Jennifer McDonough

Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients.

Degeneration of chronically demyelinated axons is a major cause of irreversible neurological disability in multiple sclerosis (MS) patients. Development of neuroprotective therapies will require elucidation of the molecular mechanisms by which neurons and axons degenerate.

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.

Dysmyelinated Lower Motor Neurons Retract and Regenerate Dysfunctional Synaptic Terminals

Axonal degeneration is the major cause of permanent neurological disability in individuals with inherited diseases of myelin. Axonal and neuronal changes that precede axonal degeneration, however, are not well characterized. We show here that dysmyelinated lower motor neurons retract and regenerate dysfunctional presynaptic terminals, leading to severe neurological disability before axonal degeneration. In addition, dysmyelination led to a decreased synaptic quantal content, an indicator of synaptic dysfunction. The amplitude and rise time of miniature endplate potentials were also increased, but these changes were primarily consistent with an increase in the passive membrane properties of the transgenic muscle fibers. Maintenance of synaptic connections should be considered as a therapeutic target for slowing progression of neurological disability in primary diseases of myelin.

Impaired regulation of electron transport chain subunit genes by nuclear respiratory factor 2 in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease. Recently, decreased expression of nuclear encoded electron transport chain genes was found in neurons in MS cortex. To understand the transcriptional mechanisms responsible for the coordinate down regulation of these genes, we performed electrophoretic mobility shifts with nuclear extracts isolated from gray matter from nonlesion areas of postmortem MS and control cortex. Nine tissue blocks from eight different MS brains and six matched control blocks from five control brains were analyzed. We identified a decrease in a transcription factor complex containing nuclear respiratory factor 2 (NRF-2) in nuclear extracts isolated from MS cortex. This decrease is correlated with decreased expression of electron transport chain subunit genes and increased oxidative damage measured by increased anti-nitrotyrosine immunoreactivity. We conclude that in MS cortex a chronic increase in oxidative stress leads to aberrant regulation of transcription of genes involved in energy metabolism.

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.

Ablating N-Acetylaspartate Prevents Leukodystrophy in a Canavan Disease Model

Canavan disease is caused by inactivating ASPA (aspartoacylase) mutations that prevent cleavage of N‐acetyl‐L‐aspartate (NAA), resulting in marked elevations in central nervous system (CNS) NAA and progressively worsening leukodystrophy. We now report that ablating NAA synthesis by constitutive genetic disruption of Nat8l (N‐acetyltransferase‐8 like) permits normal CNS myelination and prevents leukodystrophy in a murine Canavan disease model. Ann Neurol 2015;77:884–888

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 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.