Robert G. Farrer

Recovery and brain reorganization after stroke in adult and aged rats

Stroke is a prevalent and devastating disorder, and no treatment is currently available to restore lost neuronal function after stroke. One unique therapy that improves recovery after stroke is neutralization of the neurite inhibitory protein Nogo‐A. Here, we show, in a clinically relevant model, improved functional recovery and brain reorganization in the aged and adult rat when delayed anti–Nogo‐A therapy is given after ischemic injury. These results support the efficacy of Nogo‐A neutralization as treatment for ischemic stroke, even in the aged animal and after a 1‐week delay, and implicate neuronal plasticity from unlesioned areas of the central nervous system as a mechanism for recovery. Ann Neurol 2005;58:950–953

Biochemical and Morphometric Analyses Show that Myelination in the Insulin-like Growth Factor 1 Null Brain Is Proportionate to Its Neuronal Composition

To elucidate the role of insulin-like growth factor 1 (IGF1) in the normal development of brain myelination, we used behavioral, biochemical, and histological analyses to compare the myelination of brains from Igf1−/− and wild-type (WT) littermate mice. The studies were conducted at postnatal day 40, at which time the Igf1−/− mice weighed ∼66% less than wild-type mice. However, theIgf1−/− brain weight was only reduced by ∼34%. Formal neurological testing showed no sign of central or peripheral myelinopathy inIgf1−/− mice. Myelin composition was not significantly different, and myelin concentration, normalized to brain weight or protein, was equal inIgf1−/− and WT mice. Likewise, concentrations of myelin-specific proteins (MBP, myelin proteolipid protein, MAG, and 2′,3′-cyclic nucleotide,3′-phosphodiesterase) were not significantly different inIgf1−/− and WT mice. The myelin-associated lipids galactocerebroside and sulfatide were modestly reduced in Igf1−/− brains. Regional oligodendrocyte populations and myelin staining patterns were comparable in Igf1−/− and WT brains, with the notable exception of the olfactory system. TheIgf1−/− olfactory bulb was profoundly reduced in size and was depleted of mitral neurons and oligodendrocytes, and its efferent tracts were depleted of myelin. In summary, this study shows that myelination of theIgf1−/− brain is proportionate to its neuronal composition. Where projection neurons are preserved despite the deletion of IGF1, as in the cerebellar system, oligodendrocytes and myelination are indistinguishable from wild type. Where projection neurons are depleted, as in the olfactory bulb, oligodendrocytes are also depleted, and myelination is reduced in proportion to the reduced projection neuron mass. These data make a strong case for the primacy of axonal factors, not including IGF1, in determining oligodendrocyte survival and myelination.

GT3 and its O-acetylated derivative are the principal A2B5-reactive gangliosides in cultured O2A lineage cells and are down-regulated along with O-acetyl GD3 during differentiation to oligodendrocytes.

Among the developmentally regulated antigens expressed on the surface of bipotential glial precursors isolated from neonatal rat brain are the gangliosides recognized by the monoclonal antibody A2B5. Immunostaining of thin layer chromatograms showed that oligodendrocyte‐type 2 astrocyte (O2A) progenitors in culture express two ganglioside antigens that react strongly with the A2B5 antibody. Both ganglioside antigens are down‐regulated as the cells differentiate to oligodendrocytes, corresponding to the disappearance of cell surface immunostaining by A2B5 in mature oligodendrocytes. By contrast, both gangliosides continue to be expressed when the cells differentiate to type‐2 astrocytes. These two A2B5‐reactive gangliosides in O2A lineage cells were identified as GT3 and O‐acetyl GT3 by using the monoclonal antibody 18B8 that recognizes GT3 and an influenza C virus esterase that specifically removes O‐acetyl moieties from sialic acids. Thin‐layer chromatographic immunostaining with the JONES monoclonal antibody demonstrated that the progenitor cells in culture also express O‐acetyl GD3, which is similarly down‐regulated in oligodendrocytes, but retained in type‐2 astrocytes. The 18B8 and JONES antibodies also immunostained the surface of O2A progenitors. Therefore, expression of GT3 and O‐acetylation of GT3 and GD3 occur during the proliferative and migratory stages of glial cell development. J. Neurosci. Res. 57:371–380, 1999. Published 1999 Wiley‐Liss, Inc.

Expression of Glycolipids and Myelin Associated Glycoprotein during the Differentiation of Oligodendrocytes: Comparison of the CG-4 Glial Cell Line to Primary Cultures

The expression of cerebrosides, sulfatides, gangliosides and the myelin-associated glycoprotein (MAG) during differentiation of the CG-4 line of oligodendrocyte progenitors [Louis et al.: J. Neurosci Res 31: 193, 1992] was compared with their expression in primary cultures of oligodendrocyte precursors [McCarthy and de Vellis: J Cell Biol 85: 890, 1980]. When the CG-4 cells differentiated from bipolar progenitors to oligodendrocytes, there was a decrease of glucosylcerebroside synthesis and an increase in galactosylcerebroside and sulfatide synthesis. However, even after differentiation, the incorporation of [3H]galactose into these glycolipids, the amounts of galactosylcerebroside and sulfatide, and the galactocerebroside/sulfatide ratio were all much less than in primary cultures of differentiating oligodendrocytes. The major gangliosides in differentiated primary oligodendrocyte cultures were GM3 and GD3, and GD3 was also a major ganglioside in the CG-4 line. However, unlike primary cultures of O-2A lineage cells in which GM3 synthesis increased dramatically during differentiation to oligodendrocytes, the CG-4 cells expressed very little GM3. Also, the CG-4 cells expressed larger amounts of more complex gangliosides, e.g. GD1b and GT1b, which were almost entirely restricted to the b-series. The amount of MAG expressed by the CG-4 cells increased substantially when they differentiated to oligodendrocytes, and it was almost all the large immature isoform. However, even after differentiation, the amount expressed was less than in differentiated primary oligodendrocyte cultures. Overall, the lower expression of myelin-related glycolipids and MAG by the CG-4 line suggests a lesser degree of differentiation in comparison to primary oligodendrocytes under the culture conditions of these experiments, but the larger amounts of cells available from the CG-4 line should be useful for investigating glycolipid and MAG function related to the early stages of myelinogenesis.

Dendritic Spine Alterations in Neocortical Pyramidal Neurons following Postnatal Neuronal Nogo-A Knockdown

The myelin-associated protein Nogo-A is a well-known inhibitor of axonal regeneration and compensatory plasticity, yet functions of neuronal Nogo-A are not as clear. The present study examined the effects of decreased levels of neuronal Nogo-A on dendritic spines of developing neocortical neurons. Decreased Nogo-A levels in these neurons resulted in lowered spine density and an increase in filopodial type protrusions. These results suggest a role for neuronal Nogo-A in maintaining a spine phenotype in neocortical pyramidal cells.

Evidence for Fibroblast Growth Factor-2 as a Mediator of Amphetamine-Enhanced Motor Improvement following Stroke

Previously we have shown that addition of amphetamine to physical therapy results in enhanced motor improvement following stroke in rats, which was associated with the formation of new motor pathways from cortical projection neurons of the contralesional cortex. It is unclear what mechanisms are involved, but amphetamine is known to induce the neuronal release of catecholamines as well as upregulate fibroblast growth factor-2 (FGF-2) expression in the brain. Since FGF-2 has been widely documented to stimulate neurite outgrowth, the present studies were undertaken to provide evidence for FGF-2 as a neurobiological mechanism underlying amphetamine-induced neuroplasticity. In the present study rats that received amphetamine plus physical therapy following permanent middle cerebral artery occlusion exhibited significantly greater motor improvement over animals receiving physical therapy alone. Amphetamine plus physical therapy also significantly increased the number of FGF-2 expressing pyramidal neurons of the contralesional cortex at 2 weeks post-stroke and resulted in significant axonal outgrowth from these neurons at 8 weeks post-stroke. Since amphetamine is a known releaser of norepinephrine, in vitro analyses focused on whether noradrenergic stimulation could lead to neurite outgrowth in a manner requiring FGF-2 activity. Primary cortical neurons did not respond to direct stimulation by norepinephrine or amphetamine with increased neurite outgrowth. However, conditioned media from astrocytes exposed to norepinephrine or isoproterenol (a beta adrenergic agonist) significantly increased neurite outgrowth when applied to neuronal cultures. Adrenergic agonists also upregulated FGF-2 expression in astrocytes. Pharmacological analysis indicated that beta receptors and alpha1, but not alpha2, receptors were involved in both effects. Antibody neutralization studies demonstrated that FGF-2 was a critical contributor to neurite outgrowth induced by astrocyte-conditioned media. Taken together the present results suggest that noradrenergic activation, when combined with physical therapy, can improve motor recovery following ischemic damage by stimulating the formation of new neural pathways in an FGF-2-dependent manner.

Effects of brefeldin A on galactosphingolipid synthesis in an immortalized Schwann cell line: evidence for different intracellular locations of galactosylceramide sulfotransferase and ceramide galactosyltransferase activities.

Abstract: Brefeldin A (BFA) has been used extensively to study the intracellular transport and processing of proteins and sphingolipids because of its dramatic alteration of the structural and functional organization of the Golgi. We have examined the effect of BFA on the synthesis of galactosylceramide sulfate (SGalCer) and its immediate precursor galactosylceramide (GalCer) in an immortalized Schwann cell line (S16) to determine the intracellular sites of synthesis of these two related glycolipids. During a 6‐h labeling period, a dose‐dependent inhibition of [35S]sulfate incorporation into SGalCer was observed with 95% inhibition occurring at 0.5 µg/ml BFA. Labeling of newly synthesized galactosphingolipids with [3H]‐palmitic acid for 6 h in the presence of BFA resulted in increased incorporation of label into GalCer containing nonhydroxy fatty acids (NFA‐GalCer) to 162% of control values, whereas labeling of GalCer containing 2‐hydroxy fatty acids (HFA‐GalCer) was reduced to 63% of control. After 24 h, these values were at 366 and 91%, respectively. These results indicate that at least some of the HFA‐GalCer was initially synthesized at a location distal to the BFA block and separate from the site of NFA‐GalCer synthesis. Examination of [3H]palmitic acid incorporation into free ceramides showed an increase of 133 and 161% for hydroxy and nonhydroxy fatty acid ceramides, respectively, in cells treated for 6 h with BFA in comparison with levels found in untreated control cells, indicating that BFA did not block fatty acid 2‐hydroxylation or the formation of HFA ceramide. Incorporation of [3H]palmitic acid into glucosylceramide and GM3 was increased over control levels whereas labeling of GM2 was inhibited, consistent with what has been reported previously for the effect of BFA on these glycolipids in other cell types. These results suggest that there are at least two separate intracellular sites for the galactosylation of HFA and NFA ceramide, respectively, which can be distinguished by their sensitivity to BFA. Our results also indicate that the site of GalCer sulfation is not redistributed to the endoplasmic reticulum in the presence of BFA and therefore may be localized to the distal Golgi or trans‐Golgi network.

Expression of sulfated gangliosides in the central nervous system.

Abstract: Several sulfated lipids were detected in the ganglioside fraction isolated from a cell line of oligodendrocyte progenitors that had been metabolically labeled with [35S]sulfate. Separation of the ganglioside fraction by two‐dimensional TLC showed that, except for galactosylceramide‐sulfate, none of the sulfate‐labeled lipids comigrated with those glycosphingolipids visualized by orcinol staining, indicating that these sulfolipids were quantitatively minor components. At least eight sulfate‐labeled lipid bands were susceptible to desialylation by Arthrobacter ureafaciens neuraminidase, which resulted in the formation of three new bands that retained the labeled sulfate. Six of the sulfate‐labeled lipid bands containing sialic acid were also susceptible to Vibrio cholerae neuraminidase, which generated two labeled bands that appeared identical to the two major products formed after treatment with A. ureafaciens neuraminidase. In vivo labeling of lipids from 14‐day‐old rat brain with [35S]sulfate demonstrated that the synthesis of sulfated lipids containing sialic acid also occurred in intact brain tissue. These results show that sulfated gangliosides are synthesized in the CNS and that oligodendrocytes are one cell type that contributes to this synthesis.

Platelet-derived growth factor-BB activates calcium/calmodulin-dependent and -independent mechanisms that mediate Akt phosphorylation in the neurofibromin-deficient human Schwann cell line ST88-14

Background: Malignant peripheral nerve sheath tumor (MPNST) cells overexpress PDGF receptor-β, which increases intracellular calcium when activated. Results: Calcium/calmodulin (CaM) is involved in sustained phosphorylation of Akt and promotion of cell survival in an MPNST cell line. Conclusion: MPNST cells evade normal cell death through CaM-dependent sustained activation of Akt. Significance: Activation of CaM by abnormally expressed growth factor receptors may contribute to NF1 tumor formation. Neurofibromatosis type 1-derived Schwann cells isolated from malignant peripheral nerve sheath tumors (MPNSTs) overexpress PDGF receptor-β and generate an aberrant intracellular calcium increase in response to PDGF-BB. Using the human MPNST Schwann cell line ST88-14, we demonstrate that, in addition to a transient phosphorylation of Akt, PDGF-BB stimulation produces an atypical sustained phosphorylation of Akt that is dependent on calcium and calmodulin (CaM). The sustained Akt phosphorylation did not occur in PDGF-BB-stimulated normal human Schwann cells or ST88-14 cells stimulated with stem cell factor, whose receptor is also overexpressed in ST88-14 cells. The sustained Akt phosphorylation induced by PDGF-BB was inhibited by pretreatment of the cells with either the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) or the CaM antagonist W7, whereas the transient portion was not inhibited. Akt also co-immunoprecipitated with CaM in a PDGF-BB-dependent manner, suggesting that direct interaction between Akt and CaM is involved in the sustained phosphorylation of Akt. Furthermore, we provide evidence that anti-apoptotic effects of PDGF-BB on serum-deprived ST88-14 cells can be inhibited by W7, implicating the PDGF-BB-induced activation of calcium/CaM in promoting cell survival, presumably through sustained Akt activation. We conclude that the activation of the calcium/CaM/Akt pathway resulting from stimulation of overexpressed PDGF receptor-β may contribute to the survival and tumorigenicity of MPNST cells.

Anti-Nogo-A Immunotherapy Does Not Alter Hippocampal Neurogenesis after Stroke in Adult Rats

Ischemic stroke is a leading cause of adult disability, including cognitive impairment. Our laboratory has previously shown that treatment with function-blocking antibodies against the neurite growth inhibitory protein Nogo-A promotes functional recovery after stroke in adult and aged rats, including enhancing spatial memory performance, for which the hippocampus is critically important. Since spatial memory has been linked to hippocampal neurogenesis, we investigated whether anti-Nogo-A treatment increases hippocampal neurogenesis after stroke. Adult rats were subject to permanent middle cerebral artery occlusion followed 1 week later by 2 weeks of antibody treatment. Cellular proliferation in the dentate gyrus was quantified at the end of treatment, and the number of newborn neurons was determined at 8 weeks post-stroke. Treatment with both anti-Nogo-A and control antibodies stimulated the accumulation of new microglia/macrophages in the dentate granule cell layer, but neither treatment increased cellular proliferation or the number of newborn neurons above stroke-only levels. These results suggest that anti-Nogo-A immunotherapy does not increase post-stroke hippocampal neurogenesis.