TMEM106B Puncta Is Increased in Multiple Sclerosis Plaques, and Reduced Protein in Mice Results in Delayed Lipid Clearance Following CNS Injury.

During inflammatory, demyelinating diseases such as multiple sclerosis (MS), inflammation and axonal damage are prevalent early in the course. Axonal damage includes swelling, defects in transport, and failure to clear damaged intracellular proteins, all of which affect recovery and compromise neuronal integrity. The clearance of damaged cell components is important to maintain normal turnover and restore homeostasis. 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 relapsing-remitting MS (RRMS). We determined that the transmembrane protein 106B (TMEM106B), normally lysosome-associated, is insoluble in RRMS plaques relative to normal-appearing white matter from individuals with Alzheimer's disease and non-neurologic controls. Relative to wild-type mice, hypomorphic mice with a reduction in TMEM106B have increased axonal damage and lipid droplet accumulation in the spinal cord following myelin-oligodendrocyte-glycoprotein-induced experimental autoimmune encephalomyelitis. Additionally, the corpora callosa from cuprizone-challenged hypomorphic mice fail to clear lipid droplets efficiently during remyelination, suggesting that when TMEM106B is compromised, protein and lipid clearance by the lysosome is delayed. As TMEM106B contains putative lipid- and LC3-binding sites, further exploration of these sites is warranted.

Defective myelination in an RNA polymerase III mutant leukodystrophic mouse.

RNA polymerase (Pol) III synthesizes abundant short noncoding 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. These phenotypes reflect a subset of clinical features seen in patients. 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. The findings suggest unique sensitivities of oligodendrogenesis and myelination to perturbations of Pol III transcription.

Anti-Axl antibody treatment reduces the severity of experimental autoimmune encephalomyelitis.

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.

Akt3-Mediated Protection Against Inflammatory Demyelinating Disease.

Akt is a serine/threonine protein kinase that plays a major role in regulating multiple cellular processes. While the isoforms Akt1 and Akt2 are involved in apoptosis and insulin signaling, respectively, the role for Akt3 remains uncertain. Akt3 is predominantly expressed in the brain, and total deletion of Akt3 in mice results in a reduction in brain size and neurodegeneration following injury. Previously, we found that Akt3-/- mice have a significantly worse clinical course during myelin-oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model in which autoreactive immune cells enter the CNS, resulting in inflammation, demyelination, and axonal injury. Spinal cords of Akt3-/- mice are severely demyelinated and have increased inflammation compared to WT, suggesting a neuroprotective role for Akt3 during EAE. To specifically address the role of Akt3 in neuroinflammation and maintaining neuronal integrity, we used several mouse strains with different manipulations to Akt3. During EAE, Akt3 Nmf350 mice (with enhanced Akt3 kinase activity) had lower clinical scores, a lag in disease onset, a delay in the influx of inflammatory cells into the CNS, and less axonal damage compared to WT mice. A significant increased efficiency of differentiation toward FOXP3 expressing iTregs was also observed in Akt3 Nmf350 mice relative to WT. Mice with a conditional deletion of Akt3 in CD4+ T-cells had an earlier onset of EAE symptoms, increased inflammation in the spinal cord and brain, and had fewer FOXP3+ cells and FOXP3 mRNA expression. No difference in EAE outcome was observed when Akt3 expression was deleted in neurons (Syn1-CKO). These results indicate that Akt3 signaling in T-cells and not neurons is necessary for maintaining CNS integrity during an inflammatory demyelinating disease.

Autophagy Is a Tolerance-Avoidance Mechanism that Modulates TCR-Mediated Signaling and Cell Metabolism to Prevent Induction of T Cell Anergy.

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.

The role of TAM family receptors and ligands in the nervous system: From development to pathobiology.

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.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

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 naïve 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.

The control of reactive oxygen species production by SHP-1 in oligodendrocytes.

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.

Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis.

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.

Gas6 enhances axonal ensheathment by MBP+ membranous processes in human DRG/OL promyelinating co-cultures.

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.

Suppression of inflammatory responses during myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis is regulated by AKT3 signaling.

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.

Mice devoid of Tau have increased susceptibility to neuronal damage in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis.

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.

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 IFNß 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.

Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during experimental autoimmune encephalomyelitis.

BACKGROUND: Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. METHODS: WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays. RESULTS: Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals. CONCLUSIONS: These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.

GAS6 enhances repair following cuprizone-induced demyelination.

Growth arrest-specific protein 6 (gas6) activities are mediated through the Tyro3, Axl, and Mer family of receptor tyrosine kinases. Gas6 is expressed and secreted by a wide variety of cell types, including cells of the central nervous system (CNS). In this study, we tested the hypothesis that administration of recombinant human Gas6 (rhGas6) protein into the CNS improves recovery following cuprizone withdrawal. After a 4-week cuprizone diet, cuprizone was removed and PBS or rhGas6 (400 ng/ml, 4 µg/ml and 40 µg/ml) was delivered by osmotic mini-pump into the corpus callosum of C57Bl6 mice for 14 days. Nine of 11 (82%) PBS-treated mice had abundant lipid-associated debris in the corpus callosum by Oil-Red-O staining while only 4 of 19 (21%) mice treated with rhGas6 had low Oil-Red-O positive droplets. In rhGas6-treated mice, SMI32-positive axonal spheroids and APP-positive deposits were reduced in number relative to PBS-treated mice. Compared to PBS, rhGas6 enhanced remyelination as revealed by MBP immunostaining and electron microscopy. The rhGas6-treated mice had more oligodendrocytes expressing Olig1 in the cytoplasm, indicative of oligodendrocyte progenitor cell maturation. Relative to PBS-treated mice, rhGas6-treated mice had fewer activated microglia in the corpus callosum by Iba1 immunostaining. The data show that rhGas6 treatment resulted in more efficient repair following cuprizone-induced injury.

Up-regulation of soluble Axl and Mer receptor tyrosine kinases negatively correlates with Gas6 in established multiple sclerosis lesions.

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.

Axl-/- mice have delayed recovery and prolonged axonal damage following cuprizone toxicity.

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.

In brain, Axl recruits Grb2 and the p85 regulatory subunit of PI3 kinase; in vitro mutagenesis defines the requisite binding sites for downstream Akt activation.

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.

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.

Gas6/Axl signaling activates the phosphatidylinositol 3-kinase/Akt1 survival pathway to protect oligodendrocytes from tumor necrosis factor alpha-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-alpha (TNFalpha) 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 TNFalpha-only-treated cultures. In cultures treated with TNFalpha (100 ng/ml), the oligodendrocyte survival rate was 18% compared with cultures treated with TNFalpha 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 TNFalpha to oligodendrocyte cultures but not in TNFalpha-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 TNFalpha-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 TNFalpha-mediated cell death.