Adult-onset depletion of sulfatide leads to axonal degeneration with relative myelin sparing.

3-O-sulfogalactosylceramide (sulfatide) constitutes a class of sphingolipids that comprise about 4% of myelin lipids in the central nervous system. Previously, our group characterized a mouse with sulfatide's synthesizing enzyme, cerebroside sulfotransferase (CST), constitutively disrupted. Using these mice, we demonstrated that sulfatide is required for establishment and maintenance of myelin, axoglial junctions, and axonal domains and that sulfatide depletion results in structural pathologies commonly observed in Multiple Sclerosis (MS). Interestingly, sulfatide is reduced in regions of normal appearing white matter (NAWM) of MS patients. Sulfatide reduction in NAWM suggests depletion occurs early in disease development and consistent with functioning as a driving force of disease progression. To closely model MS, an adult-onset disease, our lab generated a "floxed" CST mouse and mated it against the PLP-creERT mouse, resulting in a double transgenic mouse that provides temporal and cell-type specific ablation of the Cst gene (Gal3st1). Using this mouse, we demonstrate adult-onset sulfatide depletion has limited effects on myelin structure but results in the loss of axonal integrity including deterioration of domain organization accompanied by axonal degeneration. Moreover, structurally preserved myelinated axons progressively lose the ability to function as myelinated axons, indicated by the loss of the N1 peak. Together, our findings indicate that sulfatide depletion, which occurs in the early stages of MS progression, is sufficient to drive the loss of axonal function independent of demyelination and that axonal pathology, which is responsible for the irreversible loss of neuronal function that is prevalent in MS, may occur earlier than previously recognized.

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.

Nfasc155H and MAG are specifically susceptible to detergent extraction in the absence of the myelin sphingolipid sulfatide.

Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

Adult CST-null mice maintain an increased number of oligodendrocytes.

The galactolipids galactocerebroside and sulfatide have been implicated in oligodendrocyte (OL) development and myelin formation. Much of the early evidence for myelin galactolipid function has been derived from antibody and chemical perturbation of OLs in vitro. To determine the role of these lipids in vivo, we previously characterized mice lacking galactocerebroside and sulfatide and observed abundant, unstable myelin and an increased number of OLs. We have also reported that mice incapable of synthesizing sulfatide (CST-null) while maintaining normal levels of galactocerebroside generate relatively stable myelin with unstable paranodes. Additionally, Hirahara et al. (2004; Glia 45:269-277) reported that these CST-null mice also contain an increased number of OLs in the forebrain, medulla, and cerebellum at 7 days of age. Here, we further the findings of Hirahara et al. by demonstrating that the number of OLs in the CST-null mice is also increased in the spinal cord and that this elevated OL population is maintained through, at least, 7 months of age. Moreover, we show that the enhanced OL population is accompanied by increased proliferation and decreased apoptosis of oligodendrocytic-lineage cells. Finally, through ultrastructural analysis, we show that the CST-null OLs exhibit decreased morphological complexity, a feature that may result in decreased OL competition and increased OL survival.

Sulfatide is essential for the maintenance of CNS myelin and axon structure.

Galactocerebroside (GalC) and sulfatide are abundant myelin lipids. In mice incapable of synthesizing these lipids, myelin is thin and regionally unstable and exhibits several subtle structural abnormalities. Although galactolipid-null mice have been beneficial in the analysis of galactolipid function, it has not been possible to differentiate between the functions of GalC and sulfatide with these mice alone. In the present work, we have analyzed a murine model that forms normal levels of GalC but is incapable of synthesizing sulfatide. By comparing a plethora of morphological features between the galactolipid-null and the sulfatide-null mice, we have begun to differentiate between the specific functions of these closely related lipids. The most striking difference between these two mutants is the reduction of myelin developmental abnormalities (e.g., redundant and uncompacted myelin sheaths) in young adult sulfatide-null mice as compared with the galactolipid-null animals. Although sulfatide appears to play a limited role in myelin development, this lipid is essential for myelin maintenance, as the prevalence of redundant, uncompacted, and degenerating myelin sheaths as well as deteriorating nodal/paranodal structure is increased significantly in aged sulfatide-null mice as compared with littermate wildtype mice. Finally, we show that the role played by sulfatide in CNS maintenance is not limited to the myelin sheath, as axonal caliber and circularity are normal in young adult mutant mice but are significantly altered in aged sulfatide-null animals.

Effects of galactolipid elimination on oligodendrocyte development and myelination.

The galactolipids galactocerebroside and sulfatide, which require the enzyme UDP-galactose:ceramide galactosyltransferase (CGT) for their synthesis, are among the most prevalent molecules in the myelin sheath. Numerous studies, mainly using antibody perturbation methods in vitro, have suggested that these molecules are crucial mediators of oligodendrocyte differentiation and myelin formation. Although we have previously demonstrated that myelin formation occurs in CGT null mutant mice, which are incapable of synthesizing the myelin galactolipids, here we show that there are developmental alterations in the CNS of these animals. There is a significant decrease in the number of myelinated axon segments in the mutant spinal cord despite normal levels of myelin gene-specific mRNAs and proteins. Also, there is an increased cellularity in the mature mutant spinal cord and the distinctive morphology of the additional cells suggests that they are actively myelinating oligodendrocytes. Using in situ hybridization techniques, we show that there is a 50% increase in the number of oligodendrocytes in the mutant spinal cord. The data suggest that galactolipids play an important developmental role in regulating the maturation program and final number of oligodendrocytes.

Axo-glial interactions regulate the localization of axonal paranodal proteins.

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.

Morphogenic role for acetylcholinesterase in axonal outgrowth during neural development.

Acetylcholinesterase (AChE) is the enzyme that hydrolyzes the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions. However, results from our laboratory and others indicate that AChE has an extrasynaptic, noncholinergic role during neural development. This article is a review of our findings demonstrating the morphogenic role of AChE, using a neuronal cell culture model. We also discuss how these data suggest that AChE has a cell adhesive function during neural development. These results could have additional significance as AChE is the target enzyme of agricultural organophosphate and carbamate pesticides as well as the commonly used household organophosphate chlorpyrifos (Dursban). Prenatal exposure to these agents could have adverse effects on neural development by interfering with the morphogenic function of AChE.

Genetic analysis of myelin galactolipid function.

The CGT enzyme is responsible for catalyzing the final step in GalC synthesis. The isolation of the CGT cDNA has allowed for the genetic analysis of galactolipid function by providing the opportunity to generate null mutants deficient in CGT enzymatic activity. The detailed analyses of CGT mutant mice demonstrate that the galactolipids are essential for the formation and maintenance of normal CNS myelin, but neither GalC or sulfatide appear to be required for the development of structurally normal PNS myelin. These studies also show that the differentiation of myelinating cells is not dependent on galactolipid function, in contrast to the conclusions drawn from prior antibody perturbation studies. The abnormal node of Ranvier formations present in the CNS likely explain the disrupted electrophysiological properties displayed by mutant spinal cord axons and the tremoring phenotype of these mice. The abnormal myelin structures present in the mutant animals are consistent with the possibility that the galactolipids play a role in regulating or mediating proper axo-glial interactions. The further detailed analysis of these animals should help refine our understanding of galactolipid function in the myelination process.

Genetic dissection of myelin galactolipid function.

The roles that the myelin galactolipids galactocerebroside (GalC) and sulfatide play in cellular differentiation, myelin formation and maintenance have been investigated for nearly 3 decades. During that time the primary approach has been to perturb lipid activity using antibodies and chemical agents in artificial systems. Recently, the isolation of the gene that encodes UDP-galactose:ceramide galactosyltransferase (CGT), the enzyme that catalyzes an essential step in the synthetic pathway of GalC and sulfatide, has enabled the generation of mice that lack myelin galactolipids. These mice display a severe tremor, hindlimb paralysis and electrophysiological defects. In addition, the CGT null mutants exhibit: 1) impaired oligodendrocyte differentiation, 2) myelin sheaths that are thin, incompletely compacted and unstable, and 3) structural abnormalities in the nodal and paranodal regions including disrupted axo-glial junctions. Collectively, these findings suggest that GalC and sulfatide are essential in myelin formation and maintenance, possibly by mediating intra- and intercellular interactions.

Demyelination and altered expression of myelin-associated glycoprotein isoforms in the central nervous system of galactolipid-deficient mice.

Vertebrate myelin is enriched in the lipid galactocerebroside (GalC) and its sulfated derivated sulfatide. To understand the in vivo function of these lipids, we analyzed myelination in mice that contain a null mutation in the gene encoding UDP-galactose:ceramide galactosyltransferase, the enzyme responsible for catalyzing the final step in GalC synthesis. Galactolipid-deficient myelin is regionally unstable and progressively degenerates. At postnatal day 30, demyelination is restricted to the midbrain and hindbrain, but by postnatal day 90, it spreads throughout the central nervous system. Activated microglial cells and reactive astrocytes appear with the loss of myelin in older animals. Nonetheless, major myelin protein gene mRNA levels are normal throughout the life of these animals, suggesting that widespread oligodendrocyte death is not the primary cause of demyelination. The developmental switch in myelin-associated glycoprotein isoform expression, however, does not occur normally in these mice, suggesting an alteration in oligodendrocyte maturation. Taken together, these findings indicate that GalC and sulfatide are required for the long-term maintenance of myelin and that their absence may have subtle effects on the development of oligodendrocytes.

Galactolipids in the formation and function of the myelin sheath.

Among the most abundant components of myelin are the galactolipids galactocerebroside (GalC) and sulfatide. In spite of this abundance, the roles that these molecules play in the myelin sheath are not well understood. Until recently, our concept of GalC and sulfatide functions had been principally defined by immunological and chemical perturbation studies that implicate these lipids in oligodendrocyte differentiation, myelin formation, and myelin stability. Recently, however, genetic studies have allowed us to re-analyze the functions of these lipids. Two laboratories have independently generated mice that are incapable of synthesizing either GalC or sulfatide by inactivating the gene encoding the enzyme UDP-galactose:ceramide galactosyltransferase (CGT), which is required for myelin galactolipid synthesis. These galactolipid-deficient animals exhibit a severe tremor, hindlimb paralysis, and display electrophysiological deficits in both the central and peripheral nervous systems. In addition, ultrastructural studies have revealed hypomyelinated white matter tracts with unstable myelin sheaths and a variety of myelin abnormalities including altered node length, reversed lateral loops, and compromised axo-oligodendrocytic junctions. Collectively, these observations indicate that cell-cell interactions, which are essential in the formation and maintenance of a properly functioning myelin sheath, are compromised in these galactolipid-deficient mice.

Myelin galactolipids are essential for proper node of Ranvier formation in the CNS.

The vertebrate myelin sheath is greatly enriched in the galactolipids galactocerebroside (GalC) and sulfatide. Mice with a disruption in the gene that encodes the biosynthetic enzyme UDP-galactose:ceramide galactosyl transferase (CGT) are incapable of synthesizing these lipids yet form myelin sheaths that exhibit major and minor dense lines with spacing comparable to controls. These CGT mutant mice exhibit a severe tremor that is accompanied by hindlimb paralysis. Furthermore, electrophysiological studies reveal nerve conduction deficits in the spinal cord of these mutants. Here, using electron microscopic techniques, we demonstrate ultrastructural myelin abnormalities in the CNS that are consistent with the electrophysiological deficits. These abnormalities include altered nodal lengths, an abundance of heminodes, an absence of transverse bands, and the presence of reversed lateral loops. In contrast to the CNS, no ultrastructural abnormalities and only modest electrophysiological deficits were observed in the peripheral nervous system. Taken together, the data presented here indicate that GalC and sulfatide are essential in proper CNS node and paranode formation and that these lipids are important in ensuring proper axo-oligodendrocyte interactions.

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide.

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analyses to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.

Acetylcholinesterase inhibitor treatment delays recovery from axotomy in cultured dorsal root ganglion neurons.

We have previously reported that dorsal root ganglion neurons cultured in the presence of the highly specific, reversible acetylcholinesterase inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51), showed significantly reduced neurite outgrowth and contained massive perikaryal inclusions of neurofilaments. In the present report we have more closely examined these changes in a time course study over a 21-day culture period using a combined morphological, immunocytochemical and enzymatic approach and additionally, describe, the effects of acetylcholinesterase inhibitor treatment on the state of neurofilament phosphorylation. Finally, we have examined the effects of co-administration of N6,2'-0-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) with BW284c51. At 1 day in culture, both control and treated cells displayed eccentrically located nuclei, numerous polysomes and perikaryal accumulations of neurofilaments which were immunoreactive with both phosphorylation- and nonphosphorylation-dependent neurofilament antibodies. These cytological changes, which are common features of the chromatolytic reaction following axotomy in vivo, rapidly resolved in the control neurons, where by 7 days in culture, the neurofilament accumulations had completely disappeared and neurite outgrowth was robust. In contrast, inhibitor-treated neurons retained the post-axotomy features up to 21 days and had significantly reduced neurite outgrowth. In addition, we have investigated a possible role of cyclic adenosine monophosphate (cAMP) in the recovery process since it has been shown to enhance neuritic outgrowth in cultured neurons. Our results demonstrate that the addition of dbcAMP, a membrane permeable analog of cAMP, significantly enhanced neuritic outgrowth and accelerated the recovery of BW284c51-treated dorsal root ganglion cells, as gauged by the disappearance of the axotomy-related cytological changes. Treatment with dbcAMP also increased acetylcholinesterase activity which has been positively correlated with neurite outgrowth both in vivo and in vitro. Together, these observations suggest that acetylcholinesterase has a non-cholinolytic, neurotrophic role in neuronal regeneration and development.

Inverse correlation of acetylcholinesterase (AChE) activity with the presence of neurofilament inclusions in dorsal root ganglion neurons cultured in the presence of a reversible inhibitor of AChE.

We have previously shown that treatment of cultured dorsal root ganglion neurons (DRGN) with a highly specific, reversible acetylcholinesterase (AChE) inhibitor, BW284c51, retards neuritic outgrowth in a dose dependent manner and is accompanied by the presence of abnormal, perikaryal neurofilament (NF) inclusions in approximately 40% of the cells. Since subpopulations of DRGN have been classified according to their levels of AChE activity, we have combined immunocytochemical and enzyme histochemical techniques to investigate a possible correlation between AChE activity and the presence of NF inclusion formation. Our results show that after inhibitor treatment, cells with low levels of AChE activity have a greater percentage of inclusions, with nearly 75% of cells with undetectable levels of AChE activity containing inclusions. In contrast, inclusions were present in only 3.2% of cells with high levels of AChE activity. This inverse relationship between AChE activity and the presence of NF inclusions supports our previous observations that this enzyme may have extra-synaptic functions which could affect neuronal development and regeneration.

Retardation of neuritic outgrowth and cytoskeletal changes accompany acetylcholinesterase inhibitor treatment in cultured rat dorsal root ganglion neurons.

Over the past two decades acetylcholinesterase (AChE) has been shown to be present in numerous non-cholinergic and non-cholinoceptive tissues. Interestingly, transient expression of AChE in developing nervous tissue corresponds temporally with neuronal migration and neuritic outgrowth. This observation has led our laboratory to investigate a possible novel, non-cholinergic role for AChE in the development of the nervous system. In a previous study, we demonstrated that the activity of AChE in cultured dorsal root ganglion neurons (DRGN) can be modulated by the substratum. In our current study, we have examined the effects of AChE inhibitor treatment on neuritic outgrowth on the highly permissive substratum Matrigel and the less permissive substratum Collagen Type I. DRGN received serial dilutions of the AChE-specific inhibitor 1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) ranging from 10(-4) to 10(-7) M. Results showed that neuritic outgrowth was significantly reduced in DRGN grown on Matrigel at 10(-5) and 10(-4) M BW284c51, while outgrowth on Collagen Type I was significantly reduced at 10(-6), 10(-5), and 10(-4) M concentrations of BW284c51. Inhibitor treatment did not affect cell survival and neuritic outgrowth from BW284c51-treated cells recovered to control levels after removal of the inhibitor from the medium. In addition, massive spiraling accumulations of 10 nm filaments were observed in the cell bodies of treated neurons, which resemble neurofibrillary inclusions observed in neuropathological diseases such as Pick's disease. This study demonstrates that AChE inhibitor treatment retards neuritic outgrowth and neuronal migration of cultured DRGN which is accompanied by cytoskeletal abnormalities in the cell body.(ABSTRACT TRUNCATED AT 250 WORDS)