Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.

Transcallosal Projections Require Glycoprotein M6-Dependent Neurite Growth and Guidance.

The function of mature neurons critically relies on the developmental outgrowth and projection of their cellular processes. It has long been postulated that the neuronal glycoproteins M6a and M6b are involved in axon growth because these four-transmembrane domain-proteins of the proteolipid protein family are highly enriched on growth cones, but in vivo evidence has been lacking. Here, we report that the function of M6 proteins is required for normal axonal extension and guidance in vivo. In mice lacking both M6a and M6b, a severe hypoplasia of axon tracts was manifested. Most strikingly, the corpus callosum was reduced in thickness despite normal densities of cortical projection neurons. In single neuron tracing, many axons appeared shorter and disorganized in the double-mutant cortex, and some of them were even misdirected laterally toward the subcortex. Probst bundles were not observed. Upon culturing, double-mutant cortical and cerebellar neurons displayed impaired neurite outgrowth, indicating a cell-intrinsic function of M6 proteins. A rescue experiment showed that the intracellular loop of M6a is essential for the support of neurite extension. We propose that M6 proteins are required for proper extension and guidance of callosal axons that follow one of the most complex trajectories in the mammalian nervous system.

Critical time window of neuronal cholesterol synthesis during neurite outgrowth.

Cholesterol is an essential membrane component enriched in plasma membranes, growth cones, and synapses. The brain normally synthesizes all cholesterol locally, but the contribution of individual cell types to brain cholesterol metabolism is unknown. To investigate whether cortical projection neurons in vivo essentially require cholesterol biosynthesis and which cell types support neurons, we have conditionally ablated the cholesterol biosynthesis in these neurons in mice either embryonically or postnatally. We found that cortical projection neurons synthesize cholesterol during their entire lifetime. At all stages, they can also benefit from glial support. Adult neurons that lack cholesterol biosynthesis are mainly supported by astrocytes such that their functional integrity is preserved. In contrast, microglial cells support young neurons. However, compensatory efforts of microglia are only transient leading to layer-specific neuronal death and the reduction of cortical projections. Hence, during the phase of maximal membrane growth and maximal cholesterol demand, neuronal cholesterol biosynthesis is indispensable. Analysis of primary neurons revealed that neurons tolerate only slight alteration in the cholesterol content and plasma membrane tension. This quality control allows neurons to differentiate normally and adjusts the extent of neurite outgrowth, the number of functional growth cones and synapses to the available cholesterol. This study highlights both the flexibility and the limits of horizontal cholesterol transfer in vivo and may have implications for the understanding of neurodegenerative diseases.

Survival of adult neurons lacking cholesterol synthesis in vivo.

BACKGROUND: Cholesterol, an essential component of all mammalian plasma membranes, is highly enriched in the brain. Both during development and in the adult, brain cholesterol is derived from local cholesterol synthesis and not taken up from the circulation. However, the contribution of neurons and glial cells to total brain cholesterol metabolism is unknown. RESULTS: Using conditional gene inactivation in the mouse, we disrupted the squalene synthase gene (fdft1), which is critical for cholesterol synthesis, in cerebellar granule cells and some precerebellar nuclei. Mutant mice showed no histological signs of neuronal degeneration, displayed ultrastructurally normal synapses, and exhibited normal motor coordination. This revealed that these adult neurons do not require cell-autonomous cholesterol synthesis for survival or function. CONCLUSION: We conclude that at least some adult neurons no longer require endogenous cholesterol synthesis and can fully meet their cholesterol needs by uptake from their surrounding. Glia are a likely source of cholesterol in the central nervous system.

TrkC kinase expression in distinct subsets of cutaneous trigeminal innervation and nonneuronal cells.

Neurotrophin-activated receptor tyrosine kinases (Trks) regulate sensory neuron survival, differentiation, and function. To permanently mark cells that ever express TrkC-kinase, mice with lacZ and GFP reporters of Cre recombinase activity were crossed with mice having IRES-cre inserted into the kinase-containing exon of the TrkC gene. Prenatal reporter expression matched published locations of TrkC-expression. Postnatally, more trigeminal neurons and types of mystacial pad innervation expressed reporter than immunodetectable TrkC, indicating that some innervation transiently expresses TrkC-kinase. Reporter-tagged neurons include all those that immunolabel for TrkC, a majority for TrkB, and a small proportion for TrkA. TrkA neurons expressing TrkC-reporter range from small to large size and supply well-defined types of mystacial pad innervation. Virtually all small neurons and C-fiber innervation requires TrkA to develop, but TrkC-reporter is present in only a small proportion that uniquely innervates piloneural complexes of guard hairs and inner conical bodies of vibrissa follicle-sinus complexes. TrkC-reporter is expressed in nearly all presumptive Adelta innervation, which is all eliminated in TrkA knockouts and partially eliminated in TrkC knockouts. Many types of Abeta-fiber innervation express TrkC-reporter including all Merkel, spiny, and circumferentially oriented lanceolate endings, and some reticular and longitudinally oriented lanceolate endings. Only Merkel endings require TrkC to develop and survive, whereas the other endings require TrkA and/or TrkB. Thus, TrkC is required for the existence of some types of innervation that express TrkC, but may have different functions in others. Many types of nonneuronal cells affiliated with hair follicles and blood vessels also express TrkC-reporter but lack immunodetectable TrkC.

Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast.

Ribosome-associated complex (RAC) consists of the Hsp40 homolog Zuo1 and the Hsp70 homolog Ssz1. The chaperone participates in the biogenesis of newly synthesized polypeptides. Here we have identified yeast Rpl31, a component of the large ribosomal subunit, as a contact point of RAC at the polypeptide tunnel exit. Rpl31 is encoded by RPL31a and RPL31b, two closely related genes. Delta rpl31a Delta rpl31b displayed slow growth and sensitivity to low as well as high temperatures. In addition, Delta rpl31a Delta rpl31b was highly sensitive toward aminoglycoside antibiotics and suffered from defects in translational fidelity. With the exception of sensitivity at elevated temperature, the phenotype resembled yeast strains lacking one of the RAC subunits or Rpl39, another protein localized at the tunnel exit. Defects of Delta rpl31a Delta rpl31b Delta zuo1 did not exceed that of Delta rpl31a Delta rpl31b or Delta zuo1. However, the combined deletion of RPL31a, RPL31b, and RPL39 was lethal. Moreover, RPL39 was a multicopy suppressor, whereas overexpression of RAC failed to rescue growth defects of Delta rpl31a Delta rpl31b. The findings are consistent with a model in that Rpl31 and Rpl39 independently affect a common ribosome function, whereas Rpl31 and RAC are functionally interdependent. Rpl31, while not essential for binding of RAC to the ribosome, might be involved in proper function of the chaperone complex.

Cre/loxP-mediated inactivation of the bHLH transcription factor gene NeuroD/BETA2.

NeuroD/Beta2 is a basic helix-loop-helix (bHLH) transcription factor with important functions during development of the pancreas and the nervous system. NeuroD null mutant mice die perinatally due to diabetes caused by impaired differentiation of pancreatic endocrine cells. Additionally, null mutants display severe defects in the formation of cerebellar and hippocampal granule cells, inner ear sensory neurons, and retinal photoreceptor cells. For spatio-temporally restricted inactivation of the NeuroD gene, we generated conditional mouse mutants by flanking the NeuroD coding region with loxP sites. Homozygous NeuroD(loxP) mutant mice are fully viable and express normal levels of NeuroD mRNA and protein. Breeding NeuroD(loxP) mice to Tg(malpha6-Cre)B1LFR mice that express Cre recombinase under control of the GABA(A) receptor alpha6 subunit promoter resulted in efficient inactivation of the NeuroD gene in post-migratory cerebellar granule cells and a subset of brainstem nuclei. The NeuroD(loxP) mouse mutant will be a valuable tool to study the developmental and adult function of NeuroD in nervous system and pancreas.

Cre-mediated recombination in rhombic lip derivatives.

To study the development of the cerebellum, we generated a transgenic mouse line Tg(malpha6-cre)B1LFR that expresses CRE recombinase under the GABA(A) receptor alpha6 subunit promoter. In this line, recombination of an R26R reporter allele occurred postnatally in granule cells of the cerebellum and dorsal cochlear nucleus, as well as in a subset of precerebellar nuclei in the brainstem. All neurons in which recombination occurred originated during embryogenesis from the rhombic lip. This might be explained by a very early specification event at the rhombic lip that primes cells derived from this structure to express the transgene during neuronal maturation. As no recombination occurred in the inferior olive, it may be derived from a distinct subset of precursors at the rhombic lip. No recombination occurred in any of the interneurons in the cerebellum (stellate cells, basket cells, and Golgi cells), consistent with the hypothesis that they are not derived from the same embryonic tissue as the granule cells.