carrying selective cysteine mutations and mass spectrometry data about the redox state of such sulfur containing amino acids are consistent with a putative involvement of isoform 3 in mitochondrial ROS homeostasis. Here, we thoroughly examined this issue and provided for the first time direct evidence of the role of VDAC3 in cellular response to oxidative stress. Depletion of isoform 3 but not isoform 1 significantly exacerbated the cytotoxicity of redox cyclers such as menadione and paraquat, and respiratory complex I inhibitors like rotenone, promoting uncontrolled accumulation of mitochondrial free radicals. High-resolution respirometry of transiently transfected HAP1-ΔVDAC3 cells expressing the wild type or the cysteine-null mutant VDAC3 protein, unequivocally confirmed that VDAC3 cysteines are indispensable for protein ability to counteract ROS-induced oxidative stress.

VDACs Post-Translational Modifications Discovery by Mass Spectrometry: Impact on Their Hub Function.

VDAC (voltage-dependent anion selective channel) proteins, also known as mitochondrial porins, are the most abundant proteins of the outer mitochondrial membrane (OMM), where they play a vital role in various cellular processes, in the regulation of metabolism, and in survival pathways. There is increasing consensus about their function as a cellular hub, connecting bioenergetics functions to the rest of the cell. The structural characterization of VDACs presents challenging issues due to their very high hydrophobicity, low solubility, the difficulty to separate them from other mitochondrial proteins of similar hydrophobicity and the practical impossibility to isolate each single isoform. Consequently, it is necessary to analyze them as components of a relatively complex mixture. Due to the experimental difficulties in their structural characterization, post-translational modifications (PTMs) of VDAC proteins represent a little explored field. Only in recent years, the increasing number of tools aimed at identifying and quantifying PTMs has allowed to increase our knowledge in this field and in the mechanisms that regulate functions and interactions of mitochondrial porins. In particular, the development of nano-reversed phase ultra-high performance liquid chromatography (nanoRP-UHPLC) and ultra-sensitive high-resolution mass spectrometry (HRMS) methods has played a key role in this field. The findings obtained on VDAC PTMs using such methodologies, which permitted an in-depth characterization of these very hydrophobic trans-membrane pore proteins, are summarized in this review.

N-Glycomics of Human Erythrocytes.

Glycosylation is a complex post-translational modification that conveys functional diversity to glycoconjugates. Cell surface glycosylation mediates several biological activities such as induction of the intracellular signaling pathway and pathogen recognition. Red blood cell (RBC) membrane N-glycans determine blood type and influence cell lifespan. Although several proteomic studies have been carried out, the glycosylation of RBC membrane proteins has not been systematically investigated. This work aims at exploring the human RBC N-glycome by high-sensitivity MALDI-MS techniques to outline a fingerprint of RBC N-glycans. To this purpose, the MALDI-TOF spectra of healthy subjects harboring different blood groups were acquired. Results showed the predominant occurrence of neutral and sialylated complex N-glycans with bisected N-acetylglucosamine and core- and/or antennary fucosylation. In the higher mass region, these species presented with multiple N-acetyllactosamine repeating units. Amongst the detected glycoforms, the presence of glycans bearing ABO(H) antigens allowed us to define a distinctive spectrum for each blood group. For the first time, advanced glycomic techniques have been applied to a comprehensive exploration of human RBC N-glycosylation, providing a new tool for the early detection of distinct glycome changes associated with disease conditions as well as for understanding the molecular recognition of pathogens.

VDAC Genes Expression and Regulation in Mammals.

VDACs are pore-forming proteins, coating the mitochondrial outer membrane, and playing the role of main regulators for metabolites exchange between cytosol and mitochondria. In mammals, three isoforms have evolutionary originated, VDAC1, VDAC2, and VDAC3. Despite similarity in sequence and structure, evidence suggests different biological roles in normal and pathological conditions for each isoform. We compared Homo sapiens and Mus musculus VDAC genes and their regulatory elements. RNA-seq transcriptome analysis shows that VDAC isoforms are expressed in human and mouse tissues at different levels with a predominance of VDAC1 and VDAC2 over VDAC3, with the exception of reproductive system. Numerous transcript variants for each isoform suggest specific context-dependent regulatory mechanisms. Analysis of VDAC core promoters has highlighted that, both in a human and a mouse, VDAC genes show features of TATA-less ones. The level of CG methylation of the human VDAC genes revealed that VDAC1 promoter is less methylated than other two isoforms. We found that expression of VDAC genes is mainly regulated by transcription factors involved in controlling cell growth, proliferation and differentiation, apoptosis, and bioenergetic metabolism. A non-canonical initiation site termed "the TCT/TOP motif," the target for translation regulation by the mTOR pathway, was identified in human VDAC2 and VDAC3 and in every murine VDACs promoter. In addition, specific TFBSs have been identified in each VDAC promoter, supporting the hypothesis that there is a partial functional divergence. These data corroborate our experimental results and reinforce the idea that gene regulation could be the key to understanding the evolutionary specialization of VDAC isoforms.