Lipids in regulating oligodendrocyte structure and function.

Oligodendrocytes enwrap central nervous system axons with myelin, a lipid enriched highly organized multi-layer membrane structure that allows for fast long-distance saltatory conduction of neuronal impulses. Myelin has an extremely high lipid content (∼80 % of its dry weight) and a peculiar lipid composition, with a 2:2:1 cholesterol:phospholipid:glycolipid ratio. Inherited neurodegenerative diseases of the lipids (caused by mutations in lipogenic enzymes) often present oligodendrocyte and/or myelin defects which contribute to the overall disease pathophysiology. These phenomena triggered an increasing number of studies over the functions lipid exert to shape and maintain myelin, and brought to the finding that lipids are more than only structural building blocks. They act as signaling molecules to drive proliferation and differentiation of oligodendrocyte progenitor cells, as well as proliferation of premyelinating oligodendrocytes, and their maturation into myelinating ones. Here, we summarize key findings in these areas, while presenting the main related human diseases. Despite many advances in the field, various questions remain open which we briefly discuss. This article is part of a special issue entitled "Role of Lipids in CNS Cell Physiology and Pathology".

CNS myelination and remyelination depend on fatty acid synthesis by oligodendrocytes.

Oligodendrocytes (OLs) support neurons and signal transmission in the central nervous system (CNS) by enwrapping axons with myelin, a lipid-rich membrane structure. We addressed the significance of fatty acid (FA) synthesis in OLs by depleting FA synthase (FASN) from OL progenitor cells (OPCs) in transgenic mice. While we detected no effects in proliferation and differentiation along the postnatal OL lineage, we found that FASN is essential for accurate myelination, including myelin growth. Increasing dietary lipid intake could partially compensate for the FASN deficiency. Furthermore, FASN contributes to correct myelin lipid composition and stability of myelinated axons. Moreover, we depleted FASN specifically in adult OPCs to examine its relevance for remyelination. Applying lysolecithin-induced focal demyelinating spinal cord lesions, we found that FA synthesis is essential to sustain adult OPC-derived OLs and efficient remyelination. We conclude that FA synthesis in OLs plays key roles in CNS myelination and remyelination.

De novo fatty acid synthesis by Schwann cells is essential for peripheral nervous system myelination.

Myelination calls for a remarkable surge in cell metabolism to facilitate lipid and membrane production. Endogenous fatty acid (FA) synthesis represents a potentially critical process in myelinating glia. Using genetically modified mice, we show that Schwann cell (SC) intrinsic activity of the enzyme essential for de novo FA synthesis, fatty acid synthase (FASN), is crucial for precise lipid composition of peripheral nerves and fundamental for the correct onset of myelination and proper myelin growth. Upon FASN depletion in SCs, epineurial adipocytes undergo lipolysis, suggestive of a compensatory role. Mechanistically, we found that a lack of FASN in SCs leads to an impairment of the peroxisome proliferator-activated receptor (PPAR) γ-regulated transcriptional program. In agreement, defects in myelination of FASN-deficient SCs could be ameliorated by treatment with the PPARγ agonist rosiglitazone ex vivo and in vivo. Our results reveal that FASN-driven de novo FA synthesis in SCs is mandatory for myelination and identify lipogenic activation of the PPARγ transcriptional network as a putative downstream functional mediator.

Profilin 1 is required for peripheral nervous system myelination.

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin ß1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.

Infusion of anti-Nogo-A antibodies in adult rats increases growth and synapse related proteins in the absence of behavioral alterations.

Restricted structural re-growth in the adult CNS is a major limitation to fully functional recovery following extensive CNS trauma. This limitation is partly due to the presence of growth inhibitory proteins, in particular, Nogo-A. Pre-clinical studies have demonstrated that intrathecally infused anti-Nogo-A antibodies are readily distributed via the cerebrospinal fluid penetrating throughout the spinal cord and brain, where they promote sprouting, axonal regeneration and improved functional recovery after CNS injury. Whether anti-Nogo-A treatments of intact animals might induce behavioral alterations has not been systematically tested. This is addressed here in an adult rat model of chronic intrathecal infusion of function-blocking anti-Nogo-A antibodies for 2 to 4weeks. We observed by proteomic and immunohistochemical techniques that chronic Nogo-A neutralization in the intact CNS increased expression of cytoskeletal, fiber-growth-related, and synaptic proteins in the hippocampus, a brain region which might be particularly sensitive to Nogo-A depletion due to the high expression level of Nogo-A. Despite such molecular and proteomic changes, Nogo-A blockade was not associated with any pronounced cognitive-behavioral changes indicative of hippocampal functional deficiency across several critical tests. Our results suggest that the plastic changes induced by Nogo-A blockade in the adult hippocampus are counter-balanced by homeostatic mechanisms in the intact and the injured CNS. The data indicate that anti-Nogo-A therapy appears safe in the adult CNS over 4weeks of continuous administration.

Upregulation of axon guidance molecules in the adult central nervous system of Nogo-A knockout mice restricts neuronal growth and regeneration.

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo-A/Nogo receptor (NgR1) signaling pathway. Nogo-A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody-mediated neutralization of Nogo-A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo-A KO vs. wild-type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4-positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth-inhibitory than WT but more than Nogo-A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo-A KO myelin as compared with WT neurons, supporting increased EphA4-mediated growth inhibition in Nogo-A KO mice. Consistently, in vivo, Nogo-A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo-A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo-A.

Defeating inhibition of regeneration by scar and myelin components.

Axon regeneration and the sprouting processes that underlie plasticity are blocked by inhibitory factors in the central nervous system (CNS) environment, several of which are upregulated after injury. The major inhibitory molecules are those associated with myelin and those associated with the glial scar. In myelin, NogoA, MAG, and OMgp are present on normal oligodendrocytes and on myelin debris. They act partly via the Nogo receptor, partly via an unidentified amino-Nogo receptor. In the glial scar, chondroitin sulphate proteoglycans, semaphorins, and the formation of a collagen-based membrane are all inhibitory. Methods to counteract these forms of inhibition have been identified, and these treatments promote axon regeneration in the damaged spinal cord, and in some cases recovery of function through enhanced plasticity.

Identification of new interacting partners for atypical Rho GTPases: a SILAC-based approach.

In contrast to typical Rho GTPases the regulation of atypical Rho GTPases, such as the members of the RhoBTB subfamily, rarely depends on GEFs and/or GAPs. Instead, they are regulated at the level of their expression, by post-translational modifications, by their rate of degradation as well as through binding of diverse cell-specific interactors. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a powerful cutting-edge mass-spectrometry-based technology allowing for protein-interaction studies in vitro with removal of false-positive identifications. In this chapter, we describe how the SILAC technology can be applied to the identification of new interacting partners for atypical - constitutively active - Rho GTPases, i.e. RhoBTB3.

Neuronal Nogo-A regulates neurite fasciculation, branching and extension in the developing nervous system.

Wiring of the nervous system is a multi-step process involving complex interactions of the growing fibre with its tissue environment and with neighbouring fibres. Nogo-A is a membrane protein enriched in the adult central nervous system (CNS) myelin, where it restricts the capacity of axons to grow and regenerate after injury. During development, Nogo-A is also expressed by neurons but its function in this cell type is poorly known. Here, we show that neutralization of neuronal Nogo-A or Nogo-A gene ablation (KO) leads to longer neurites, increased fasciculation, and decreased branching of cultured dorsal root ganglion neurons. The same effects are seen with antibodies against the Nogo receptor complex components NgR and Lingo1, or by blocking the downstream effector Rho kinase (ROCK). In the chicken embryo, in ovo injection of anti-Nogo-A antibodies leads to aberrant innervation of the hindlimb. Genetic ablation of Nogo-A causes increased fasciculation and reduced branching of peripheral nerves in Nogo-A KO mouse embryos. Thus, Nogo-A is a developmental neurite growth regulatory factor with a role as a negative regulator of axon-axon adhesion and growth, and as a facilitator of neurite branching.

Neuronal Nogo-A modulates growth cone motility via Rho-GTP/LIMK1/cofilin in the unlesioned adult nervous system.

Nogo-A has been extensively studied as a myelin-associated neurite outgrowth inhibitor in the lesioned adult central nervous system. However, its role in the intact central nervous system has not yet been clarified. Analysis of the intact adult nervous system of C57BL/6 Nogo-A knock-out (KO) versus wild-type (WT) mice by a combined two-dimensional gel electrophoresis and isotope-coded affinity tagging approach revealed regulation of cytoskeleton-, transport-, and signaling growth-related proteins, pointing to regulation of the actin cytoskeleton, the neuronal growth machinery, and in particular the Rho-GTPase/LIMK1/cofilin pathway. Nogo-A KO adult neurons showed enlarged, more motile growth cones compared with WT neurons. The phenotype was reproduced by acute in vitro neutralization of neuronal Nogo-A. LIMK1 phosphorylation was increased in Nogo-A KO growth cones, and its reduction caused the decrease of KO growth cone motility to WT levels. Our study suggests that in the unlesioned adult nervous system, neuronal Nogo-A can restrict neuronal growth through negative modulation of growth cone motility.

Neutralization of the membrane protein Nogo-A enhances growth and reactive sprouting in established organotypic hippocampal slice cultures.

The reduced ability of central axons to regenerate after injury is significantly influenced by the presence of several molecules that inhibit axonal growth. Nogo-A is one of the most studied and most potent of the myelin-associated growth inhibitory molecules. Its neutralization, as well as interference with its signalling, allows for enhanced axonal sprouting and growth following injury. Using differentiated rat organotypic hippocampal slice cultures treated for 5 days with either of two different function-blocking anti-Nogo-A antibodies, we show an increase in CA3 fibre regeneration after lesion. In intact slices, 5 days of anti-Nogo-A antibody treatment led to increased sprouting of intact CA3 fibres that are positive for neurofilament 68. A transcriptomic approach confirmed the occurrence of a growth response on the molecular level upon Nogo-A neutralization in intact cultures. Our results demonstrate that Nogo-A neutralization for 5 days is sufficient for the induction of growth in mature CNS tissue without the prerequisite of an injury. Nogo-A may therefore act as a tonic growth suppressor/stabilizer in the adult intact hippocampus.

Intrathecally infused antibodies against Nogo-A penetrate the CNS and downregulate the endogenous neurite growth inhibitor Nogo-A.

Neutralizing antibodies against the neurite growth inhibitory protein Nogo-A are known to induce regeneration, enhance compensatory growth, and enhance functional recovery. In intact adult rats and monkeys or spinal cord injured adult rats, antibodies reached the entire spinal cord and brain through the CSF circulation from intraventricular or intrathecal infusion sites. In the tissue, anti-Nogo antibodies were found inside Nogo-A expressing oligodendrocytes and neurons. Intracellularly, anti-Nogo-A antibodies were colocalized with endogenous Nogo-A in large organels, some of which containing the lysosomal marker cathepsin-D. This suggests antibody-induced internalization of cell surface Nogo-A. Total Nogo-A tissue levels in spinal cord were decreased in intact adult rats following 7 days of antibody infusion. This mechanism was confirmed in vitro; cultured oligodendrocytes and neurons had lower Nogo-A contents in the presence of anti-Nogo-A antibodies. These results demonstrate that antibodies against a CNS cell surface protein reach their antigen through the CSF and can induce its downregulation.

Nogo-A-deficient mice reveal strain-dependent differences in axonal regeneration.

Nogo-A, a membrane protein enriched in myelin of the adult CNS, inhibits neurite growth and regeneration; neutralizing antibodies or receptor blockers enhance regeneration and plasticity in the injured adult CNS and lead to improved functional outcome. Here we show that Nogo-A-specific knock-outs in backcrossed 129X1/SvJ and C57BL/6 mice display enhanced regeneration of the corticospinal tract after injury. Surprisingly, 129X1/SvJ Nogo-A knock-out mice had two to four times more regenerating fibers than C57BL/6 Nogo-A knock-out mice. Wild-type newborn 129X1/SvJ dorsal root ganglia in vitro grew a much higher number of processes in 3 d than C57BL/6 ganglia, confirming the stronger endogenous neurite growth potential of the 129X1/SvJ strain. cDNA microarrays of the intact and lesioned spinal cord of wild-type as well as Nogo-A knock-out animals showed a number of genes to be differentially expressed in the two mouse strains; many of them belong to functional categories associated with neurite growth, synapse formation, and inflammation/immune responses. These results show that neurite regeneration in vivo, under the permissive condition of Nogo-A deletion, and neurite outgrowth in vitro differ significantly in two widely used mouse strains and that Nogo-A is an important endogenous inhibitor of axonal regeneration in the adult spinal cord.