Robert J. Clements

Analysis of the mitochondrial proteome in multiple sclerosis cortex

Mitochondrial dysfunction has been proposed to play a role in the neuropathology of multiple sclerosis (MS). Previously, we reported significant alterations in the transcription of nuclear-encoded electron transport chain genes in MS and confirmed translational alterations for components of Complexes I and III that resulted in reductions in their activity. To more thoroughly and efficiently elucidate potential alterations in the expression of mitochondrial and related proteins, we have characterized the mitochondrial proteome in postmortem MS and control cortex using Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS). Using principal component analysis (PCA) and hierarchical clustering techniques we were able to analyze the differential patterns of SELDI-TOF spectra to reveal clusters of peaks which distinguished MS from control samples. Four proteins in particular were responsible for distinguishing disease from control. Peptide fingerprint mapping unambiguously identified these differentially expressed proteins. Three proteins identified are involved in respiration including cytochrome c oxidase subunit 5b (COX5b), the brain specific isozyme of creatine kinase, and hemoglobin β-chain. The fourth protein identified was myelin basic protein (MBP). We then investigated whether these alterations were consistent in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. We found that MBP was similarly altered in EAE but the respiratory proteins were not. These data indicate that while the EAE mouse model may mimic aspects of MS neuropathology which result from inflammatory demyelinating events, there is another distinct mechanism involved in mitochondrial dysfunction in gray matter in MS which is not modeled in EAE.

Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis post-mortem tissue

Parvalbumin (PV) and calretinin (CR) are calcium binding proteins (CBP’s) expressed in discrete GABAergic interneuron populations in the human cortex. CBP’s are known to buffer calcium concentrations and protect neurons from increases in intracellular calcium. Perturbations in intracellular calcium can activate proteolytic enzymes including calpain, leading to deleterious effects to axons. Ca++-mediated mechanisms have been found to be associated with axonal pathology in MS and the restructuring of calcium channels has been shown to occur in experimental autoimmune encephalomyelitis (EAE) as well as multiple sclerosis tissue. Previous data indicates a reduction in the expression of the parvalbumin gene as well as reduced extension of neurites on parvalbumin expressing interneurons within multiple sclerosis normal appearing grey matter (NAGM). Modifications in interneuron parvalbumin or calretinin levels could change calcium buffering capacity, as well as the way these cells respond to neuronal insults. The present study was designed to compare CBP immunoreactive neurons in normal and multiple sclerosis post-mortem NAGM. To this end, we utilized immunofluorescent staining and high resolution confocal microscopy to map regions of the human motor cortex, and characterize layer specific CBP distribution in the normal and multiple sclerosis motor cortex. Our results indicate a significant reduction in the number of PV interneurons within layer 2 of the multiple sclerosis primary motor cortex with no concurrent change in number of calretinin positive neurons.

Changes in Methionine Metabolism and Histone H3 Trimethylation Are Linked to Mitochondrial Defects in Multiple Sclerosis

Mitochondrial changes, including decreased expression of electron transport chain subunit genes and impaired energetic, have been reported in multiple sclerosis (MS), but the mechanisms involved in these changes are not clear. To determine whether epigenetic mechanisms are involved, we measured the concentrations of methionine metabolites by liquid chromatography tandem mass spectrometry, histone H3 methylation patterns, and markers of mitochondrial respiration in gray matter from postmortem MS and control cortical samples. We found decreases in respiratory markers as well as decreased concentrations of the methionine metabolites S-adenosylmethionine, betaine, and cystathionine in MS gray matter. We also found expression of the enzyme betaine homocysteine methyltransferase in cortical neurons. This enzyme catalyzes the remethylation of homocysteine to methionine, with betaine as the methyl donor, and has previously been thought to be restricted to liver and kidney in the adult human. Decreases in the concentration of the methyl donor betaine were correlated with decreases in histone H3 trimethylation (H3K4me3) in NeuN+ neuronal nuclei in MS cortex compared with controls. Mechanistic studies demonstrated that H3K4me3 levels and mitochondrial respiration were reduced in SH-SY5Y cells after exposure to the nitric oxide donor sodium nitroprusside, and betaine was able to rescue H3K4me3 levels and respiratory capacity in these cells. Chromatin immunoprecipitation experiments showed that betaine regulates metabolic genes in human SH-SY5Y neuroblastoma cells. These data suggest that changes to methionine metabolism may be mechanistically linked to changes in neuronal energetics in MS cortex. SIGNIFICANCE STATEMENT For decades, it has been observed that vitamin B12 deficiency and multiple sclerosis (MS) share certain pathological changes, including conduction disturbances. In the present study, we have found that vitamin B12-dependent methionine metabolism is dysregulated in the MS brain. We found that concentrations of the methyl donor betaine are decreased in MS cortex and are correlated with reduced levels of the histone H3 methyl mark H3K4me3 in neurons. Cell culture and chromatin immunoprecipitation-seq data suggest that these changes may lead to defects in mitochondria and impact neuronal energetics. These data have uncovered a novel pathway linking methionine metabolism with mitochondrial respiration and have important implications for understanding mechanisms involved in neurodegeneration in MS.

Liquid Crystal Elastomer Microspheres as Three-Dimensional Cell Scaffolds Supporting the Attachment and Proliferation of Myoblasts

We report that liquid crystal elastomers (LCEs), often portrayed as artificial muscles, serve as scaffolds for skeletal muscle cell. A simultaneous microemulsion photopolymerization and cross-linking results in nematic LCE microspheres 10-30 μm in diameter that when conjoined form a LCE construct that serves as the first proof-of-concept for responsive LCE muscle cell scaffolds. Confocal microscopy experiments clearly established that LCEs with a globular, porous morphology permit both attachment and proliferation of C2C12 myoblasts, while the nonporous elastomer morphology, prepared in the absence of a microemulsion, does not. In addition, cytotoxicity and proliferation assays confirm that the liquid crystal elastomer materials are biocompatible promoting cellular proliferation without any inherent cytotoxicity.

Neuronal Hemoglobin Expression and Its Relevance to Multiple Sclerosis Neuropathology

Multiple sclerosis (MS) is characterized by demyelination and progressive neurological disability. Previous studies have reported defects to mitochondria in MS including decreased expression of nuclear encoded electron transport chain subunit genes and inhibition of respiratory complexes. We previously reported increased levels of the hemoglobin β subunit (Hbb) in mitochondrial fractions isolated from postmortem MS cortex compared to controls. In the present study, we performed immunohistochemistry to determine the distribution of Hbb in postmortem MS cortex and identified proteins which interact with Hbb by liquid chromatography tandem mass spectrometry (LC-MS/MS). We found that Hbb was enriched in pyramidal neurons in internal layers of the cortex and interacts with subunits of ATP synthase, histones, and a histone lysine demethylase. We also found that Hbb is present in the nucleus and that expression of Hbb in SH-SY5Y neuroblastoma cells increased trimethylation of histone H3 on lysine 4 (H3K4me3), a histone mark that regulates cellular metabolism. These data suggest that Hbb may be a part of a mechanism linking neuronal energetics with epigenetic changes to histones in the nucleus and may provide neuroprotection in MS by supporting neuronal metabolism.

Effects of intrauterine 2,3,7,8-tetrachlorodibenzo-p-dioxin on the development and function of the gonadotrophin releasing hormone neuronal system in the male rat ☆

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental disrupter that continues to be generated from numerous industrial processes. In utero and lactational exposure of rats to levels of TCDD similar to those encountered by human populations have profound and persistent effects on growth, the reproductive axis and spatial learning and memory. TCDD is thought to act through the aryl hydrocarbon receptor, which displays crosstalk with estrogen-mediated genomic activation. An in vitro superfusion system was used to evaluate the effects of TCDD exposure on GnRH-release from hypothalamic explants at three developmental time points in male rats. Pregnant dams were treated with 5 microg/kg TCDD on gestational day 15, and male offspring displayed a marked reduction in GnRH release. However, total mediobasal hypothalamus/preoptic area (MBH/POA) GnRH content was significantly greater in dioxin-exposed animals. These results suggest deficits in release rather than production of GnRH. Confocal microscopy was used to characterize anatomical features of individual GnRH-positive neurons, as well as the organization of the neuronal network governing GnRH release. Differences in cellular structures were apparent in discrete regions of the GnRH neural network, specifically the lateral preoptic area and septal region. We propose that developmental reproductive effects in males treated in utero and lactionally with dioxin, results from a suppressive effect of TCDD on mechanisms governing GnRH release. These effects coincide with changes in growth and development, indicating that TCDD may induce a syndrome of effects by modifying hypothalamic structures regulating growth and reproductive development.

Effects of Structural Variations on the Cellular Response and Mechanical Properties of Biocompatible, Biodegradable, and Porous Smectic Liquid Crystal Elastomers

The authors report on series of side-chain smectic liquid crystal elastomer (LCE) cell scaffolds based on star block-copolymers featuring 3-arm, 4-arm, and 6-arm central nodes. A particular focus of these studies is placed on the mechanical properties of these LCEs and their impact on cell response. The introduction of diverse central nodes allows to alter and custom-modify the mechanical properties of LCE scaffolds to values on the same order of magnitude of various tissues of interest. In addition, it is continued to vary the position of the LC pendant group. The central node and the position of cholesterol pendants in the backbone of ε-CL blocks (alpha and gamma series) affect the mechanical properties as well as cell proliferation and particularly cell alignment. Cell directionality tests are presented demonstrating that several LCE scaffolds show cell attachment, proliferation, narrow orientational dispersion of cells, and highly anisotropic cell growth on the as-synthesized LCE materials.

Angiotensin II-dependent growth of vascular smooth muscle cells requires transactivation of the epidermal growth factor receptor via a cytosolic phospholipase A2-mediated release of arachidonic acid

Angiotensin (Ang) II stimulates vascular smooth muscle cell (VSMC) growth via activation of cytosolic phospholipase A(2) (cPLA(2)), release of arachidonic acid (ArAc) and activation of mitogen-activated protein kinase (MAPK). The mechanism linking AT(1) receptor stimulation of ArAc release with MAPK activation may involve transactivation of the epidermal growth factor receptor (EGFR). In this study, Ang II increased phosphorylation of the EGFR and MAPK in cultured VSMC and these effects were attenuated by the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)), and restored by addition of ArAc. Ang II- or ArAc-induced phosphorylation of the EGFR and MAPK were abolished by the EGFR kinase inhibitor AG1478. Ang II or ArAc also stimulated VSMC growth that was blocked by AG1478 or the MAPK kinase (MEK) inhibitor PD98059. Thus, it appears that the cPLA(2)-dependent release of ArAc may provide a mechanism for the transactivation between the AT(1) receptor and the EGFR signaling cascade.

The neuronal metabolite NAA regulates histone H3 methylation in oligodendrocytes and myelin lipid composition

The neuronal mitochondrial metabolite N-acetylaspartate (NAA) is decreased in the multiple sclerosis (MS) brain. NAA is synthesized in neurons by the enzyme N-acetyltransferase-8-like (NAT8L) and broken down in oligodendrocytes by aspartoacylase (ASPA) into acetate and aspartate. We have hypothesized that NAA links the metabolism of axons with oligodendrocytes to support myelination. To test this hypothesis, we performed lipidomic analyses using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and high-performance thin-layer chromatography (HPTLC) to identify changes in myelin lipid composition in postmortem MS brains and in NAT8L knockout (NAT8L−/−) mice which do not synthesize NAA. We found reduced levels of sphingomyelin in MS normal appearing white matter that mirrored decreased levels of NAA. We also discovered decreases in the amounts of sphingomyelin and sulfatide lipids in the brains of NAT8L−/− mice compared to controls. Metabolomic analysis of primary cultures of oligodendrocytes treated with NAA revealed increased levels of α-ketoglutarate, which has been reported to regulate histone demethylase activity. Consistent with this, NAA treatment resulted in alterations in the levels of histone H3 methylation, including H3K4me3, H3K9me2, and H3K9me3. The H3K4me3 histone mark regulates cellular energetics, metabolism, and growth, while H3K9me3 has been linked to alterations in transcriptional repression in developing oligodendrocytes. We also noted the NAA treatment was associated with increases in the expression of genes involved in sulfatide and sphingomyelin synthesis in cultured oligodendrocytes. This is the first report demonstrating that neuronal-derived NAA can signal to the oligodendrocyte nucleus. These data suggest that neuronal-derived NAA signals through epigenetic mechanisms in oligodendrocytes to support or maintain myelination.

Role of Surfactant during Microemulsion Photopolymerization for the Creation of Three-Dimensional Liquid Crystal Elastomer Microsphere Spatial Cell Scaffolds

Three-dimensional (3D) cell scaffolds based on connected nematic liquid crystal elastomer microsphere architectures support the attachment and proliferation of C2C12 myoblasts, neuroblastomas (SHSY5Y) and human dermal fibroblasts (hDF). The microsphere spatial cell scaffolds were prepared by an oil-in-water microemulsion photopolymerization of reactive nematic mesogens in the presence of various surfactants, and the as-prepared scaffold constructs are composed of smooth surface microspheres with diameter ranging from 10 to 30 μm. We here investigate how the nature and type of surfactant used during the microemulsion photopolymerization impacts both the size and size distribution of the resulting microspheres as well as their surface morphology, i.e. the surface roughness.