Biochemical Alterations in White Matter Tracts of the Aging Mouse Brain Revealed by FTIR Spectroscopy Imaging.

White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and biochemical changes that lead to myelin degeneration. In this study, we investigate myelination across the lifespan in C57BL/6 mice using electron microscopy and Fourier transform infrared (FTIR) spectroscopic imaging to better understand the relationship between structural and biochemical changes in CNS white matter tracts. A decrease in the number of myelinated axons was associated with altered lipid profiles in the corpus callosum of aged mice. FTIR spectroscopic imaging revealed alterations in functional groups associated with phospholipids, including the lipid acyl, lipid ester and phosphate vibrations. Biochemical changes in white matter were observed prior to structural changes and most predominant in the anterior regions of the corpus callosum. This was supported by biochemical analysis of fatty acid composition that demonstrated an overall trend towards increased monounsaturated fatty acids and decreased polyunsaturated fatty acids with age. To further explore the molecular mechanisms underlying these biochemical alterations, gene expression profiles of lipid metabolism and oxidative stress pathways were investigated. A decrease in the expression of several genes involved in glutathione metabolism suggests that oxidative damage to lipids may contribute to age-related white matter degeneration.

Iron Heterogeneity in Early Active Multiple Sclerosis Lesions.

OBJECTIVE: Multiple sclerosis (MS) is a heterogeneous inflammatory demyelinating disease. Iron distribution is altered in MS patients' brains, suggesting iron liberation within active lesions amplifies demyelination and neurodegeneration. Whether the amount and distribution of iron are similar or different among different MS immunopatterns is currently unknown. METHODS: We used synchrotron X-ray fluorescence imaging, histology, and immunohistochemistry to compare the iron quantity and distribution between immunopattern II and III early active MS lesions. We analyzed archival autopsy and biopsy tissue from 21 MS patients. RESULTS: Immunopattern II early active lesions contain 64% more iron (95% confidence interval [CI] = 17-127%, p = 0.004) than immunopattern III lesions, and 30% more iron than the surrounding periplaque white matter (95% CI = 3-64%, p = 0.03). Iron in immunopattern III lesions is 28% lower than in the periplaque white matter (95% CI = -40 to -14%, p < 0.001). When normalizing the iron content of early active lesions to that of surrounding periplaque white matter, the ratio is significantly higher in immunopattern II (p < 0.001). Microfocused X-ray fluorescence imaging shows that iron in immunopattern II lesions localizes to macrophages, whereas macrophages in immunopattern III lesions contain little iron. INTERPRETATION: Iron distribution and content are heterogeneous in early active MS lesions. Iron accumulates in macrophages in immunopattern II, but not immunopattern III lesions. This heterogeneity in the two most common MS immunopatterns may be explained by different macrophage polarization, origin, or different demyelination mechanisms, and paves the way for developing new or using existing iron-sensitive magnetic resonance imaging techniques to differentiate among immunopatterns in the general nonbiopsied MS patient population. ANN NEUROL 2021;89:498-510.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Sirt2 epigenetically down-regulates PDGFRα expression and promotes CG4 cell differentiation.

We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.

Does Sirt2 Regulate Cholesterol Biosynthesis During Oligodendroglial Differentiation In Vitro and In Vivo?

Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2 -/- mice, primary OL cells and CG4-OL cells. Our results demonstrate that expression of cholesterol biosynthetic genes in the CNS white matter or cholesterol content in purified myelin fractions did not differ between Sirt2 -/- and age-matched wild-type mice. Cholesterol biosynthetic gene expression profiles and total cholesterol content were not altered in primary OLs from Sirt2 -/- mice and in CG4-OLs when Sirt2 was either down-regulated with RNAi or overexpressed. In addition, Sirt2 knockdown or overexpression in CG4-OLs had no effect on SREBP-2 nuclear translocation. Our results indicate that Sirt2 does not impact the expression of genes encoding enzymes involved in cholesterol biosynthesis, total cholesterol content, or nuclear translocation of SREBP-2 during OL differentiation and myelination.

Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation.

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv ) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3' untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation.

Advances in the treatment of relapsing-remitting multiple sclerosis: the role of pegylated interferon ß-1a.

Multiple sclerosis (MS) is a progressive, neurodegenerative disease with unpredictable phases of relapse and remission. The cause of MS is unknown, but the pathology is characterized by infiltration of auto-reactive immune cells into the central nervous system (CNS) resulting in widespread neuroinflammation and neurodegeneration. Immunomodulatory-based therapies emerged in the 1990s and have been a cornerstone of disease management ever since. Interferon ß (IFNß) was the first biologic approved after demonstrating decreased relapse rates, disease activity and progression of disability in clinical trials. However, frequent dosing schedules have limited patient acceptance for long-term therapy. Pegylation, the process by which molecules of polyethylene glycol are covalently linked to a compound, has been utilized to increase the half-life of IFNß and decrease the frequency of administration required. To date, there has been one clinical trial evaluating the efficacy of pegylated IFN. The purpose of this article is to provide an overview of the role of IFN in the treatment of MS and evaluate the available evidence for pegylated IFN therapy in MS.

Pseudogenes regulate parental gene expression via ceRNA network.

The concept of competitive endogenous RNA (ceRNA) was first proposed by Salmena and colleagues. Evidence suggests that pseudogene RNAs can act as a 'sponge' through competitive binding of common miRNA, releasing or attenuating repression through sequestering miRNAs away from parental mRNA. In theory, ceRNAs refer to all transcripts such as mRNA, tRNA, rRNA, long non-coding RNA, pseudogene RNA and circular RNA, because all of them may become the targets of miRNA depending on spatiotemporal situation. As binding of miRNA to the target RNA is not 100% complementary, it is possible that one miRNA can bind to multiple target RNAs and vice versa. All RNAs crosstalk through competitively binding to miRNAvia miRNA response elements (MREs) contained within the RNA sequences, thus forming a complex regulatory network. The ratio of a subset of miRNAs to the corresponding number of MREs determines repression strength on a given mRNA translation or stability. An increase in pseudogene RNA level can sequester miRNA and release repression on the parental gene, leading to an increase in parental gene expression. A massive number of transcripts constitute a complicated network that regulates each other through this proposed mechanism, though some regulatory significance may be mild or even undetectable. It is possible that the regulation of gene and pseudogene expression occurring in this manor involves all RNAs bearing common MREs. In this review, we will primarily discuss how pseudogene transcripts regulate expression of parental genes via ceRNA network and biological significance of regulation.

Secretory vesicle cholesterol: Correlating lipid domain organization and Ca2+ triggered fusion.

Membrane organization has received substantial research interest since the degree of ordering in membrane regions is relevant in many biological processes. Here we relate the impact of varying cholesterol concentrations on native secretory vesicle fusion and the lateral domain organization of membrane extracts from these vesicles. Membranes of isolated cortical secretory vesicles were either depleted of cholesterol, had cholesterol loaded to excess of native levels, or were depleted of cholesterol but subsequently reloaded to restore native cholesterol levels. Lipid analyses confirmed cholesterol was the only species significantly altered by these treatments. Treated vesicles were characterized for their ability to undergo fusion. Cholesterol depletion resulted in a decrease of Ca2+ sensitivity and the extent of fusion, while cholesterol loading had no effect on fusion parameters. Membrane extracts were characterized in terms of lipid packing by surface pressure-area isotherms whereas the lateral membrane organization was analyzed by Brewster angle microscopy. While no differences in the isotherms were observed, imaging revealed drastic differences in domain size, shape and frequency between the various conditions. Cholesterol depletion induced larger but fewer domains, suggesting that domain coalescence into larger structures may disrupt the native temporal-spatial organization of the fusion machinery and thus inhibit vesicle docking, priming, and fusion. In contrast, adding excess cholesterol, or rescuing with exogenous cholesterol after sterol depletion, resulted in more but smaller domains. Therefore, cholesterol is an important membrane organizer in the process of Ca2+ triggered vesicular fusion, which can be related to specific physical effects on native membrane substructure.

Dissecting the mechanism of Ca2+-triggered membrane fusion: probing protein function using thiol reactivity.

1. Ca(2+)-triggered membrane fusion involves the coordinated actions of both lipids and proteins, but the specific mechanisms remain poorly understood. The urchin cortical vesicle model is a stage-specific native preparation fully enabling the directly coupled functional-molecular analyses necessary to identify critical components of fast triggered membrane fusion. 2. Recent work on lipidic components has established a direct role for cholesterol in the fusion mechanism via local contribution of negative curvature to readily enable the formation of transient lipidic fusion intermediates. In addition, cholesterol- and sphingomyelin-enriched domains regulate the efficiency of fusion by focally organizing other components to ensure an optimized response to the triggering Ca(2+) transient. 3. There is less known about the identity of proteins involved in the Ca(2+)-triggering steps of membrane fusion. Thiol reagents can be used as unbiased tools to probe protein functions. Comparisons of several thiol-reactive reagents have identified different effects on Ca(2+) sensitivity and the extent of fusion, suggesting that there are at least two distinct thiol sites that participate in the fusion mechanism: one that regulates the efficiency of Ca(2+) sensing/triggering and one that may function during the membrane merger event itself. 4. To identify the proteins that regulate Ca(2+) sensitivity, the fluorescent thiol reagent Lucifer yellow iodoacetamide was used to potentiate fusion and simultaneously tag the proteins involved. Ongoing work involves the isolation of cholesterol-enriched membrane fractions to reduce the complexity of the labelled proteome, narrowing the number of candidate proteins.

Enhancement of the Ca(2+)-triggering steps of native membrane fusion via thiol-reactivity.

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca(2+) triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca(2+) sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr(2+), a weak Ca(2+) mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca(2+), suggesting that IA acts at the Ca(2+)-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.

Identifying critical components of native Ca2+-triggered membrane fusion. Integrating studies of proteins and lipids.

Ca(2+)-triggered membrane fusion is the defining step of exocytosis. Despite realization that the fusion machinery must include lipids and proteins working in concert, only of late has work in the field focused more equally on both these components. Here we use isolated sea urchin egg cortical vesicles (CV), a stage-specific preparation of Ca(2+)-sensitive release-ready vesicles that enables the tight coupling of molecular and functional analyses necessary to dissect molecular mechanisms. The stalk-pore hypothesis proposes that bilayer merger proceeds rapidly via transient, high-negative curvature, intermediate membrane structures. Consistent with this, cholesterol, a major component of the CV membrane, contributes to a critical local negative curvature that supports formation of lipidic fusion intermediates. Following cholesterol depletion, structurally dissimilar lipids having intrinsic negative curvature greater than or equal to cholesterol recover the ability of CV to fuse but do not recover fusion efficiency (Ca(2+) sensitivity and kinetics). Conversely, cholesterol- and sphingomyelin-enriched microdomains regulate the efficiency of the fusion mechanism, presumably by contributing spatial and functional organization of other critical lipids and proteins at the fusion site. Critical proteins are thought to participate in Ca(2+) sensing, initiating membrane deformations, and facilitating fusion pore expansion. Capitalizing on a novel effect of the thiol-reactive reagent iodoacetamide (IA), potentiation of the Ca(2+) sensitivity and kinetics, a fluorescently tagged IA has been used to enhance fusion efficiency and simultaneously label the proteins involved. Isolation of cholesterol-enriched CV membrane fractions, using density gradient centrifugation, is being used to narrow the list of protein candidates potentially critical to the mechanism of fast Ca(2+)-triggered membrane fusion.