Oligodendrogliopathy in Multiple Sclerosis: Low Glycolytic Metabolic Rate Promotes Oligodendrocyte Survival.

UNLABELLED: Multiple sclerosis (MS) lesions feature demyelination with limited remyelination. A distinct injury phenotype of MS lesions features dying back of oligodendrocyte (OL) terminal processes, a response that destabilizes myelin/axon interactions. This oligodendrogliopathy has been linked with local metabolic stress, similar to the penumbra of ischemic/hypoxic states. Here, we developed an in vitro oligodendrogliopathy model using human CNS-derived OLs and related this injury response to their distinct bioenergetic properties. We determined the energy utilization properties of adult human surgically derived OLs cultured under either optimal or metabolic stress conditions, deprivation of growth factors, and glucose and/or hypoxia using a Seahorse extracellular flux analyzer. Baseline studies were also performed on OL progenitor cells derived from the same tissue and postnatal rat-derived cells. Under basal conditions, adult human OLs were less metabolically active than their progenitors and both were less active than the rat cells. Human OLs and progenitors both used aerobic glycolysis for the majority of ATP production, a process that contributes to protein and lipid production necessary for myelin biosynthesis. Under stress conditions that induce significant process retraction with only marginal cell death, human OLs exhibited a significant reduction in overall energy utilization, particularly in glycolytic ATP production. The stress-induced reduction of glycolytic ATP production by the human OLs would exacerbate myelin process withdrawal while favoring cell survival, providing a potential basis for the oligodendrogliopathy observed in MS. The glycolytic pathway is a potential therapeutic target to promote myelin maintenance and enhance repair in MS. SIGNIFICANCE STATEMENT: The neurologic deficits that characterize multiple sclerosis (MS) reflect disruption of myelin (demyelination) within the CNS and failure of repair (remyelination). We define distinct energy utilization properties of human adult brain-derived oligodendrocytes and oligodendrocyte progenitor cells under conditions of metabolic stress that model the initial relapsing and subsequent progressive phases of MS. The observed changes in energy utilization affect both cell survival and myelination capacity. These processes may be amenable to therapeutic interventions to limit the extent of cumulative tissue injury and to promote repair in MS.

Peripheral nerve injury induces persistent vascular dysfunction and endoneurial hypoxia, contributing to the genesis of neuropathic pain.

Nerve injury is associated with microvascular disturbance; however, the role of the vascular system has not been well characterized in the context of neuropathic pain. Furthermore, ischemia is thought to play a role in a number of neuropathic pain conditions, and yet the role of hypoxia has also not been characterized in neuropathic pain conditions. In this study, we observed the presence of persistent endoneurial hypoxia in a mouse model of traumatic peripheral nerve injury, causing painful mononeuropathy. We attribute the ongoing hypoxia to microvascular dysfunction, endoneurial fibrosis, and increased metabolic requirements within the injured nerve. Increased lactate levels were observed in injured nerves, as well as increased oxygen consumption and extracellular acidification rates, suggesting that anaerobic glycolysis is required to maintain cellular ATP levels. Hypoxia causes a reduction in levels of the Na(+)/K(+) ATPase ion transporter in both cultured primary dorsal root ganglion neurons and injured peripheral nerve. A reduction of Na(+)/K(+) ATPase ion transporter levels likely contributes to the hyperexcitability of injured nerves. Physiological antagonism of hypoxia with hyperbaric oxygen alleviated mechanical allodynia in nerve-injured animals. These results suggest that hypoxia and the Na(+)/K(+) ATPase ion transporter may be a novel mechanistic target for the treatment of neuropathic pain. In addition, the findings support the possibility of using hypoxia activated pro-drugs to localize treatments for neuropathic pain and nerve injury to injured nerves.

Mitochondrial and bioenergetic dysfunction in trauma-induced painful peripheral neuropathy.

BACKGROUND: Mitochondrial dysfunction is observed in various neuropathic pain phenotypes, such as chemotherapy induced neuropathy, diabetic neuropathy, HIV-associated neuropathy, and in Charcot-Marie-Tooth neuropathy. To investigate whether mitochondrial dysfunction is present in trauma-induced painful mononeuropathy, a time-course of mitochondrial function and bioenergetics was characterized in the mouse partial sciatic nerve ligation model. RESULTS: Traumatic nerve injury induces increased metabolic indices of the nerve, resulting in increased oxygen consumption and increased glycolysis. Increased metabolic needs of the nerve are concomitant with bioenergetic and mitochondrial dysfunction. Mitochondrial dysfunction is characterized by reduced ATP synthase activity, reduced electron transport chain activity, and increased futile proton cycling. Bioenergetic dysfunction is characterized by reduced glycolytic reserve, reduced glycolytic capacity, and increased non-glycolytic acidification. CONCLUSION: Traumatic peripheral nerve injury induces persistent mitochondrial and bioenergetic dysfunction which implies that pharmacological agents which seek to normalize mitochondrial and bioenergetic dysfunction could be expected to be beneficial for pain treatment. Increases in both glycolytic acidification and non-glycolytic acidification suggest that pH sensitive drugs which preferentially act on acidic tissue will have the ability to preferential act on injured nerves without affecting healthy tissues.

Computational modeling and biological validation of novel non-steroidal ligands for the cholesterol recognition/interaction amino acid consensus (CRAC) motif of the mitochondrial translocator protein (TSPO).

Mitochondria play a critical role in the physiological homeostasis of the cell, contributing to numerous cellular processes, including bioenergetics, metabolism and cell life and death. Owing to their keystone role, mitochondria have gained much attention as pharmacological targets. The outer mitochondrial integral membrane translocator protein (TSPO) has attracted a significant degree of pharmacological interest owing to its ability to bind a number of classes of drugs with high affinity and specificity. In addition to its well-characterized drug binding site, TSPO possess an additional high-affinity ligand binding site, originally identified for its ability to bind the lipid cholesterol, which was named the cholesterol recognition/interaction amino acid consensus (CRAC) motif. Previous investigations from our laboratory identified additional ligands targeted to TSPO's CRAC motif which are able to potently inhibit mitochondrial cholesterol transport and steroid biosynthesis, processes for which TSPO has been well-characterized. However, all of these compounds possessed the steroidal backbone common to cholesterol and steroid hormones. In our efforts to expand our understanding of TSPO's CRAC motif, we performed studies aimed at identifying non-steroidal ligands for this motif. Molecular modeling and in silico screening of large chemical libraries identified a panel of compounds which were subsequently screened for bioactivity in a number of steroidogenic model systems. These efforts identified a family of non-steroidal CRAC ligands able to potently inhibit steroidogenesis, and at higher concentrations, promote apoptosis. In addition, the best candidate in this family was able to suppress testosterone synthesis when administered to rats, indicating that this novel family of non-steroidal CRAC ligands may serve as prototypes for the development of drugs useful for treatment of diseases of steroid overproduction, such as Cushing's syndrome and steroidogenic cell tumors in humans and animals.

Steroidogenesis in MA-10 mouse Leydig cells is altered via fatty acid import into the mitochondria.

Mitochondria are home to many cellular processes, including oxidative phosphorylation and fatty acid metabolism, and in steroid-synthesizing cells, they are involved in cholesterol import and metabolism, which is the initiating step in steroidogenesis. The formation of macromolecular protein complexes aids in the regulation and efficiency of these mitochondrial functions, though because of their dynamic nature, they are hard to identify. To overcome this problem, we used Blue-Native PAGE with whole-gel mass spectrometry on isolated mitochondria from control and hormone-treated MA-10 mouse tumor Leydig cells. The presence of multiple mitochondrial protein complexes was shown. Although these were qualitatively similar under control and human chorionic gonadotropin (hCG)-stimulated conditions, quantitative differences in the components of the complexes emerged after hCG treatment. A prominent decrease was observed with proteins involved in fatty acid import into the mitochondria, implying that mitochondrial beta-oxidation is not essential for steroidogenesis. To confirm this observation, we inhibited fatty acid import utilizing the CPT1a inhibitor etomoxir, resulting in increased steroid production. Conversely, stimulation of mitochondrial beta-oxidation with metformin resulted in a dose-dependent reduction in steroidogenesis. These changes were accompanied by changes in mitochondrial respiration and in the lactic acid formed during glycolysis. Taken together, these results suggest that upon hormonal stimulation, mitochondria efficiently import cholesterol for steroid production at the expense of other lipids necessary for energy production, specifically fatty acids required for beta-oxidation.

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells.

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.

Translocator protein 2 is involved in cholesterol redistribution during erythropoiesis.

Translocator protein (TSPO) is an 18-kDa cholesterol- and drug-binding protein conserved from bacteria to humans. While surveying for Tspo-like genes, we identified its paralogous gene, Tspo2, encoding an evolutionarily conserved family of proteins that arose by gene duplications before the divergence of avians and mammals. Comparative analysis of Tspo1 and Tspo2 functions suggested that Tspo2 has become subfunctionalized, typical of duplicated genes, characterized by the loss of diagnostic drug ligand-binding but retention of cholesterol-binding properties, hematopoietic tissue- and erythroid cell-specific distribution, and subcellular endoplasmic reticulum and nuclear membrane localization. Expression of Tspo2 in erythroblasts is strongly correlated with the down-regulation of the enzymes involved in cholesterol biosynthesis. Overexpression of TSPO2 in erythroid cells resulted in the redistribution of intracellular free cholesterol, an essential step in nucleus expulsion during erythrocyte maturation. Taken together, these data identify the TSPO2 family of proteins as mediators of cholesterol redistribution-dependent erythroblast maturation during mammalian erythropoiesis.

Cholesterol transport in steroid biosynthesis: role of protein-protein interactions and implications in disease states.

The transfer of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in hormone-induced steroid formation. To ensure that this step is achieved efficiently, free cholesterol must accumulate in excess at the outer mitochondrial membrane and then be transferred to the inner membrane. This is accomplished through a series of steps that involve various intracellular organelles, including lysosomes and lipid droplets, and proteins such as the translocator protein (18 kDa, TSPO) and steroidogenic acute regulatory (StAR) proteins. TSPO, previously known as the peripheral-type benzodiazepine receptor, is a high-affinity drug- and cholesterol-binding mitochondrial protein. StAR is a hormone-induced mitochondria-targeted protein that has been shown to initiate cholesterol transfer into mitochondria. Through the assistance of proteins such as the cAMP-dependent protein kinase regulatory subunit Ialpha (PKA-RIalpha) and the PKA-RIalpha- and TSPO-associated acyl-coenzyme A binding domain containing 3 (ACBD3) protein, PAP7, cholesterol is transferred to and docked at the outer mitochondrial membrane. The TSPO-dependent import of StAR into mitochondria, and the association of TSPO with the outer/inner mitochondrial membrane contact sites, drives the intramitochondrial cholesterol transfer and subsequent steroid formation. The focus of this review is on (i) the intracellular pathways and protein-protein interactions involved in cholesterol transport and steroid biosynthesis and (ii) the roles and interactions of these proteins in endocrine pathologies and neurological diseases where steroid synthesis plays a critical role.

Targeting and insertion of the cholesterol-binding translocator protein into the outer mitochondrial membrane.

Translocator protein (18 kDa, TSPO), previously known as the peripheral-type benzodiazepine receptor, is an outer mitochondrial membrane (OMM) protein necessary for cholesterol import and steroid production. We reconstituted the mitochondrial targeting and insertion of TSPO into the OMM to analyze the signals and mechanisms required for this process. Initial studies indicated the formation of a mitochondrial 66 kDa complex through Blue Native-PAGE analysis. The formation of this complex was found to be dependent on the presence of ATP and the cytosolic chaperone Hsp90. Through mutational analysis we identified two areas necessary for TSPO targeting, import, and function: amino acids 103-108 (Schellman motif), which provide the necessary structural orientation for import, and the cholesterol-binding C-terminus required for insertion. Although the translocase of the outer mitochondrial membrane (TOM) complex proteins Tom22 and Tom40 were present in the OMM, the TOM complex did not interact with TSPO. In search of proteins involved in TSPO import, we analyzed complexes known to interact with TSPO by mass spectrometry. Formation of the 66 kDa complex was found to be dependent on an identified protein, Metaxin 1, for formation and TSPO import. The level of import of TSPO into steroidogenic cell mitochondria was increased following treatment of the cells with cAMP. These findings suggest that the initial targeting of TSPO to mitochondria is dependent upon the presence of cytosolic chaperones interacting with the import receptor Tom70. The C-terminus plays an important role in targeting TSPO to mitochondria, whereas its import into the OMM is dependent upon the presence of the Schellman motif. Final integration of TSPO into the OMM occurs via its interaction with Metaxin 1. Import of TSPO into steroidogenic cell mitochondria is regulated by cAMP.

Potential Benefit of the Charge-Stabilized Nanostructure Saline RNS60 for Myelin Maintenance and Repair.

Myelin injury in multiple sclerosis (MS) has been attributed both to "outside-in" primary immune mediated and "inside-out" metabolic stress of oligodendrocyte (OL) related mechanisms. Subsequent remyelination is dependent on recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). RNS60 is a physically-modified saline containing charge-stabilized nanobubbles generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. Administration of RNS60 has been shown to reduce the severity of EAE by dampening the immune response and myelin loss. Additionally, RNS60 has been demonstrated to enhance mitochondrial ATP synthesis in neurons. Here, we used post-natal rat derived OLs and OPCs to assess the impact of RNS60 on the response of OLs to metabolic stress in vitro (glucose-nutrient deprivation, referred to as 'NG') and on OPC differentiation capacity. Under the NG condition, our findings indicate that RNS60 decreases caspases 3/7 activation. Respirometric analyses revealed that RNS60 increased spare glycolytic capacity (SGC) under normal culture conditions. However, RNS60 enhanced OL spare respiratory capacity (SRC) when a metabolic stress was present. Furthermore, we show that RNS60 promotes OPC differentiation under physiological conditions. Our findings provide evidence for the potential therapeutic efficacy of RNS60 through the promotion of OL survival and OPC differentiation.

Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3.

Leydig cell steroidogenesis is a multistep process that takes place in the mitochondria and endoplasmic reticulum (ER). The physical association between these 2 organelles could facilitate both steroidogenesis substrate availability and mitochondrial product passage to steroidogenic enzymes in the ER, thus regulating the rate of steroid formation. Confocal microscopy, using antisera against organelle-specific antigens, and electron microscopy studies demonstrated that there is an increase in the number of mitochondria-ER contact sites in response to hormone treatment in MA-10 mouse tumor Leydig cells. Electron tomography and 3-dimensional reconstruction allowed for the visualization of mitochondria-associated membranes (MAMs). MAMs were isolated and found to contain the 67-kDa long isoform of the adenosine triphosphatase (ATPase) family, AAA domain-containing protein 3 (ATAD3). The 67-kDa ATAD3 is anchored in the inner mitochondrial membrane and is enriched in outer-inner mitochondrial membrane contact sites. ATAD3-depleted MA-10 cells showed reduced production of steroids in response to human choriogonadotropin but not to 22R-hydroxycholesterol treatment, indicating a role of ATAD3 in the delivery of the substrate cholesterol into the mitochondria. The N terminus of ATAD3 contains 50 amino acids that have been proposed to insert into the outer mitochondrial membrane and associated organelles such as the ER. Deletion of the ATAD3 N terminus resulted in the reduction of hormone-stimulated progesterone biosynthesis, suggesting a role of ATAD3 in mitochondria-ER contact site formation. Taken together, these results demonstrate that the hormone-induced, ATAD3-mediated, MAM formation participates in the optimal transfer of cholesterol from the ER into mitochondria for steroidogenesis.

Organelle plasticity and interactions in cholesterol transport and steroid biosynthesis.

Steroid biosynthesis is a multi-step process controlled by pituitary hormones, which, via cAMP-dependent signaling pathways, drive tissue-specific steroid formation. Steroidogenesis begins with the transport of the substrate, cholesterol, from intracellular stores into the inner mitochondrial membrane, where the steroidogenic enzyme CYP11A1 converts cholesterol to pregnenolone. This process is accelerated by hormones and involves a number of proteins and protein-protein interactions. Indeed, cholesterol, stored in lipid droplets and membranes, is transferred through a hormone-induced complex of proteins derived from the cytosol, mitochondria, and other organelles termed the transduceosome to the outer mitochondrial membrane. From there, cholesterol reaches CYP11A1 through outer/inner membrane contact sites. Thus, cholesterol transfer is likely achieved through a hormone-dependent reorganization of organelles and protein distribution and interactions. The findings reviewed herein suggest the presence of a hormone-dependent organelle communication network mediated by protein-protein interactions and inter-organelle trafficking, resulting in the efficient and timely delivery of cholesterol into mitochondria for steroid synthesis.

Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones.

Steroid hormones are critical for organismal development and health. The rate-limiting step in steroidogenesis is the transport of cholesterol from the outer mitochondrial membrane (OMM) to the cytochrome P450 enzyme CYP11A1 in the inner mitochondrial membrane (IMM). Cholesterol transfer occurs through a complex termed the "transduceosome," in which cytosolic steroidogenic acute regulatory protein interacts with OMM proteins translocator protein and voltage-dependent anion channel (VDAC) to assist with the transfer of cholesterol to OMM. It has been proposed that cholesterol transfer from OMM to IMM occurs at specialized contact sites bridging the two membranes composed of VDAC and IMM adenine nucleotide translocase (ANT). Blue native PAGE of Leydig cell mitochondria identified two protein complexes that were able to bind cholesterol at 66- and 800-kDa. Immunoblot and mass spectrometry analyses revealed that the 800-kDa complex contained the OMM translocator protein (18-kDa) and VDAC along with IMM CYP11A1, ATPase family AAA domain-containing protein 3A (ATAD3A), and optic atrophy type 1 proteins, but not ANT. Knockdown of ATAD3A, but not ANT or optic atrophy type 1, in Leydig cells resulted in a significant decrease in hormone-induced, but not 22R-hydroxycholesterol-supported, steroid production. Using a 22-phenoxazonoxy-5-cholene-3-beta-ol CYP11A1-specific probe, we further demonstrated that the 800-kDa complex offers the microenvironment needed for CYP11A1 activity. Addition of steroidogenic acute regulatory protein to the complex mobilized the cholesterol bound at the 800-kDa complex, leading to increased steroid formation. These results identify a bioactive, multimeric protein complex spanning the OMM and IMM unit that is responsible for the hormone-induced import, segregation, targeting, and metabolism of cholesterol.

A novel functional translocator protein ligand for cancer imaging.

The translocator protein (TSPO) is an attractive target for tumor imaging due to its up-regulation in numerous cancer cell types. Here, we report a series of functional TSPO ligands, n-TSPOmbb732, which can be conjugated to a variety of signaling moieties and are widely applicable in TSPO-targeted molecular imaging. Two fluorescent dye-labeled 6-TSPOmbb732 displayed nanomolar binding affinities to TSPO and were successfully imaged in vitro.

Protein-protein interactions mediate mitochondrial cholesterol transport and steroid biosynthesis.

Transport of cholesterol into the mitochondria is the rate-determining, hormone-sensitive step in steroid biosynthesis. Here we report that the mechanism underlying mitochondrial cholesterol transport involves the formation of a macromolecular signaling complex composed of the outer mitochondrial membrane translocator protein (TSPO), previously known as peripheral-type benzodiazepine receptor; the TSPO-associated protein PAP7, which binds and brings to mitochondria the regulatory subunit RIalpha of the cAMP-dependent protein kinase (PKARIalpha); and the hormone-induced PKA substrate, steroidogenic acute regulatory protein (StAR). Hormone treatment of MA-10 Leydig cells induced the co-localization of TSPO, PAP7, PKARIalpha, and StAR in mitochondria, visualized by confocal microscopy, and the formation in living cells of a high molecular weight multimeric complex identified using photoactivable amino acids. The hormone-induced recruitment of exogenous TSPO in this complex was found to parallel the increased presence of 7-azi-5alpha-cholestan-3beta-ol in the samples. Co-expression of Tspo, Pap7, PkarIalpha, and Star genes resulted in the stimulation of steroid formation in both steroidogenic MA-10 and non-steroidogenic COS-F2-130 cells that were engineered to metabolize cholesterol. Disruption of these protein-protein interactions and specifically the PKARIalpha-PAP7 and PAP7-TSPO interactions, using PAP7 mutants where the N0 area homologous to dual A-kinase-anchoring protein-1 or the acyl-CoA signature motif were deleted or using the peptide Ht31 known to disrupt the anchoring of PKA, inhibited both basal and hormone-induced steroidogenesis. These results suggest that the initiation of cAMP-induced protein-protein interactions results in the formation of a multivalent scaffold in the outer mitochondrial membrane that mediates the effect of hormones on mitochondrial cholesterol transport and steroidogenesis.