Acute neuroinflammation induces AIS structural plasticity in a NOX2-dependent manner.

BACKGROUND: Chronic microglia-mediated inflammation and oxidative stress are well-characterized underlying factors in neurodegenerative disease, whereby reactive inflammatory microglia enhance ROS production and impact neuronal integrity. Recently, it has been shown that during chronic inflammation, neuronal integrity is compromised through targeted disruption of the axon initial segment (AIS), the axonal domain critical for action potential initiation. AIS disruption was associated with contact by reactive inflammatory microglia which wrap around the AIS, increasing association with disease progression. While it is clear that chronic microglial inflammation and enhanced ROS production impact neuronal integrity, little is known about how acute microglial inflammation influences AIS stability. Here, we demonstrate that acute neuroinflammation induces AIS structural plasticity in a ROS-mediated and calpain-dependent manner. METHODS: C57BL/6J and NOX2-/- mice were given a single injection of lipopolysaccharide (LPS; 5 mg/kg) or vehicle (0.9% saline, 10 mL/kg) and analyzed at 6 h-2 weeks post-injection. Anti-inflammatory Didox (250 mg/kg) or vehicle (0.9% saline, 10 mL/kg) was administered beginning 24 h post-LPS injection and continued for 5 days; animals were analyzed 1 week post-injection. Microglial inflammation was assessed using immunohistochemistry (IHC) and RT-qPCR, and AIS integrity was quantitatively analyzed using ankyrinG immunolabeling. Data were statistically compared by one-way or two-way ANOVA where mean differences were significant as assessed using Tukey's post hoc analysis. RESULTS: LPS-induced neuroinflammation, characterized by enhanced microglial inflammation and increased expression of ROS-producing enzymes, altered AIS protein clustering. Importantly, inflammation-induced AIS changes were reversed following resolution of microglial inflammation. Modulation of the inflammatory response using anti-inflammatory Didox, even after significant AIS disruption occurred, increased the rate of AIS recovery. qPCR and IHC analysis revealed that expression of microglial NOX2, a ROS-producing enzyme, was significantly increased correlating with AIS disruption. Furthermore, ablation of NOX2 prevented inflammation-induced AIS plasticity, suggesting that ROS drive AIS structural plasticity. CONCLUSIONS: In the presence of acute microglial inflammation, the AIS undergoes an adaptive change that is capable of spontaneous recovery. Moreover, recovery can be therapeutically accelerated. Together, these findings underscore the dynamic capabilities of this domain in the presence of a pathological insult and provide evidence that the AIS is a viable therapeutic target.

Developmental changes in myelin-induced proliferation of cultured Schwann cells.

Schwann cell proliferation induced by a myelin-enriched fraction was examined in vitro. Although nearly all the Schwann cells contained material that was recognized by antisera to myelin basic protein after 24 h, only 1% of the cells were synthesizing DNA. 72 h after the addition of the mitogen a maximum of 10% of the cells incorporated [3H]thymidine. If the cultures were treated with the myelin-enriched fraction for 24 h and then washed, the number of proliferating Schwann cells decreased by 75% when compared with those cells that were incubated with the mitogen continuously. When Schwann cells were labeled with [14C]thymidine followed by a pulse of [3H]thymidine 24 h later, every Schwann cell labeled with [3H]thymidine was also labeled with [14C]thymidine. Although almost every Schwann cell can metabolize the myelin membranes within 24 h of exposure, a small population of cell initially utilizes the myelin as a mitogen, and this population continues to divide only if myelin is present in the extracellular media. The percentage of the Schwann cells that initially recognize the myelin-enriched fraction as a mitogen is dependent upon the age of the animal from which the cells were prepared.

Cerebellar granule cells contain a membrane mitogen for cultured Schwann cells.

Proliferation of Schwann cells is one of the first events that occurs after contact with a growing axon. To further define the distribution and properties of this axonal mitogen, we have (a) cocultured cerebellar granule cells, which lack glial ensheathment in vivo with Schwann cells; and (b) exposed Schwann cell cultures to isolated granule cell membranes. Schwann cells cocultured with granule cells had a 30-fold increase in the labeling index over Schwann cells cultured alone, suggesting that the mitogen is located on the granule cell surface. Inhibition of granule cell proteoglycan synthesis caused a decrease in the granule cells' ability to stimulate Schwann cell proliferation. Membranes isolated from cerebellar granule cells when added to Schwann cell cultures caused a 45-fold stimulation in [3H]thymidine incorporation. The granule cell mitogenic signal was heat and trypsin sensitive and did not require lysosomal processing by Schwann cells to elicit its proliferative effect. The ability of granule cells and their isolated membranes to stimulate Schwann cell proliferation suggests that the mitogenic signal for Schwann cells is a ubiquitous factor present on all axons regardless of their ultimate state of glial ensheathment.

Differential proliferative responses of cultured Schwann cells to axolemma- and myelin-enriched fractions. I. Biochemical studies.

Cultured rat Schwann cells were treated for 72 h with axolemma- and myelin-enriched fractions prepared from rat brainstem. [3H]Thymidine was added to the cultures 48 h before the termination of the experiment. Although, both fractions produced a dose-dependent uptake of label into Schwann cells, the shape of the dose response curves and rates at which [3H]thymidine was incorporated were different. The axolemma-enriched fraction produced a sigmoid dose response curve with a Hill coefficient of 2.05. The dose response curve for myelin rose sharply and saturated at a level that was approximately 50% of the maximal response observed with axolemma. Schwann cells that had been treated with axolemma exhibited little change in the rate of [3H]thymidine incorporation from 36-72 h after the addition of the membranes. In contrast, Schwann cells accumulated label three times faster during the 48-72-h period following the addition of myelin to the cultures when compared with the rate during the preceding 12-h interval. Furthermore, the mitogenic activity of the myelin-enriched fraction was decreased by the addition of ammonium chloride, a lysosomal inhibitor, whereas the activity of the axolemmal fraction was not impaired.

Transfection of neonatal rat Schwann cells with SV-40 large T antigen gene under control of the metallothionein promoter.

Secondary cultures of Schwann cells were transfected with a plasmid containing the SV-40 T antigen gene expressed under the control of the mouse metallothionein-I promoter. We used the calcium phosphate method for transfection and obtained a transfection efficiency of 0.01%. The colonies were cloned by limited dilution, and these cloned cell lines were carried in medium containing zinc chloride (100 microM). One cloned cell line, which has now been carried for 180 doublings, appears to have a transformed phenotype with a doubling time of 20 h. These cells express SV-40 T antigen while maintaining established Schwann cell properties (positive staining for 217c, Ran-2, A5E3, glial fibrillary acidic protein, presence of 2',3'-cyclic nucleotide phosphohydrolase [CNPase] activity, and the ability to synthesize sulfogalactosylceramide and mRNA for the myelin protein, P0). Removal of zinc chloride from the medium resulted in reduced expression of T antigen and a change in the appearance of the cells to a more bipolar shape, although they still did not exhibit contact inhibition and maintained a doubling time of 20 h. These cells now became Ran-2-negative and showed increases in CNPase activity and in their ability to synthesize sulfogalactosylceramide. The amount of P0 mRNA remained unchanged. Transfected Schwann cells, however, stopped dividing when they contacted either basal lamina or neurites and became bipolar in appearance. The Schwann cells in contact with the neurites then extended processes to wrap around bundles of neurites. Transfection with the SV-40 T antigen gene therefore provides a method for obtaining Schwann cell lines that continue to express properties associated with untransfected cells in culture and may be used to study axon-Schwann cell interaction.

Axons: isolation from mammalian central nervous system.

Centrifugation of a homogenate of white matter, in a solution of buffered sucrose containing salt, produces a floating layer of myelinated axons. When these are suspended in hypotonic buffer, the mnyelin swells and strips away from the axon. Axons are then separated from the myelin by centrifugation. The resulting preparation consists of a variable population of processes with lengths up to 200 micrometers and diameters between 0.3 and 5.0 micrometers. The axons contain neurofilaments and mitochondria, although no axolemma or neurotubules are evident. The preparation contains cerebroside and sulfatide, yet is essentially free of myelin.

Isolation of filaments from brain.

A method is presented for the isolation of filaments of 90-angstrom diameter from the white matter of bovine brain by first floating the myelinated axons in a centrifugal field and then fractionating the axons on a series of density gradients. This results in a fraction that contains two types of bundles of filaments but few other constituents. The filaments are stable over a wide range of temperatures and at both low and high ionic strength. Their density and their resistance to digestion by ribonuclease and deoxyribonuclease indicate that they are primarily protein. The molecular weight of the subunit is approximately 60,000. The protein does not comigrate with microtubule protein and does not bind cholcicine or nucleotides.

Suppressor cell activity in multiple sclerosis.

Patients with multiple sclerosis and matched controls were tested for lymphocyte stimulation response and induction of suppressor cell activity in response to concanavalin A (Con A) and antigens from axolemma or myelin. Of 17 stable patients, 6 failed to have a suppressor cell response activated by one of these brain cell antigens. Among the patients who lacked these suppressor responses, five had lymphocyte stimulation responses to the same antigens. All matched controls except for one had suppressor cell responses to these antigens and none responded with a positive cellular immune reaction. We found no difference in lymphoproliferative responses to Con A in patients and controls. The level of suppressor cell activity induced by Con A in the stable MS patients varied but did not differ significantly from that of controls.

Identification of a 58-kDa antigen with increased immunoreactivity in the cerebella of multiple sclerosis patients.

An increase in immunoreactivity associated with a 58-kDa antigen was found in a majority of MS cerebellar homogenates examined by Western blot analysis using antisera obtained by selective immunization of rabbits with autopsy cerebella. Two-dimensional immunoblotting demonstrated that the majority of the increased immunoreactivity observed in MS cerebella was associated with the highest apparent pI of three immunoreactive species at 58 kDa. Immuno-crossreaction with rat cerebellar homogenates demonstrated that the 58-kDa antigen was developmentally regulated, showing the greatest immunoreactivity at embryonic day 15. The 58-kDa cerebellar antigen may represent a membrane protein which is re-expressed as part of the onset of MS.

Characterization of an antiserum against an axolemma-enriched fraction.

An antiserum was raised to rat central nervous system (CNS) axolemma-enriched fractions (AEF), which showed no cross-reactivity with myelin proteins or liver microsomes yet gave an endpoint titer of 1:51 200 to CNS AEF by the enzyme-linked immunosorbent assay (ELISA). Immunochemical staining of electroblotted proteins from rat CNS and peripheral nervous system (PNS) AEFs separated by gel electrophoresis identified a major reactive band at 38.5 kD. CNS AEF also showed major immunoreactivity at 91 kD (+/- 3 kD) and a broad band from 110 kD to 130 kD. By immunoperoxidase staining the antiserum specifically recognized the axolemma of peripheral nerve and synaptic terminals in the CNS. The significance of the specificity is discussed with respect to anti-synaptosome antisera.

Role of adult oligodendrocytes in remyelination after neural injury.

Traumatic neural injury is often accompanied by demyelination. The factors that determine the ability of the CNS to remyelinate are examined as well as the origin of the cells responsible for the remyelination. Evidence is presented that suggests that both a precursor-type oligodendrocyte as well as an oligodendrocyte that previously formed a myelin sheath are able to remyelinate in the CNS. A key determinate of the success of remyelination is the ability for either cell type to proliferate before remyelination. We have developed a method to isolate the oligodendrocyte from the mature CNS and studied the mitotic potential of these adult oligodendrocytes in vitro. In contrast to neonatal oligodendrocytes, the adult oligodendrocytes respond weakly to soluble and particulate mitogens. However, when cocultured with neurites, the adult oligodendrocytes demonstrate a more vigorous mitotic response, which may be related to the ability of the neurite to upregulate receptors which transduce the mitotic signal. Since we have identified fibroblast growth factor as a mitogen associated with the axonal plasma membrane that stimulates neonatal oligodendrocytes to divide, we have examined the redistribution of membrane-associated fibroblast growth factor in an in vitro model of neuronal injury. After neuritic injury, fibroblast growth factor containing membrane vesicles were redistributed to the surface of cocultured oligodendrocytes. After invasion of macrophages to a site of neural injury, enzymes secreted by macrophages can release extracellular stores of fibroblast growth factor into the vicinity. This burst of mitotic potential may preferentially stimulate astrocyte rather than oligodendrocyte division, leading to glial scarring and a subsequent failure of remyelination. Other factors to be considered in the potential for remyelination after injury are astrocyte-derived factors that inhibit myelination and the proteins in oligodendrocytes that prevent axonal regrowth indirectly influencing remyelination potential. Thus, provided the oligodendrocyte can gain access to relevant mitogens either in the axonal plasma membrane or in a soluble form and undergo a wave of proliferation, there is good potential for remyelination after neural injury. However, if axonal regrowth is inhibited and astrocytes preferentially are stimulated to divide and form a glial scar, the prognosis for remyelination is poor.

An automated method for autoradiographic analysis of cultured Schwann cells.

A semi-automated analysis system based on video image analysis was developed to count labelled and unlabelled nuclei of Schwann cells which had been exposed to tritiated thymidine followed by processing for autoradiography. A Model 3000 Image Analysis system (Image Technology Corporation, Deer Park, NY) was used to acquire and process the images and provide quantitative measurements based on the distinctive size and shape of the Schwann cell nucleus. The maximum and minimum dimensions for the labelled and unlabelled nuclei were determined. These stored dimensional parameters were then compared with the dimensions of a given field of cell nuclei by the image analysis system. The counts from various fields were collected until a total of 1000 labelled and unlabelled nuclei had been analyzed. A labelling index (LI = ratio of labelled cells to total cells X 100) was then calculated and printed by the system. LIs of autoradiographs determined by automated analysis correlated well with those determined by visual cell counting. The principle of the image analysis program as described here is applicable to other systems for the measurement of LIs of a particular cell type in a mixed population. This automated process eliminates both the subjectivity and fatigue of visual counting and facilitates the rapid measurement of the LI of large numbers of autoradiographs with precision.

Quantitation of changes in P0 mRNA by polymerase chain reaction in primary cultured Schwann cells stimulated by axolemma-enriched fraction.

A requirement for large numbers of primary culture cells has frequently restricted investigations of gene expression in glial cells. We have developed a non-radioactive method based on reverse transcription-polymerase chain reaction (RT-PCR) to accurately assess small changes in the expression of the myelin specific gene P0 in Schwann cells. Using axolemma-enriched fraction (AEF) as an inducing agent, we demonstrate that RT-PCR can be used to detect 4-8-fold increases in P0 mRNA levels occurring in a time and dose dependent manner, utilizing only 250000 cells per assay. Initial experiments used an in vitro transcribed RNA for P0 constructed with a 300 bp deletion for quantitation by competitive RT-PCR. Relative quantitation by co-amplification of the housekeeping gene glyceraldehyde-phosphate dehydrogenase was established and provided similar results. Product evaluation was enhanced 50-100-fold by the incorporation of primers labelled with biotin at the 5' end, allowing for the sensitive detection of PCR product by enhanced chemiluminescence and autoradiography. This technique provides sensitivity to detect and evaluate picogram amounts of DNA. Our results validate the assay for P0 gene expression and indicate that the technique should facilitate the study of multiple genes of interest in glial cell systems.

The subcellular localization of lipid in myelin-free axonal preparations.

Bovine myelin-free axonal preparations were subjected to a series of washes designed to partition membranes from other cellular components initially present in these preparations. These washes were composed entirely of membranous structures, essentially free of neurofilament protein subunits, and contained high specific activity of acetylcholinesterase, an axolemma-specific enzyme. The distribution of acetylcholinesterase in the washes paralleled the distribution of lipid and the lipid composition of these washes closely resembled that of bovine axolemma-enriched fractions. In addition, acetylcholinesterase, lipid and galactocerebroside were histo- and immunohistochemically localized on similar structures in the starting material. Our results demonstrate that some of the lipid in myelin-free axonal preparations may be accounted for by axolemma.

Further studies on the mitogenic response of cultured Schwann cells to rat CNS axolemma-enriched fractions.

Axolemma-enriched fractions isolated from rat CNS stimulated cultured Schwann cells to divide without changing their morphology. Fluorescent activated cell sorter analysis of the axolemma-stimulated cells demonstrated an approximate 3-fold increase in the number of Schwann cells in the S-phase of the cell cycle. This increase correlated well with increases in the number of [3H]thymidine-labeled nuclei observed by light level radioautography. The membrane-bound mitogen was relatively heat-stable, but trypsin-sensitive, and was inactivated both by lipid extraction and sonication. Liver plasma membranes did not increase the mitotic index over that of untreated cells, indicating the axolemma-induced mitosis was not a general response to exogenous membranes. Increasing serum concentrations in the presence of a constant level of axolemma did not change the mitotic index, suggesting that the axolemma did not cause mitosis by removal of an inhibitory factor in serum. Potential mitogens such as gangliosides, myelin basic protein, heparin, an axolemmal lipid extract, and cGMP had no effect on the cultured Schwann cells. The characteristics of the axolemma-related mitogenic factor are discussed relative to other known mitogens for cultured Schwann cells.

Localization of 2',3'-cyclic nucleotide 3'-phosphodiesterase on cultured Schwann cells.

Primary cultures of Schwann cells were labeled by indirect immunofluorescence using an antibody directed against 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). Schwann cells which had been maintained in culture for 8 weeks were labeled with this antibody. Immunoblot analysis of Schwann cell homogenates revealed a single band with a molecular weight of 54,000 daltons which corresponded to a single immunoreactive polypeptide present in myelin prepared from rat sciatic nerve. The subcellular localization of CNPase was examined by fractionation of cultured Schwann cell homogenates with linear sucrose gradients. The distribution of CNPase paralleled that of 5'-nucleotidase, a putative marker for plasma membranes. These results suggest that CNPase is localized on the plasma membranes of Schwann cells and is expressed by the cells in the absence of an axonal stimulus.

Isolation and partial characterization of rat CNS axolemma enriched fractions.

Axolemma-enriched fractions were prepared from rat brain by osmotic shock of a purified preparation of myelinated axons and subsequent separation of myelin, two axolemma-enriched fractions and myelin-free axons by density gradient centrifugation. Compared with the starting whole homogenate, the fractions were enriched in specific activity of Na+K+ ATPase, acetylcholinesterase, 5'nucleotidase as well as 2',3'-cyclic nucleotide 3'phosphohydrolase. Compared with myelin, the axolemmal fractions are greatly enriched in high molecular weight proteins. The 1.0/1.2 fraction has a predominant peak of fucose-labeled glycoprotein with a molecular weight between that of the myelin associated glycoprotein and the Wolfgram protein which is absent from the myelin glycoprotein profile. Polyacrylamide gel electrophoresis showed that the protein profile of myelin isolated by this procedure was similar to that of myelin isolated by other procedures and that the myelin specific basic and proteolipid proteins were virtually absent in the axolemma-enriched fractions. Both axolemma fractions were enriched in higher MW proteins, some of which resembled proteins in the myelin protein profile. Both axolemma-enriched fractions specifically bind between 2 and 3 pmoles of [3H]tetrodotoxin per mg protein. The axolemma-enriched fractions incorporated [3H]leucine and [14C]fucose exclusively into high molecular weight proteins and glycoproteins. In contrast myelin concomitantly isolated with the axolemma-enriched fractions had a significant amount of [3H]leucine labeled protein in myelin proteolipid and basic proteins. In addition to the myelin associated g-ycoprotein the [14C]fucose labeled a glycoprotein of slightly larger apparent molecular weight than proteolipid protein was found in the myelin fraction while the comparable labeled glycoprotein was absent in the axolemma-enriched fractions. The possible extent of contamination of these fractions by myelin or myelin subfractions and relationship of these axolemma-enriched fractions to other axolemma preparations are discussed.

Antisera to an axolemma-enriched fraction inhibit neurite outgrowth and destroy axons in vitro.

Antisera prepared to an axolemma-enriched fraction derived from rat brain inhibited neurite outgrowth and destroyed mature axons in spinal cord-dorsal root ganglia cultures. Similar antibody-mediated anti-axon effects may be important in some diseases of the human nervous system.

Enzyme-linked immunosorbent assay (ELISA) for antibodies to human myelin and axolemma-enriched fractions.

The Enzyme-Linked Immunosorbent Assay (ELISA) is a well established procedure for antibody determination which has gained wide acceptance, particularly in diagnostic virology. We have adapted the method for use with the lipid rich antigens of human myelin and axolemma enriched fractions. Adsorption of the antigen onto the assay plates was rapid and relatively independent of pH. Antibodies to myelin and axolemma cross-reacted extensively. Little antibody reaction was noted using human liver microsomes, indicating the antibodies were specific but that myelin and axolemma shared at least one strong common antigen. With further purification of the antigen, this method should be useful in evaluating immunogenicity and antigenic purity of these membrane fractions.

In vitro use of Schwann cells to elucidate neurotoxic injury.

Schwann cells are responsible for the maintenance of the peripheral myelin sheath and neurotoxic insult directed against these cells can result in demyelination with a concomitant loss of neural function. We have utilized several in vitro techniques to investigate the effects of neurotoxins on the complex interactions between SC and axons. SC may be isolated from fresh neonatal sciatic nerves and used to examine the effect of neurotoxins on the axonal membrane induction of SC proliferation and specific myelin protein mRNA expression. We have recently devised a method to obtain SC from frozen sciatic nerves. This method allows pooling of neonatal nerves to generate enough cells for subsequent study. We have also transfected primary Schwann cells with a plasmid containing the large T antigen to obtain a SC line suitable for neurotoxicology studies. The functional status of cultured SC may also be studied via expression of SC specific antigens such as glial fibrillary acidic protein, CNPase, S100, laminin, P0 and myelin basic protein. We also propose culturing SC with dorsal root ganglion neurons to investigate the effect of neurotoxins on all stages of SC maturation, from proliferation to the in vitro synthesis of a compact myelin sheath. These strategies will allow us to investigate the cellular mechanisms of neurotoxicity in the PNS.