MCT1 Deletion in Oligodendrocyte Lineage Cells Causes Late-Onset Hypomyelination and Axonal Degeneration.

Oligodendrocytes (OLs) are important for myelination and shuttling energy metabolites lactate and pyruvate toward axons through their expression of monocarboxylate transporter 1 (MCT1). Recent studies suggest that loss of OL MCT1 causes axonal degeneration. However, it is unknown how widespread and chronic loss of MCT1 in OLs specifically affects neuronal energy homeostasis with aging. To answer this, MCT1 conditional null mice were generated that allow for OL-specific MCT1 ablation. We observe that MCT1 loss from OL lineage cells is dispensable for normal myelination and axonal energy homeostasis early in life. By contrast, loss of OL lineage MCT1 expression with aging leads to significant axonal degeneration with concomitant hypomyelination. These data support the hypothesis that MCT1 is important for neuronal energy homeostasis in the aging central nervous system (CNS). The reduction in OL MCT1 that occurs with aging may enhance the risk for axonal degeneration and atrophy in neurodegenerative diseases.

Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase.

Glutamate has been implicated in a wide range of brain pathologies and is thought to be metabolized via the astrocyte-specific enzyme glutamine synthetase (GS). We show here that oligodendrocytes, the myelinating glia of the central nervous system, also express high levels of GS in caudal regions like the midbrain and the spinal cord. Selective removal of oligodendrocyte GS in mice led to reduced brain glutamate and glutamine levels and impaired glutamatergic synaptic transmission without disrupting myelination. Furthermore, animals lacking oligodendrocyte GS displayed deficits in cocaine-induced locomotor sensitization, a behavior that is dependent on glutamatergic signaling in the midbrain. Thus, oligodendrocytes support glutamatergic transmission through the actions of GS and may represent a therapeutic target for pathological conditions related to brain glutamate dysregulation.

Protective role of the lipid phosphatase Fig4 in the adult nervous system.

The signaling lipid phosphatidylinositol 3,5-bisphosphate, PI(3,5)P2, functions in vesicular trafficking through the endo-lysosomal compartment. Cellular levels of PI(3,5)P2 are regulated by an enzyme complex comprised of the kinase PIKFYVE, the phosphatase FIG4, and the scaffold protein VAC14. Mutations of human FIG4 cause inherited disorders including Charcot-Marie-Tooth disease type 4J, polymicrogyria with epilepsy, and Yunis-Varón syndrome. Constitutive Fig4-/- mice exhibit intention tremor, spongiform degeneration of neural tissue, hypomyelination, and juvenile lethality. To determine whether PI(3,5)P2 is required in the adult, we generated Fig4flox/-; CAG-creER mice and carried out tamoxifen-induced gene ablation. Global ablation in adulthood leads to wasting, tremor, and motor impairment. Death follows within 2 months of tamoxifen treatment, demonstrating a life-long requirement for Fig4. Histological examinations of the sciatic nerve revealed profound Wallerian degeneration of myelinated fibers, but not C-fiber axons in Remak bundles. In optic nerve sections, myelinated fibers appear morphologically intact and carry compound action potentials at normal velocity and amplitude. However, when iKO mice are challenged with a chemical white matter lesion, repair of damaged CNS myelin is significantly delayed, demonstrating a novel role for Fig4 in remyelination. Thus, in the adult PNS Fig4 is required to protect myelinated axons from Wallerian degeneration. In the adult CNS, Fig4 is dispensable for fiber stability and nerve conduction, but is required for the timely repair of damaged white matter. The greater vulnerability of the PNS to Fig4 deficiency in the mouse is consistent with clinical observations in patients with Charcot-Marie-Tooth disease.

Oligodendrocytes control potassium accumulation in white matter and seizure susceptibility.

The inwardly rectifying K+ channel Kir4.1 is broadly expressed by CNS glia and deficits in Kir4.1 lead to seizures and myelin vacuolization. However, the role of oligodendrocyte Kir4.1 channels in controlling myelination and K+ clearance in white matter has not been defined. Here, we show that selective deletion of Kir4.1 from oligodendrocyte progenitors (OPCs) or mature oligodendrocytes did not impair their development or disrupt the structure of myelin. However, mice lacking oligodendrocyte Kir4.1 channels exhibited profound functional impairments, including slower clearance of extracellular K+ and delayed recovery of axons from repetitive stimulation in white matter, as well as spontaneous seizures, a lower seizure threshold, and activity-dependent motor deficits. These results indicate that Kir4.1 channels in oligodendrocytes play an important role in extracellular K+ homeostasis in white matter, and that selective loss of this channel from oligodendrocytes is sufficient to impair K+ clearance and promote seizures.

LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair.

Low-density lipoprotein receptor-related protein-1 (LRP1) is a large endocytic and signaling molecule broadly expressed by neurons and glia. In adult mice, global inducible (Lrp1flox/flox;CAG-CreER) or oligodendrocyte (OL)-lineage specific ablation (Lrp1flox/flox;Pdgfra-CreER) of Lrp1 attenuates repair of damaged white matter. In oligodendrocyte progenitor cells (OPCs), Lrp1 is required for cholesterol homeostasis and differentiation into mature OLs. Lrp1-deficient OPC/OLs show a strong increase in the sterol-regulatory element-binding protein-2 yet are unable to maintain normal cholesterol levels, suggesting more global metabolic deficits. Mechanistic studies revealed a decrease in peroxisomal biogenesis factor-2 and fewer peroxisomes in OL processes. Treatment of Lrp1-/- OPCs with cholesterol or activation of peroxisome proliferator-activated receptor-γ with pioglitazone alone is not sufficient to promote differentiation; however, when combined, cholesterol and pioglitazone enhance OPC differentiation into mature OLs. Collectively, our studies reveal a novel role for Lrp1 in peroxisome biogenesis, lipid homeostasis, and OPC differentiation during white matter development and repair.

PI(3,5)P2 biosynthesis regulates oligodendrocyte differentiation by intrinsic and extrinsic mechanisms.

Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1(+) and Rab7(+) vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1(+)perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

Rescue of neurodegeneration in the Fig4 null mouse by a catalytically inactive FIG4 transgene.

The lipid phosphatase FIG4 is a subunit of the protein complex that regulates biosynthesis of the signaling lipid PI(3,5)P2. Mutations of FIG4 result in juvenile lethality and spongiform neurodegeneration in the mouse, and are responsible for the human disorders Charcot-Marie-Tooth disease, Yunis-Varon syndrome and polymicrogyria with seizures. We previously demonstrated that conditional expression of a wild-type FIG4 transgene in neurons is sufficient to rescue most of the abnormalities of Fig4 null mice, including juvenile lethality and extensive neurodegeneration. To evaluate the contribution of the phosphatase activity to the in vivo function of Fig4, we introduced the mutation p.Cys486Ser into the Sac phosphatase active-site motif CX5RT. Transfection of the Fig4(Cys486Ser) cDNA into cultured Fig4(-/-) fibroblasts was effective in preventing vacuolization. The neuronal expression of an NSE-Fig4(Cys486Ser) transgene in vivo prevented the neonatal neurodegeneration and juvenile lethality seen in Fig4 null mice. These observations demonstrate that the catalytically inactive FIG4 protein provides significant function, possibly by stabilization of the PI(3,5)P2 biosynthetic complex and/or localization of the complex to endolysosomal vesicles. Despite this partial rescue, later in life the NSE-Fig4(Cys486Ser) transgenic mice display significant abnormalities that include hydrocephalus, defective myelination and reduced lifespan. The late onset phenotype of the NSE-Fig4(Cys486Ser) transgenic mice demonstrates that the phosphatase activity of FIG4 has an essential role in vivo.

Th Cell Diversity in Experimental Autoimmune Encephalomyelitis and Multiple Sclerosis.

Multiple sclerosis (MS) is believed to be initiated by myelin-reactive CD4(+) Th cells. IL-12-polarized Th1 cells, IL-23-polarized Th17 cells, and Th17 cells that acquire Th1 characteristics were each implicated in autoimmune pathogenesis. It is debated whether Th cells that can drive the development of demyelinating lesions are phenotypically diverse or arise from a single lineage. In the current study, we assessed the requirement of IL-12 or IL-23 stimulation, as well as Th plasticity, for the differentiation of T cells capable of inducing CNS axon damage. We found that stable murine Th1 and Th17 cells independently transfer experimental autoimmune encephalomyelitis (widely used as an animal model of MS) in the absence of IL-23 and IL-12, respectively. Plastic Th17 cells are particularly potent mediators of demyelination and axonopathy. In parallel studies, we identified MS patients who consistently mount either IFN-γ- or IL-17-skewed responses to myelin basic protein over the course of a year. Brain magnetic resonance imaging revealed that patients with mixed IFN-γ and IL-17 responses have relatively high T1 lesion burden, a measure of permanent axon damage. Our data challenge the dogma that IL-23 and Th17 plasticity are universally required for the development of experimental autoimmune encephalomyelitis. This study definitively demonstrates that autoimmune demyelinating disease can be driven by distinct Th-polarizing factors and effector subsets, underscoring the importance of a customized approach to the pharmaceutical management of MS.

Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy.

Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P2 levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement. However, it is unclear whether FIG4 has cell autonomous roles in both motor neurons and Schwann cells, and how loss of FIG4/PtdIns(3,5)P2-mediated functions contribute to the pathogenesis of CMT4J. Here, we report that mice with conditional inactivation of Fig4 in motor neurons display neuronal and axonal degeneration. In contrast, conditional inactivation of Fig4 in Schwann cells causes demyelination and defects in autophagy-mediated degradation. Moreover, Fig4-regulated endolysosomal trafficking in Schwann cells is essential for myelin biogenesis during development and for proper regeneration/remyelination after injury. Our data suggest that impaired endolysosomal trafficking in both motor neurons and Schwann cells contributes to CMT4J neuropathy.

Semaphorin 5A inhibits synaptogenesis in early postnatal- and adult-born hippocampal dentate granule cells.

Human SEMAPHORIN 5A (SEMA5A) is an autism susceptibility gene; however, its function in brain development is unknown. In this study, we show that mouse Sema5A negatively regulates synaptogenesis in early, developmentally born, hippocampal dentate granule cells (GCs). Sema5A is strongly expressed by GCs and regulates dendritic spine density in a cell-autonomous manner. In the adult mouse brain, newly born Sema5A-/- GCs show an increase in dendritic spine density and increased AMPA-type synaptic responses. Sema5A signals through PlexinA2 co-expressed by GCs, and the PlexinA2-RasGAP activity is necessary to suppress spinogenesis. Like Sema5A-/- mutants, PlexinA2-/- mice show an increase in GC glutamatergic synapses, and we show that Sema5A and PlexinA2 genetically interact with respect to GC spine phenotypes. Sema5A-/- mice display deficits in social interaction, a hallmark of autism-spectrum-disorders. These experiments identify novel intra-dendritic Sema5A/PlexinA2 interactions that inhibit excitatory synapse formation in developmentally born and adult-born GCs, and they provide support for SEMA5A contributions to autism-spectrum-disorders.

Where no synapses go: gatekeepers of circuit remodeling and synaptic strength.

Growth inhibitory molecules in the adult mammalian central nervous system (CNS) have been implicated in the blocking of axonal sprouting and regeneration following injury. Prominent CNS regeneration inhibitors include Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and chondroitin sulfate proteoglycans (CSPGs), and a key question concerns their physiological role in the naïve CNS. Emerging evidence suggests novel functions in dendrites and at synapses of glutamatergic neurons. CNS regeneration inhibitors target the neuronal actin cytoskeleton to regulate dendritic spine maturation, long-term synapse stability, and Hebbian forms of synaptic plasticity. This is accomplished in part by antagonizing plasticity-promoting signaling pathways activated by neurotrophic factors. Altered function of CNS regeneration inhibitors is associated with mental illness and loss of long-lasting memory, suggesting unexpected and novel physiological roles for these molecules in brain health.

NgR1 and NgR3 are receptors for chondroitin sulfate proteoglycans.

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin-associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. We found that NgR1 and NgR3 bind with high affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (Ngr1(-/-); Ngr2(-/-); Ngr3(-/-); which are also known as Rtn4r, Rtn4rl2 and Rtn4rl1, respectively), but not single mutants, showed enhanced axonal regeneration following retro-orbital optic nerve crush injury. The combined loss of Ngr1 and Ngr3 (Ngr1(-/-); Ngr3(-/-)), but not Ngr1 and Ngr2 (Ngr1(-/-); Ngr2(-/-)), was sufficient to mimic the triple mutant regeneration phenotype. Regeneration in Ngr1(-/-); Ngr3(-/-) mice was further enhanced by simultaneous ablation of Rptpσ (also known as Ptprs), a known CSPG receptor. Collectively, our results identify NgR1 and NgR3 as CSPG receptors, suggest that there is functional redundancy among CSPG receptors, and provide evidence for shared mechanisms of MAI and CSPG inhibition.

Preventing flow-metabolism uncoupling acutely reduces axonal injury after traumatic brain injury.

We have previously presented evidence that the development of secondary traumatic axonal injury is related to the degree of local cerebral blood flow (LCBF) and flow-metabolism uncoupling. We have now tested the hypothesis that augmenting LCBF in the acute stages after brain injury prevents further axonal injury. Data were acquired from rats with or without acetazolamide (ACZ) that was administered immediately following controlled cortical impact injury to increase cortical LCBF. Local cerebral metabolic rate for glucose (LCMRglc) and LCBF measurements were obtained 3 h post-trauma in the same rat via ¹8F-fluorodeoxyglucose and ¹4C-iodoantipyrine co-registered autoradiographic images, and compared to the density of damaged axonal profiles in adjacent sections, and in additional groups at 24 h used to assess different populations of injured axons stereologically. ACZ treatment significantly and globally elevated LCBF twofold above untreated-injured rats at 3 h (p<0.05), but did not significantly affect LCMRglc. As a result, ipsilateral LCMRglc:LCBF ratios were reduced by twofold to sham-control levels, and the density of ß-APP-stained axons at 24 h was significantly reduced in most brain regions compared to the untreated-injured group (p<0.01). Furthermore, early LCBF augmentation prevented the injury-associated increase in the number of stained axons from 3-24 h. Additional robust stereological analysis of impaired axonal transport and neurofilament compaction in the corpus callosum and cingulum underlying the injury core confirmed the amelioration of ß-APP axon density, and showed a trend, but no significant effect, on RMO14-positive axons. These data underline the importance of maintaining flow-metabolism coupling immediately after injury in order to prevent further axonal injury, in at least one population of injured axons.

Chondroitinase ABC enhances pericontusion axonal sprouting but does not confer robust improvements in behavioral recovery.

Traumatic brain injury (TBI) results in enduring functional deficits. Strategies aimed at promoting plasticity within the injured brain may aid in enhancing functional outcome. We have previously shown that spontaneous pericontusional axon sprouting occurs within 7-14 days after controlled cortical impact injury in the adult rat, but ultimately fails due to an increasingly growth-inhibitory environment. We therefore sought to determine whether acute infusion of chondroitinase ABC into the site of the cortical contusion, to further reduce pericontusional growth-inhibitory chondroitin sulfate proteoglycans (CSPGs), would enhance and prolong the sprouting response. We also wanted to determine if chondroitinase-enhanced sprouting would ameliorate the behavioral deficits in forelimb function that occur in this model. Acute chondroitinase infusion decreased intact CSPGs and significantly increased pericontusional cortical grey and white matter growth-associated protein 43 (GAP43)-positive axon sprouting at 7 days post-injury. A return of intact CSPGs at later time points likely contributed to the absence of persistently increased levels of axon sprouting by 14-21 days post-injury. There was no overall benefit on forelimb function during the time of maximal sprouting or at any subsequent times in three of four behavioral outcome measures. However, there was a chondroitinase-induced improvement in recovery from unskilled limb use deficits on the staircase forelimb reaching test toward sham-injured values at 28 days, which was not achieved by the vehicle-treated rats, indicating that there is some minor functional benefit of the increased sprouting induced by chondroitinase treatment. The current results, together with data from spinal cord injury models after chondroitinase intervention, suggest that a combinatorial approach with the addition of neurotrophins and rehabilitation would result in more robust axon sprouting and consequently improve behavioral outcome.

Pericontusion axon sprouting is spatially and temporally consistent with a growth-permissive environment after traumatic brain injury.

We previously reported that pericontusional extracellular chondroitin sulfate proteoglycans (CSPGs) are profoundly reduced for 3 weeks after experimental traumatic brain injury, indicating a potential growth-permissive window for plasticity. Here, we investigate the extracellular environment of sprouting neurons after controlled cortical impact injury in adult rats to determine the spatial and temporal arrangement of inhibitory and growth-promoting molecules in relation to growth-associated protein 43-positive (GAP43+) neurons. Spontaneous cortical sprouting was maximal in pericontused regions at 7 and 14 days after injury but absent by 28 days. Perineuronal nets containing CSPGs were reduced at 7 days after injury in the pericontused region (p < 0.05), which was commensurate with a reduction in extracellular CSPGs. Sprouting was restricted to the perineuronal nets and CSPG-deficient regions at 7 days, indicating that the pericontused region is temporarily and spatially permissive to new growth. At this time point,GAP43+ neurons were associated with brain regions containing cells positive for polysialic acid neural cell adhesion molecule but not with fibronectin-positive cells. Brain-derived neurotrophic factor was reduced in the immediate pericontused region at 7 days. Along with prior Western blot evidence, these data suggest that a lowered intrinsic growth stimulus, together with a later return of growth-inhibitory CSPGs, may contribute to the ultimate disappearance of sprouting neurons after traumatic brain injury.