Search

During inflammatory, demyelinating diseases such as multiple sclerosis (MS) axonal damage is prevalent early in the disease course. Axonal damage includes swellings, defects in transport, and failure to clear damaged intracellular proteins, all of which affect recovery and compromise the integrity of neurons and remyelination. Autophagy and the clearance of damaged cell components by the proteasome are important for the maintenance of normal cellular turnover; and the restoration of cellular homeostasis. The gradual accumulation of insoluble proteins in the brain is known to impair recovery from several neurodegenerative diseases. In this study, we used mass spectrometry to identify insoluble proteins within high-speed, mercaptoethanol/sarcosyl-insoluble pellets from purified white matter plaques isolated from the brains of individuals with MS. We determined that insoluble transmembrane protein106B (TMEM106B), expressed in neurons and glia and normally lysosomal-associated, is increased in MS plaques relative to normal-appearing white matter from individuals with Alzheimer’s disease and non-neurologic controls. We found that decreased TMEM106B protein in mice results in significant axonal damage and lipid droplet accumulation in the spinal cord following chronic myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. When TMEM106Bt/t mice were treated with cuprizone to experimentally induce demyelination, a significant increase in lipid deposition was observed in the corpus callosum of TMEM106Bt/t mice post-cuprizone withdrawal. Our study shows that the brain and spinal cord from challenged TMEM106Bt/t mice accumulate OilRedO+/Perilipin2+ lipid droplets. We postulate that increased insolubility of TMEM106B in MS plaques limits debris clearance by the lysosome which over time contributes to failed remyelination and axonal defects.Abbreviated AbstractTransmembrane protein106B (TMEM106B), a lysosome-associated protein, is significantly less soluble in multiple sclerosis plaques than in white matter controls. Decreased TMEM106B produces significant axonal damage and lipid accumulation in mouse models of demyelinating diseases. TMEM106B insolubility and likely loss of function may limit lysosome transport and contribute to CNS pathology.

RNA polymerase (Pol) III synthesizes abundant short non-coding RNAs that have essential functions in protein synthesis, secretion and other processes. Despite the ubiquitous functions of these RNAs, mutations in Pol III subunits cause Pol III-related leukodystrophy, an early-onset neurodegenerative disease. The basis of this neural sensitivity and the mechanisms of disease pathogenesis are unknown. Here we show that mice expressing pathogenic mutations in the largest Pol III subunit, Polr3a, specifically in Olig2-expressing cells, have impaired growth and developmental delay, deficits in cognitive, sensory and fine sensorimotor function, and hypomyelination in multiple regions of the cerebrum and spinal cord. In contrast, the gross motor defects and cerebellar hypomyelination that are common features of severely affected patients are absent in the mice, suggesting a relatively mild form of the disease in this conditional model. Our results show that disease pathogenesis in the mice involves defects that reduce both the number of mature myelinating oligodendrocytes and the ability of these cells to produce a myelin sheath of normal thickness. Thus, the findings suggest cell-specific roles for Pol III in the development and/or survival of oligodendrocytes as well as their function in myelination.Significance StatementPathogenic mutations in subunits of RNA polymerase (Pol) III cause a prevalent autosomal recessive form of leukodystrophy. However, understanding of the mechanisms of pathogenesis, including how ubiquitously-expressed Pol III mutations affect primarily the central nervous system, has been limited by the absence of an animal model of the disease. We show that conditional knock-in of pathogenic Polr3a mutations in the Olig2 lineage in mice results in growth, neurobehavioral and hypomyelination phenotypes reflecting a subset of clinical features of Pol III-related leukodystrophy patients. Myelination defects in the mice identify neural-specific roles for Pol III transcription. The phenotypes of Pol III-related leukodystrophic mice enable genetic and pharmacological approaches aimed at mitigating the consequences of this disease in humans.

Background Multiple sclerosis is an immune-mediated disease of the central nervous system (CNS) characterized by inflammation, oligodendrocytes loss, demyelination, and damaged axons. Tyro3, Axl, and MerTK belong to a family of receptor tyrosine kinases that regulate innate immune responses and CNS homeostasis. During experimental autoimmune encephalomyelitis (EAE), the mRNA expression of MerTK, Gas6, and Axl significantly increase, whereas Tyro3 and ProS1 remain unchanged. We have shown that Gas6 is neuroprotective during EAE, and since Gas6 activation of Axl may be necessary for conferring neuroprotection, we sought to determine whether α-Axl or α-MerTK antibodies, shown by others to activate their respective receptors in vivo, could effectively reduce inflammation and neurodegeneration. Methods Mice received either α-Axl, α-MerTK, IgG isotype control, or PBS before the onset of EAE symptoms. EAE clinical course, axonal damage, demyelination, cytokine production, and immune cell activation in the CNS were used to determine the severity of EAE. Results α-Axl antibody treatment significantly decreased the EAE clinical indices of female mice during chronic EAE and of male mice during both acute and chronic phases. The number of days mice were severely paralyzed also significantly decreased with α-Axl treatment. Inflammatory macrophages/microglia and the extent of demyelination significantly decreased in the spinal cords of α-Axl-treated mice during chronic EAE, with no differences in the production of pro-inflammatory cytokines. α-MerTK antibody did not influence EAE induction or progression. Conclusion Our data suggests that the beneficial effect of Gas6/Axl signaling observed in mice administered with Gas6 can be partially preserved by administering an activating α-Axl antibody, but not α-MerTK.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naive young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

In Brief Mocholi et al. show that, following T cell activation, activation of autophagy constitutes a tolerance-avoidance mechanism that, through modulation of cell metabolism and specific signaling pathways, allows T cells to engage in effector responses and avoid anergy. In vivo inhibition of autophagy in T cells induces tolerance and prevents autoimmunity. In response to activation, CD4+ T cells upregulate autophagy. However, the functional consequences of that upregulation have not been fully elucidated. In this study, we identify autophagy as a tolerance-avoidance mechanism. Our data show that inhibition of autophagy during CD4+ T cell activation induces a long-lasting state of hypo-responsiveness that is accompanied by the expression of an anergic gene signature. Cells unable to induce autophagy after T cell receptor (TCR) engagement show inefficient mitochondrial respiration and decreased turnover of the protein tyrosine phosphatase PTPN1, which translates into defective TCR-mediated signaling. In vivo, inhibition of autophagy during antigen priming induces T cell anergy and decreases the severity of disease in an experimental autoimmune encephalomyelitis mouse model. Interestingly, CD4+ T cells isolated from the synovial fluid of juvenile idiopathic arthritis patients, while resistant to suboptimal stimulation-induced anergy, can be tolerized with autophagy inhibitors. We propose that autophagy constitutes a tolerance-avoidance mechanism, which determines CD4+ T cell fate.

Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K–dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K–dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6−/− and TAM mutant mice models.

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP-1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP-1-depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS-induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP-1-deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid-2)-related factor 2 (Nrf2) responsive gene, heme oxygenase-1 (HO-1). Furthermore, we demonstrate that SHP-1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP-1 in normal-appearing white matter. These studies reveal critical pathways controlled by SHP-1 in oligodendrocytes that relate to susceptibility of SHP-1-deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases. GLIA 2015;63:1753–1771

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination., This work was supported by the National Multiple Sclerosis Society (Grant RG 4046-A6) and the National Institutes of Health (Grant R21 NS079144-01, Neuropathology Training Grant T32 NS007098, and Cellular and Molecular Biology and Genetics Training Grant T32 GM007491).

AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biologic processes. AKT3 is expressed in immune cells and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein–induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hypothesized that during EAE, deletion of AKT3 would negatively affect the CNS of AKT3−/− mice, making them more susceptible to CNS damage. During acute EAE, AKT3−/−mice were more severely affected than wild type (WT) mice. Evaluation of spinal cords showed that during acute and chronic disease, AKT3−/− spinal cords had more demyelination compared with WT spinal cords. Quantitative RT-PCR determined higher levels of IL-2, IL-17, and IFN-γ mRNA in spinal cords from AKT3−/− mice than WT. Experiments using bone marrow chimeras demonstrated that AKT3−/− mice receiving AKT3-deficient bone marrow cells had elevated clinical scores relative to control WT mice reconstituted with WT cells, indicating that altered function of both CNS cells and bone marrow–derived immune cells contributed to the phenotype. Immunohistochemical analysis revealed decreased numbers of Foxp3+ regulatory T cells in the spinal cord of AKT3−/− mice compared with WT mice, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell–mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.

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.

Neuroblastomas constitute about 10% of childhood cancers and are responsible for 15% of pediatric cancer mortality. We evaluated the efficacy and the mechanism of cell death induced by CAY10404, a selective cyclooxygenase-2 (Cox-2) inhibitor in four human neuroblastoma cell lines (SH-EP, SH-SY5Y, SK-N-MC and MSN). Treatment with CAY10404 in the range of 15-115 microM revealed a dose-dependent decrease in cell number and an average IC50 (inhibitory concentration 50%) of 60 microM. About 20-30% of the cells were terminal deoxynucleotidyltransferase-mediated UTP nick-end-labeling (TUNEL) positive 48 h after treatment. Western blot analysis of CAY10404-treated cells showed poly(ADP-ribose) polymerase (PARP) cleavage and cleaved caspase-3 signifying caspase activity and apoptotic cell death. Inhibitor-of-apoptosis proteins including X-linked inhibitor-of-apoptosis protein (XIAP) and survivin did not change significantly after CAY10404 treatment. Fluorescence activated cell sorter (FACS) analysis performed in two different cell lines 48 h following CAY10404 treatment showed a reduction in the number of cells in the G1 phase of the cell cycle and an increase in the number of cells in the G2 phase. When radioresistant SH-EP cells were treated with CAY10404, a 49% decrease in cell viability was observed relative to DMSO-treated cells; pretreatment with CAY10404 followed by ortho-voltage irradiation further enhanced cell death (58%) suggesting radiosensitization by CAY10404.

In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.

Microtubule-associated protein-2 (MAP-2) functions to maintain neuronal morphology by promoting the assembly of microtubules. MAP-2c is an alternately spliced form of MAP-2, containing the first 151 amino acids of high-molecular-weight (HMW) MAP-2 joined to the last 321 amino acids, eliminating 1,352 amino acids specific to HMW MAP-2. A polyclonal antibody generated to the splice site of human MAP-2c was used to determine its cellular localization. The MAP-2c antiserum was depleted of any HMW MAP-2 reactivity by absorption with HMW MAP-2 fusion protein. Western blot analysis of human fetal spinal cord homogenates demonstrated that the antibody is specific for human MAP-2c. MAP-2c immunoreactivity was found in the perinuclear cytoplasm and processes of anterior motor neurons and large processes of the posterior column in sections from 22–24-week human fetal spinal cord. Double-label confocal microscopy was performed using the MAP-2c polyclonal antibody and either a HMW MAP-2 or a neurofilament protein (highly phosphorylated 160- and 200-kDa protein) monoclonal antibody to identify these processes as dendrites or axons, respectively. HMW MAP-2 and MAP-2c colocalized in cell bodies and dendrites of anterior motor neurons, demonstrating for the first time the presence of native MAP-2c within dendrites. In addition, immunoelectron microscopy showed MAP-2c associated with microtubules in dendrites of motor neurons. MAP-2c and the neurofilament proteins were found in axons of the dorsal and ventral roots. The presence of MAP-2c within axons and dendrites suggests that MAP-2c contributes to neuronal plasticity during human fetal development.

In human fetal spinal cord (HFSC), six additional microtubule-associated protein-2 (MAP-2) transcripts are generated by alternative splicing of two recently described exons, exon 8 and exon 13. The following three translated proteins are detected by western blot analysis: MAP-2b expressing exon 8 (MAP-2b + 8; MAP-2a), MAP-2b expressing exon 13 (MAP-2b + 13), and MAP-2c expressing exon 8 and exon 13 (MAP-2c + 8 + 13). The finding that MAP-2b + 8 is expressed in HFSC demonstrates for the first time the presence of MAP-2a in human fetal CNS. Immunocytochemical studies show that exon 8-specific antibody and exon 13-specific antibody stain independent and overlapping populations of neurons in the lumbar region of the HFSC. Antibody 13-immunopositive neurons have predominantly cytosolic staining, whereas in the antibody 8-immunoreactive neurons staining was observed in the cytosol, dendrites, and some synapses. The prenatal expression of MAP-2a, which has been used as a marker of synaptogenesis, not only demonstrates the presence of a mature MAP-2 isoform in HFSC, but suggests that MAP-2a is important during human fetal as well as postnatal synaptogenesis.

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-beta 1 (TGF-beta 1), 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-beta 1 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.

Elucidation of the mechanisms involved in the regeneration of oligodendrocytes and remyelination is a central issue in multiple sclerosis (MS) research. We recently identified a novel alternatively spliced, developmentally regulated oligodendrocyte-specific protein designated microtubule-associated protein-2+13 [microtubule-associated protein-2 expressing exon 13 (MAP-2+13)]. MAP-2+13 is expressed in human fetal oligodendrocytes during process extension and myelination but is minimally expressed in normal mature CNS. To test the hypothesis that MAP-2+13 is reexpressed in regenerating oligodendrocytes in MS lesions, we examined the brains of MS patients for the expression of this protein. By immunocytochemistry using a series of monoclonal antibodies specific for MAP-2+13, we determined that MAP-2+13 expression was up-regulated in all 31 lesions from 10 different MS brains. MAP-2+13 was expressed in regenerating oligodendrocytes associated with demyelinated lesions, with the highest counts found in regions of extensive remyelination. By electron microscopy, MAP-2+13 was localized to oligodendrocytes engaged in remyelination, evident by their process extension and association with thinly myelinated (remyelinated) and demyelinated axons. These results suggest a hitherto unsuspected role for this microtubule-associated protein in oligodendrocyte function during development and myelin repair.

T cell activation requires the regulation of signaling pathways and metabolic programs downstream of T cell receptor and CD28 engagement. Upon activation, effector CD4+ T helper cells induce autophagy, a lysosomal-mediated protein degradation mechanism. The controlled degradation and recycling of cell components allow effector T helper cells to respond to various types of stimuli. However, it is unknown how autophagy may regulate the antigenic responses and functional capacity of CD4+T cells. We found that inhibiting activation-induced autophagy in CD4+T helper type 1 (TH1) cells induces a hyporesponsive state. This hyporesponsiveness is associated with a reduction in ATP generation and decreased glycolysis and mitochondrial respiration. Moreover, T cells that are unable to upregulate autophagy accumulate a protein tyrosine phosphatase that negatively regulates mitogen-activated protein kinase (MAPK) signaling. In vivo deletion of an essential autophagy-related gene in T cells delayed the onset of and decreased the severity of experimental autoimmune encephalomyelitis. Our data support that autophagy may be necessary for T helper cell activation by regulating signaling pathways and metabolic programs, and the inhibition of this catabolic process may induce tolerance.

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP-1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP-1-depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS-induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP-1-deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid-2)-related factor 2 (Nrf2) responsive gene, heme oxygenase-1 (HO-1). Furthermore, we demonstrate that SHP-1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP-1 in normal-appearing white matter. These studies reveal critical pathways controlled by SHP-1 in oligodendrocytes that relate to susceptibility of SHP-1-deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases.

Microtubule-associated protein 2 (MAP-2) isoforms are developmentally expressed in the nervous system and contain a number of functional domains. Adjacent to the first repeat of the microtubule-binding domain is an RTPPKSP motif for binding SH3 domains. To identify SH3-containing proteins that interact with MAP-2, transfections, filter overlay assays, glutathioneS-transferase (GST)-mediated binding assays, co-immunoprecipitations and enzyme-linked immunosorbent assays were performed. Transfections of MAP-2a, MAP-2b, and MAP-2c constructs into COS7 cells, followed by incubation of the cell lysates with SH3-GST fusion proteins, determined that the strongest interaction was between MAP-2c and the non-receptor tyrosine kinase Fyn; however, MAP-2b and MAP-2c also bound to Grb2. Co-immunoprecipitation of Fyn and MAP-2c from human fetal homogenates confirmed the interaction in vivo. MAP-2 synthetic peptides spanning the RTPPKSP motif bound to Fyn, and the interaction was regulated by phosphorylation. Co-transfections with MAP-2c and the extracellular signal-regulated kinase 2 (ERK2) demonstrated that MAP-2c is threonine/serine-phosphorylated on its RTPPKSP motif and that threonine phosphorylation abolished the MAP-2c/Fyn binding. Kinase assays and co-transfection of MAP-2c and Fyn confirmed that Fyn tyrosine kinase phosphorylates MAP-2c. Thus, the activation of signaling pathways may regulate cytoskeletal dynamics by altering the state of phosphorylation of MAP-2 by both ERK2 and Fyn kinase.

Oligodendrocyte development and survival occurs in response to signalling pathways activated by protein trophic factors. To identify novel factors that regulate human oligodendrocyte maturation/survival, we performed microarray analysis and identified genes expressed in moderate amounts in oligodendrocytes at 18–22 gestational weeks (gw). Among those characterized were the genes encoding the receptor tyrosine kinases, Axl and Mer. These receptors belong to the mammalian Axl/Mer/Rse receptor tyrosine kinase family and are developmentally regulated. The ligand for this family is the growth arrest-specific 6 gene product (Gas6). Gas6 stimulates growth and prevents apoptosis in a variety of cell types. Oligodendrocytes isolated from 20 to 23 gw human fetal spinal cord were grown for 6 days on NIH 3T3 fibroblasts engineered to stably express Gas6 or on unmodified 3T3 cells. Oligodendrocytes were identified by immunostaining for proteins expressed by mature oligodendrocytes, CNPaseI and MBP. An increase in the numbers of CNPaseI+ and MBP+ oligodendrocytes, and the extent of process branching and extension, was observed when grown on irradiated 3T3-cells secreting Gas6, but not on irradiated 3T3 cells. The effect of Gas6 was abolished in the presence of the Axl-Fc receptor, but remained unchanged in the presence of TrkA-Fc receptor. Our results suggest a role for the Gas6-Axl signalling in regulating oligodendrocyte viability and maturation.

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.

Microtubule-associated protein-2 (MAP-2) is a prominent cytoskeletal protein in the mammalian nervous system. Two high-molecular-weight (HMW) MAP-2 isoforms, MAP-2a and MAP-2b, are developmentally regulated. MAP-2b is expressed through the life of the neuron, while MAP-2a expression coincides with the time of synaptic formation. MAP-2a and MAP-2b differ in size by approximately 10 kD. Attempts to differentiate MAP-2a from MAP-2b led to the identification of additional exons; exons 7A, 8, 13, and 16. The focus of the present study was to define the complete molecular composition of MAP-2a that was prerequisite for investigating the functional characteristic of the MAP-2a protein. Detailed examination of rat brain mRNA by Northern blot analysis and RT-PCR showed that MAP-2a contains only exon 8 in addition to the exons found in the MAP-2b transcript. Exons 7A, 13, and 16 are not present in the MAP-2a transcript. Antibody generated to exon 8 expressed protein, immunoprecipitated a HMW protein from adult rat brain that co-migrated with MAP-2a and was immunopositive with other MAP-2 antibodies. Comparative transfections of full-length MAP-2a and MAP-2b cDNA into COS-7 cells demonstrated that MAP-2a influenced the microtubule network differently than MAP-2b by inducing rapid and stable microtubule bundle formation even in the presence of nocodazole.

The molecular requirements for human myelination are incompletely defined, and further study is needed to fully understand the cellular mechanisms involved during development and in demyelinating diseases. We have established a human co-culture model to study myelination. Our earlier observations showed that addition of human γ-carboxylated growth-arrest-specific protein 6 (Gas6) to human oligodendrocyte progenitor cell (OPC) cultures enhanced their survival and maturation. Therefore, we explored the effect of Gas6 in co-cultures of enriched OPCs plated on axons of human fetal dorsal root ganglia explant. Gas6 significantly enhanced the number of myelin basic protein-positive (MBP+) oligodendrocytes with membranous processes parallel with and ensheathing axons relative to co-cultures maintained in defined medium only for 14 days. Gas6 did not increase the overall number of MBP+ oligodendrocytes/culture; however, it significantly increased the length of MBP+ oligodendrocyte processes in contact with and wrapping axons. Multiple oligodendrocytes were in contact with a single axon, and several processes from one oligodendrocyte made contact with one or multiple axons. Electron microscopy supported confocal Z-series microscopy demonstrating axonal ensheathment by MBP+ oligodendrocyte membranous processes in Gas6-treated co-cultures. Contacts between the axonal and oligodendrocyte membranes were evident and multiple wraps of oligodendrocyte membrane around the axon were visible supporting a model system in which to study events in human myelination and aspects of non-compact myelin formation.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.

Macroautophagy is a catabolic process whereby cell components, which range from soluble proteins to whole organelles, are sequestered in de novo formed double-membrane autophagosomes that fuse with lysosomes to degrade their cargo. Effector T helper cells induce macroautophagy during activation to maintain cell proliferation and cytokine production. T cells that are unable to upregulate macroautophagy activity show a reduction in ATP generation, suggesting that macroautophagy is necessary to maintain the energy metabolism required to meet the demands of T cell activation. It is presently unknown whether macroautophagy regulates the activation state and functional capacity of CD4+T cells. We explored the instructive role of macroautophagy in effector T helper cell activation and tolerance. Inhibition of macroautophagy during activation of CD4+T helper type 1 (TH1) cells induces a long-lasting state of hyporesponsiveness, with a molecular signature that resembles that of anergic T cells. Moreover, inhibition of macroautophagy prevented activated TH1 cells from upregulating glycolysis and mitochondrial respiration, supporting that macroautophagy may regulate metabolic homeostasis during activation of effector T helper cells. Consequently, inhibition of macroautophagy in vivo prevents CD4+ T cell activation in response to immunization and results in decreased severity of experimental autoimmune encephalomyelitis. Our data support that macroautophagy, likely by regulating metabolism, is necessary for the activation of effector T helper cells and that its absence induces a tolerant state.

The abundant axonal microtubule-associated protein tau regulates microtubule and actin dynamics, thereby contributing to normal neuronal function. We examined whether mice deficient in tau (Tau−/−) or with high levels of human tau differ from wild-type (WT) mice in their susceptibility to neuroaxonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. After sensitization with MOG35–55, there was no difference in clinical disease course between human tau and WT mice, but Tau−/− mice had more severe clinical disease and significantly more axonal damage in spinal cord white matter than those in WT mice. Axonal damage in gray matter correlated with clinical severity in individual mice. By immunoblot analysis, the early microtubule-associated protein-1b was increased 2-fold in the spinal cords of Tau−/− mice with chronic experimental autoimmune encephalomyelitis versus naive Tau−/− mice. This difference was not detected in comparable WT animals, which suggests that there was compensation for the loss of tau in the deficient mice. In addition, levels of the growth arrest–specific protein 7b, a tau-binding protein that is stabilized when bound to tau, were higher in WT than those in Tau−/− spinal cord samples. These data indicate that loss of tau exacerbates experimental autoimmune encephalomyelitis and suggest that maintaining tau integrity might reduce the axonal damage that occurs in inflammatory neurodegenerative diseases such as multiple sclerosis.

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. © 2011 Wiley-Liss, Inc.

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.

Axl is a receptor tyrosine kinase implicated in cell survival following growth factor withdrawal and other stressors. The binding of Axl's ligand, growth arrest-specific protein 6 (Gas6), results in Axl autophosphorylation, recruitment of signaling molecules, and activation of downstream survival pathways. Pull-down assays and immunoprecipitations using wildtype and mutant Axl transfected cells determined that Axl directly binds growth factor receptor-bound protein 2 (Grb2) at pYVN and the p85 subunit of phosphatidylinositol-3 kinase (PI3 kinase) at two pYXXM sites (pY779 and pY821). Also, p85 can indirectly bind to Axl via an interaction between p85's second proline-rich region and the N-terminal SH3 domain of Grb2. Further, Grb2 and p85 can compete for binding at the pY821VNM site. Gas6-stimulation of Axl-transfected COS7 cells recruited activated PI3 kinase and phosphorylated Akt. An interaction between Axl, p85 and Grb2 was confirmed in brain homogenates, enriched populations of O4+ oligodendrocytes, and O4- flow-through prepared from day 10 mouse brain, indicating that cells with active Gas6/Axl signal through Grb2 and the PI3 kinase/Akt pathways.

Activation of the receptor tyrosine kinase Axl recruits signaling molecules that regulate cell growth and survival. To evaluate Axl's role in brain during cuprizone toxicity, mice were fed cuprizone and evaluated at 3-, 4-, and 6-week cuprizone treatment and 3- and 5-week post-cuprizone withdrawal. At 4-week cuprizone treatment, the corpora callosa of wildtype (WT) mice had robust Oil Red O+ staining indicative of ongoing phagocytosis. Axl-/- mice had minimal Oil Red O+ staining, fewer microglia, and significantly more TUNEL+/ASPA+ mature oligodendrocytes than the WT. At 6-week cuprizone treatment, there was significantly more Oil Red O+ staining in the Axl-/- corpora callosa than in the WT indicating a lag in the clearance of cellular and myelin debris. Relative to WT mice, there were fewer mature oligodendrocytes and significantly more SMI-32+ axons at 3-week post-cuprizone withdrawal, indicative of axonal damage in the Axl-/- corpora callosa. Electron microscopy determined that at 3-week post-cuprizone withdrawal the number of dystrophic axons and axons containing autophagosome-like vacuoles/mouse was increased in the Axl-/- mice relative to the WT mice. In Axl-/- corpora callosa, 5-week post-cuprizone withdrawal, the number of mature oligodendrocytes was comparable to the WT mice, but axons in the Axl-/- mice were SMI-32+, suggesting that Axl-/- mice have delayed clearance of apoptotic oligodendrocytes and myelin debris resulting in prolonged axonal damage and recovery from cuprizone toxicity.

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.

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5–2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 ± 35 μg (mean ± SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11–14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11–14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to ∼50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls. The changes in GFAP message content contrasted sharply with those in protein content, which increases slowly and does not reach maximal levels until after 4 weeks. Elevated levels of GFAP mRNA, protein, and immunostaining persisted through 65 dpi, >40 days after clinical signs had resolved.

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 inAkt1−/−oligodendrocytes. Oligodendrocyte cultures established from wild-type andRse−/−mice, but not fromAxl−/−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.

Neuroblastoma is the most common solid tumor of infants and carries a poor prognosis especially in advanced stages. The present recommended therapies carry a high risk of side effects that is associated with long-term morbidity. We evaluated the efficacy of a low dose of the selective cyclooxygenase-2 inhibitor Nimesulide in preventing human Neuroblastoma tumor growth in Severe Combined Immune-deficient mice. Mice containing established tumors (SH-SY5Y cells) treated with 20 mg/kg Nimesulide every 4th day beginning on day 1 of cell injections resulted in a 65% reduction of tumor growth compared to the DMSO treated control mice (P

We investigated the interaction of MAP-2c and Fyn in the initiation of process outgrowth in COS7 cells. Single transfections of Fyn and MAP-2c resulted in a dramatic decrease in flat, rounded COS7 cells, and a significant increase in both the number of cells with multiple short, spike-like processes, and cells with longer processes. Co-transfection of Fyn and MAP-2c resulted in an additive increase in the number of cells with more than two processes and discrete sites of co-localization within processes. When single or double transfected cells were treated with cytochalasin D or lantrunculin there was a dramatic increase in the number of cells with more than two processes. In addition, there was an increase in the length of the processes, both in single and double transfected cells, suggesting that the actin meshwork provides a barrier for MT-based process extension. When co-transfected cells were post-treated with nocodazole, Fyn was not associated with MAP-2c and acetylated, stable tubulin. Although some Fyn/MAP-2c co-localization was retained, punctate staining of MAP-2c and Fyn were observed at the cell periphery, in areas devoid of stable MTs. Mutations in either tyrosine 67 (Tyr67), a site on human MAP-2c phosphorylated by Fyn, or a second tyrosine residue (Tyr50), did not alter the ability of MAP-2c and Fyn to induce process outgrowth. These studies suggest that independent of one another MAP-2c and Fyn are able to induce process outgrowth and in concert can initiate and enhance process outgrowth in an additive manner.

The Src homology 3 (SH3) domain of Fyn binds to a conserved PXXP motif on microtubule-associated protein-2. Co-transfections into COS7 cells and in vitro kinase assays performed with Fyn and wild-type, or mutant MAP-2c, determined that Fyn phosphorylated MAP-2c on tyrosine 67. The phosphorylation generated a consensus sequence for the binding of the SH2 domain of Grb2 (pYSN). Pull-down assays with SH2-Grb2 from human fetal brain homogenates, and co-immunoprecipitation of Grb2 and MAP-2 confirmed the interaction in vivo, and demonstrated that MAP-2c is tyrosine-phosphorylated in human fetal brain. Filter overlay assays confirmed that the SH2 domain of Grb2 binds to human MAP-2c following incubation with active Fyn. Enzyme-linked immunosorbent assays confirmed the interaction between the SH2 domain of Grb2 and a tyrosine-phosphorylated MAP-2 peptide spanning the pY(67)SN motif. Thus, MAP-2c can directly recruit multiple signaling proteins important for central nervous system development.

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 isgrowth arrest-specificgene 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.

We have identified a developmentally regulated, oligodendrocyte-specific protein, designated microtubule-associated protein-2 expressing exon 13 (MAP-2+13), in the human central nervous system (CNS). Monoclonal antibodies directed against MAP-2+13 labeled oligodendrocytes in the white matter of human fetal spinal cord. Double-label immunofluorescence and confocal microscopy, and immunoelectron microscopy localized MAP-2+13 to the soma and extending processes of fetal oligodendrocytes, but not to the myelin sheath. The immunoreactivity was throughout the perikarya. Ultrastructural examination of the fetal myelin sheaths showed them to be thin and not fully compacted, indicating that myelination was in progress. There was no overlap in staining of GFAP+ astrocytes and MAP-2+13+ oligodendrocytes. MAP-2+13 was also expressed in intermediate filament-negative "radial glia" extending from the central canal to the subpial surface. In the mature CNS, MAP-2+13 also marked cells of oligodendroglial morphology, but these cells were rare. These finding demonstrate that in the human CNS, MAP-2+13 is a novel protein transiently expressed in cells of oligodendroglial lineage.

The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2 + 8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2 + 8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2 + 8 comigrated with MAP-2a. In human adult brain, HMW MAP-2 + 8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultrastructural level, HMW MAP-2 + 8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2 + 8. The presence of HMW MAP-2 + 8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.

Three unique 5′ untranslated regions (UTRs) have been characterized for human microtubule-associated protein-2 (MAP-2) transcripts. All three UTRs shared a common 171-bp sequence adjacent to the MAP-2 coding region and then diverged upstream. The size of the unique upstream sequence was 281, 146, or 104 bp. PCR of genomic DNA demonstrated that the 5′ UTRs span multiple exons. The unique region of the UTRs recognizes a 9.5- and a 6-kb MAP-2 transcript in poly(A)+ mRNA isolated from human MSN cells, and PCR analysis demonstrated that each unique UTR is contained in multiple high- and low-molecular-weight MAP-2 transcripts. Reverse transcription-PCR (RT-PCR) performed on MSN mRNA isolated from polysomes demonstrated that all three of the UTRs contained within multiple MAP-2 transcripts were associated with polysomes and hence translated. RT-PCR from human fetal spinal cord and adult brain mRNA demonstrated that all of the UTRs are expressed at these developmental time points.

Antibodies were raised to two synthetic peptides with amino acid sequences encoded by a variable region of exons 10 and 11 of the tau gene. The affinity-purified antibodies, designated E-10 and E-11, were used to determine whether PHF-tau and normal tau differ in variants containing three or four repeats in the microtubule-binding domain, respectively. Normal adult human brain was shown by gel electrophoresis to contain six isoforms of tau. All of the isoforms reacted with E-11, whereas only four of them with slower electrophoretic mobility were recognized by E-10. Fetal brain tau was readily recognized by E-11 but reacted poorly with E-10. In PHF preparations, E-11 bound to all three polypeptides of PHF-tau of 68 kD, 64 kD, and 60 kD and reacted intensely with a material smearing from the top of the gel to about the 50-kD region. In contrast, E-10 only weakly recognized the two higher molecular weight PHF-tau polypeptides of 68 kD and 64 kD, as well as smeared material, and the binding was not affected by phosphatase treatment. Using recombinant tau with four repeats as a reference, the immunoreactivity of E-10 with PHF-tau was estimated to be approximately 5% of that of E-11. By comparison, the immunoreactivity of E-10 with four isoforms of normal tau was comparable to that of E-11. These results indicate that the ratio of three vs. four repeat variants in PHF-tau is higher than in normal tau and suggest that Alzheimer disease may be associated with the disproportional expression of fetal (or juvenile) forms of tau. Alternatively, the weak reactivity of PHF-tau with E-10 antibody could be due to post-translational modifications other than phosphorylation. © 1995 Wiley-Liss, Inc.

Microtubule-associated protein 2 (MAP-2) is an abundant neuronal cytoskeletal protein that binds to tubulin and stabilizes microtubules. Using fusion protein constructs we have defined the epitopes of 10 monoclonal antibodies (mAbs) to discrete regions of human MAP-2. Proteins were expressed in pATH vectors. After electrophoresis, immunoblotting was performed. By western blot analysis five of the mAbs (AP-14, AP-20, AP-21, AP-23, and AP-25) share epitopes with only the high molecular weight isoforms (MAP-2a, MAP-2b); two of the mAbs (AP-18 and tau 46) recognize MAP-2a, MAP-2b, and MAP-2c. Although AP-18 immunoreactivity was detected within heat-stable protein homogenates isolated from a human neuroblastoma cell line MSN, fusion protein constructs encompassing human MAP-2 were negative, suggesting that the AP-18 epitope is phosphorylated. Furthermore, AP-18 immunoreactivity was lost after alkaline phosphatase treatment of heat-stable protein preparations from MSN cells. Four of the mAbs (322, 636, 635, and 39) recognize epitopes located within amino acids 169–219 of human MAP-2. AP-21 maps to a region between amino acids 553 and 645. AP-23 maps between amino acids 645 and 993, whereas AP-20, AP-14, and AP-25 map between amino acids 995 and 1332. Expression of the region of MAP-2 between amino acids 1787 and 1824 was positive to tau 46.

Microtubule-associated protein 2 (MAP-2) is an abundant component of the cytoskeleton present in dendrites and cell bodies of neurons of the CNS. To examine the biological function of MAP-2, two MAP-2 antisense (AS) oligonucleotides complementary to the 5' region of the rat MAP-2 cDNA were added to rat primary embryonic day 17-18 (E17-18) cultured cortical neurons 24 h after plating and neurite outgrowth and morphology studied. The treatment of primary cortical cultures with either of the two MAP-2 AS oligonucleotides resulted in decreased MAP-2 and reduction in the number of neuritic processes relative to the control or MAP-2 sense-treated cultures. By immunostaining and light microscopy the AS-treated neurons appeared smaller, more rounded, and less intensely stained for MAP-2 than the untreated or the MAP-2 sense-treated cultures. By electron microscopy disorganized microtubules and a reduction in the number of microtubules within neurites of the AS-treated cultures were observed. We conclude that MAP-2 continues to be required for microtubule spacing and stability within neurites once they have formed.