Bridget Shafit-Zagardo

Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation

During mammalian central nervous system (CNS) development, contact-mediated activation of Notch1 receptors on oligodendrocyte precursors by the ligand Jagged1 induces Hes5, which inhibits maturation of these cells. Here we tested whether the Notch pathway is re-expressed in the adult CNS in multiple sclerosis (MS), an inflammatory demyelinating disease in which remyelination is typically limited. We found that transforming growth factor-β1 (TGF-β1), a cytokine upregulated in MS, specifically re-induced Jagged1 in primary cultures of human astrocytes. Within and around active MS plaques lacking remyelination, Jagged1 was expressed at high levels by hypertrophic astrocytes, whereas Notch1 and Hes5 localized to cells with an immature oligodendrocyte phenotype, and TGF-β1 was associated with perivascular extracellular matrix in the same areas. In contrast, there was negligible Jagged1 expression in remyelinated lesions. Experiments in vitro showed that Jagged1 signaling inhibited process outgrowth from primary human oligodendrocytes. These data are the first to implicate the Notch pathway in the limited remyelination in MS. Thus, Notch may represent a potential target for therapeutic intervention in this disease.

Failure of neuronal maturation in Alzheimer disease dentate gyrus.

The dentate gyrus, an important anatomic structure of the hippocampal formation, is one of the major areas in which neurogenesis takes place in the adult mammalian brain. Neurogenesis in the dentate gyrus is thought to play an important role in hippocampus-dependent learning and memory. Neurogenesis has been reported to be increased in the dentate gyrus of patients with Alzheimer disease, but it is not known whether the newly generated neurons differentiate into mature neurons. In this study, the expression of the mature neuronal marker high molecular weight microtubule-associated protein (MAP) isoforms MAP2a and b was found to be dramatically decreased in Alzheimer disease dentate gyrus, as determined by immunohistochemistry and in situ hybridization. The total MAP2, including expression of the immature neuronal marker, the MAP2c isoform, was less affected. These findings suggest that newly generated neurons in Alzheimer disease dentate gyrus do not become mature neurons, although neuroproliferation is increased.

Okadaic Acid Induces Early Changes in Microtubule‐Associated Protein 2 and γ Phosphorylation Prior to Neurodegeneration in Cultured Cortical Neurons

Abstract: Microtubules and their associated proteins play a prominent role in many physiological and morphological aspects of brain function. Abnormal deposition of the microtubule‐associated proteins (MAPs), MAP2 and γ, is a prominent aspect of Alzheimers disease. MAP2 and γ are heat‐stable phosphoproteins subject to high rates of phosphorylation/dephosphorylation. The phosphorylation state of these proteins modulates their affinity for tubulin and thereby affects the structure of the neuronal cytoskeleton. The dinoflagellate toxin okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A. In cultured rat cortical neurons and a human neuroblastoma cell line (MSN), okadaic acid induces increased phosphorylation of MAP2 and γ concomitant with early changes in the neuronal cytoskeleton and ultimately leads to cell death. These results suggest that the diminished rate of MAP2 and γ dephosphorylation affects the stability of the neuronal cytoskeleton. The effect of okadaic acid was not restricted to neurons. Astrocytes stained with antibodies to glial fibrillary acidic protein (GFAP) showed increased GFAP staining and changes in astrocyte morphology from a flat shape to a stellate appearance with long processes.

Localization and characterization of the binding site for the regulatory subunit of type II cAMP-dependent protein kinase on MAP2

Microtubule-associated protein 2 (MAP2) binds, and is a substrate for, type II cAMP-dependent protein kinase. The structural domain in MAP2 that binds the regulatory subunit (RII) of protein kinase II was identified by expressing fragments of a human MAP2 cDNA in E. coli using the pATH11 vector. Fusion proteins were resolved by SDS-PAGE and transferred to nitrocellulose. The filters were probed with purified bovine heart or brain RII, anti-RII monoclonal antibodies, and 125I-labeled protein A. Binding of RII was localized to a 31 amino acid sequence near the N-terminus of the MAP2 molecule. Fusion proteins containing this fragment bound both heart and brain RIIs in a concentration-dependent manner, but bound heart RII with a higher apparent affinity than brain RII. The amino acid sequence of this fragment (DRETAEEVSARIVQVVTAEAVAVLKGEQEKE) is totally conserved between human and mouse MAP2, suggesting an important role for the RII binding site of MAP2 in neuronal function.

Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose‐dependent reduction in survivin protein. Although 74% of the SAO‐treated MSN cells were trypan blue+, PARP cleavage or activated caspase‐3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co‐administration of z‐Val‐Ala‐Asp(OMe)‐fluoromethyl ketone (zVAD‐fmk) with SAO did not inhibit cell death suggesting a caspase‐independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO‐treated TC620 cells were trypan blue+, PARP was cleaved, cells were TUNEL+ and PI‐staining revealed macronuclei and numerous apoptotic bodies. Co‐treatment of the TC620 cells with SAO and zVAD‐fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two‐fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase‐independent mechanism.

Gas6/Axl Signaling Activates the Phosphatidylinositol 3-Kinase/Akt1 Survival Pathway to Protect Oligodendrocytes from Tumor Necrosis Factorα-Induced Apoptosis

Growth arrest-specific protein 6 (gas6) activity is mediated through the receptor tyrosine kinase family members Axl, Rse, and Mer, all of which are expressed in human oligodendrocytes. In this study, we examined whether recombinant human (rh) gas6 protects oligodendrocytes from growth factor (insulin) withdrawal or tumor necrosis factor-α (TNFα) cytotoxicity. In addition, we examined whether the effect was caspase-dependent, which receptor mediated the protective effect, and whether survival required Akt1 activation. Oligodendrocyte viability was assessed by O4 staining and terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling. Addition of rhgas6 to insulin-depleted cultures resulted in a significant increase in oligodendrocyte viability. Rhgas6 and caspase inhibitors also reduced active caspase-3 immunoreactivity relative to TNFα-only-treated cultures. In cultures treated with TNFα (100 ng/ml), the oligodendrocyte survival rate was 18% compared with cultures treated with TNFα and rhgas6 (64%) or the caspase inhibitors IETD-fmk [z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone] (65%) and zVAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) (63%). Increased phosphoAkt (Ser473) immunoreactivity was detected 15 min after administration of gas6 and TNFα to oligodendrocyte cultures but not in TNFα-treated cultures. The gas6 protective effect was abrogated by the Axl decoy receptor Axl-Fc, by the phosphatidylinositol 3 (PI3) kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], and in Akt1−/− oligodendrocytes. Oligodendrocyte cultures established from wild-type and Rse−/− mice, but not from Axl−/− mice, were also protected from TNFα-induced cell death when maintained in rhgas6. We conclude that gas6 signaling through the Axl receptor and the PI3 kinase/Akt1 survival pathway protects oligodendrocytes from growth factor withdrawal and TNFα-mediated cell death.

The Growth Arrest-Specific Gene Product Gas6 Promotes the Survival of Human Oligodendrocytes via a Phosphatidylinositol 3-Kinase-Dependent Pathway

Microarray analysis revealed that transcripts for the Axl and Mer receptor tyrosine kinases are expressed at high levels in O4+-immunopanned oligodendrocytes isolated from second trimester human fetal spinal cord. In humans the sole known ligand for the Axl/Rse/Mer kinases is growth arrest-specific gene 6 (Gas6), which in the CNS is secreted by neurons and endothelial cells. We hypothesized that Gas6 is a survival factor for oligodendrocytes and receptor activation signals downstream to the phosphatidylinositol 3 (PI3)-kinase/Akt pathway to increase cell survival in the absence of cell proliferation. To test this hypothesis, we grew enriched human oligodendrocytes for 6 d on a monolayer of NIH3T3 cells stably expressing Gas6. CNP+ oligodendrocytes on Gas6-secreting 3T3 cells had more primary processes and arborizations than those plated solely on 3T3 cells. Also, a twofold increase in CNP+ and MBP+ oligodendrocytes was observed when they were plated on the Gas6-secreting cells. The effect was abolished in the presence of Axl-Fc but remained unchanged in the presence of the irrelevant receptor fusion molecule TrkA-Fc. A significant decrease in CNP+/TUNEL+ oligodendrocytes was observed when recombinant human Gas6 (rhGas6) was administered to oligodendrocytes plated on poly-l-lysine, supporting a role for Gas6 signaling in oligodendrocyte survival during a period of active myelination in human fetal spinal cord development. PI3-kinase inhibitors blocked the anti-apoptotic effect of rhGas6, whereas a MEK/ERK inhibitor had no effect. Thus Gas6 sustains human fetal oligodendrocyte viability by receptor activation and downstream signaling via the PI3-kinase/Akt pathway.

Tumor necrosis factor-induced proliferation of astrocytes from mature brain is associated with down-regulation of glial fibrillary acidic protein mRNA

Previous results from this laboratory have shown that tumor necrosis factor (TNF) is mitogenic for bovine astrocytes in chemically defined (CO) medium. The maximum mitogenic response was detected with 200 U/ml at 48 h. We have now extended these studies to assess the effect of TNF on message levels for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The results have shown that, whereas TNF had only a slight effect on vimentin mRNA, TNF induced a marked decrease to 4.3 ± 2.0% of controls in GFAP mRNA which was both time and dose dependent. The lowest effective dose was 50 U/ml and the maximal effective dose was 200 U/ml. Kinetic analysis of this response demonstrated that a marked decrease in GFAP mRNA was present at 12 h and continued to decrease through 72 h. To determine the reversibility of the TNF effect, astrocyte cultures were exposed to 200 U/ml TNF for varying periods of tee and then cultured in fresh CD medium. A 1‐h pulse with TNF was sufficient to reduce GFAP mRNA levels when measured 24 h later. However, cultures incubated with 200 U/ml TNF for 48 h followed by incubation in CD medium without TNF for 7 days showed that GFAP mRNA levels had returned to 60% of the control values. Nuclear runoff assays showed that the effect of TNF on GFAP mRNA was at the posttranscriptional level. Polyacrylamide gel electrophoretic analysis of astrocyte cytoskeletal proteins demonstrated that GFAP levels were reduced after a 5‐day incubation with 200 U/ml TNF whereas protein levels of vimentin and actin were not significantly changed. Western blots confirmed that GFAP levels were reduced to 36% of the control values. Thus the effect of TNF on GFAP mRNA expression was not due to a generalized effect on intermediate filament metabolism.

Interferon regulatory factor 3 inhibits astrocyte inflammatory gene expression through suppression of the proinflammatory miR-155 and miR-155*.

Astrocytes, together with microglia and macrophages, participate in innate inflammatory responses in the CNS. Although inflammatory mediators such as interferons generated by astrocytes may be critical in the defense of the CNS, sustained unopposed cytokine signaling could result in harmful consequences. Interferon regulatory factor 3 (IRF3) is a transcription factor required for IFNβ production and antiviral immunity. Most cells express low levels of IRF3 protein, and the transcriptional mechanism that upregulates IRF3 expression is not known. In this study, we explored the consequence of adenovirus‐mediated IRF3 gene transfer (Ad‐IRF3) in primary human astrocytes. We show that IRF3 transgene expression suppresses proinflammatory cytokine gene expression upon challenge with IL‐1/IFNγ and alters astrocyte activation phenotype from a proinflammatory to an anti‐inflammatory one, akin to an M1–M2 switch in macrophages. This was accompanied by the rescue of neurons from cytokine‐induced death in glial‐neuronal co‐cultures. Furthermore, Ad‐IRF3 suppressed the expression of microRNA‐155 and its star‐form partner miR‐155*, immunoregulatory miRNAs highly expressed in multiple sclerosis lesions. Astrocyte miR‐155/miR155* were induced by cytokines and TLR ligands with a distinct hierarchy and involved in proinflammatory cytokine gene induction by targeting suppressor of cytokine signaling 1, a negative regulator of cytokine signaling and potentially other factors. Our results demonstrate a novel proinflammatory role for miR‐155/miR‐155* in human astrocytes and suggest that IRF3 can suppress neuroinflammation through regulating immunomodulatory miRNA expression.

Making sense of the multiple MAP-2 transcripts and their role in the neuron

Microtubule-associated protein-2 (MAP-2) is a family of heat-stable, phosphoproteins expressed predominantly in the cell body and dendrites of neurons. Three major MAP-2 isoforms, (MAP-2a, MAP-2b, MAP-2c) are differentially expressed during the development of the nervous system and have an important role in microtubule dynamics. Several MAP-2 cDNA clones that correspond to the major MAP-2 transcripts and additional, novel MAP-2 transcripts expressed in the CNS and PNS have been characterized. The transcripts result from the alternative splicing of a single MAP-2 gene consisting of 20 exons. Studies are now being directed toward understanding the role of the multiple MAP-2 forms that contain novel exons in the nervous system. The expression, localization, and possible functions of the newly identified spliced forms are the focus of this review.