Masahiro Fukaya

Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain

The adult CNS contains an abundant population of oligodendrocyte precursor cells (NG2+ cells) that generate oligodendrocytes and repair myelin, but how these ubiquitous progenitors maintain their density is unknown. We generated NG2-mEGFP mice and used in vivo two-photon imaging to study their dynamics in the adult brain. Time-lapse imaging revealed that NG2+ cells in the cortex were highly dynamic; they surveyed their local environment with motile filopodia, extended growth cones and continuously migrated. They maintained unique territories though self-avoidance, and NG2+ cell loss though death, differentiation or ablation triggered rapid migration and proliferation of adjacent cells to restore their density. NG2+ cells recruited to sites of focal CNS injury were similarly replaced by a proliferative burst surrounding the injury site. Thus, homeostatic control of NG2+ cell density through a balance of active growth and self-repulsion ensures that these progenitors are available to replace oligodendrocytes and participate in tissue repair.

Degeneration and impaired regeneration of gray matter oligodendrocytes in amyotrophic lateral sclerosis

Oligodendrocytes associate with axons to establish myelin and provide metabolic support to neurons. In the spinal cord of amyotrophic lateral sclerosis (ALS) mice, oligodendrocytes downregulate transporters that transfer glycolytic substrates to neurons and oligodendrocyte progenitors (NG2+ cells) exhibit enhanced proliferation and differentiation, although the cause of these changes in oligodendroglia is unknown. We found extensive degeneration of gray matter oligodendrocytes in the spinal cord of SOD1 (G93A) ALS mice prior to disease onset. Although new oligodendrocytes were formed, they failed to mature, resulting in progressive demyelination. Oligodendrocyte dysfunction was also prevalent in human ALS, as gray matter demyelination and reactive changes in NG2+ cells were observed in motor cortex and spinal cord of ALS patients. Selective removal of mutant SOD1 from oligodendroglia substantially delayed disease onset and prolonged survival in ALS mice, suggesting that ALS-linked genes enhance the vulnerability of motor neurons and accelerate disease by directly impairing the function of oligodendrocytes.

NMDA Receptor Signaling in Oligodendrocyte Progenitors Is Not Required for Oligodendrogenesis and Myelination

Oligodendrocyte precursor cells (OPCs) express NMDA receptors (NMDARs) and form synapses with glutamatergic neurons throughout the CNS. Although glutamate influences the proliferation and maturation of these progenitors in vitro, the role of NMDAR signaling in oligodendrogenesis and myelination in vivo is not known. Here, we investigated the consequences of genetically deleting the obligatory NMDAR subunit NR1 from OPCs and their oligodendrocyte progeny in the CNS of developing and mature mice. NMDAR-deficient OPCs proliferated normally, achieved appropriate densities in gray and white matter, and differentiated to form major white matter tracts without delay. OPCs also retained their characteristic physiological and morphological properties in the absence of NMDAR signaling and were able to form synapses with glutamatergic axons. However, expression of calcium-permeable AMPA receptors (AMPARs) was enhanced in NMDAR-deficient OPCs. These results suggest that NMDAR signaling is not used to control OPC development but to regulate AMPAR-dependent signaling with surrounding axons, pointing to additional functions for these ubiquitous glial cells.

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

J. Neurochem. (2011) 116, 1122–1137.

Transient Opening of the Mitochondrial Permeability Transition Pore Induces Microdomain Calcium Transients in Astrocyte Processes

Astrocytes extend highly branched processes that form functionally isolated microdomains, facilitating local homeostasis by redistributing ions, removing neurotransmitters, and releasing factors to influencexa0blood flow and neuronal activity. Microdomains exhibit spontaneous increases in calcium (Ca2+), butxa0the mechanisms and functional significance of thisxa0localized signaling are unknown. By developing conditional, membrane-anchored GCaMP3 mice, we found that microdomain activity that occurs in the absence of inositol triphosphate (IP3)-dependent release from endoplasmic reticulum arises through Ca2+ efflux from mitochondria during brief openings of the mitochondrial permeability transition pore. These microdomain Ca2+ transients were facilitated by the production of reactive oxygen species during oxidative phosphorylation and were enhanced by expression of a mutant form of superoxide dismutase 1 (SOD1 G93A) that causes astrocyte dysfunction and neurodegeneration in amyotrophic lateral sclerosis (ALS). By localizing mitochondria to microdomains, astrocytes ensure local metabolic supportxa0for energetically demanding processes and enable coupling between metabolic demand and Ca2+ signaling events.

The synaptic targeting of mGluR1 by its carboxyl-terminal domain is crucial for cerebellar function.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca2+ release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.

ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

Abstract During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

EFA6A, a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling, Behaviors, and Short Term Plasticity

Background: Roles of the syntaxin-1A·CaMKII interaction are not physiologically understood in vivo. Results: A point mutation in syntaxin-1A caused abnormal plasticity, recycling, and behaviors in mice. Conclusion: The CaMKII/syntaxin-1A interaction is essential for maintenance of neuronal plasticity. Significance: Syntaxin-1A is involved in regulatory pathways in higher brain functions. Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

Type I phosphatidylinositol 4-phosphate 5-kinase γ is required for neuronal migration in the mouse developing cerebral cortex.

Type I phosphatidylinositol 4‐phosphate 5‐kinase (PIP5KI)γ is one of the phosphoinositide kinases that produce phosphatidylinositol 4,5‐bisphosphate, which is a critical regulator of cell adhesion formation, actin dynamics and membrane trafficking. Here, we examined the functional roles of PIP5KIγ in radial neuronal migration during cortical formation. Reverse transcription–polymerase chain reaction analysis revealed that PIP5KIγ_v2/v6 and PIP5KIγ_v3 were expressed throughout cortical development with distinct expression patterns. In situ hybridisation analysis showed that PIP5KIγ mRNA was expressed throughout the cortical layers. Immunohistochemical analysis revealed that PIP5KIγ was localised in a punctate manner in the radial glia and migrating neuroblasts. Knockdown of PIP5KIγ using in utero electroporation disturbed the radial neuronal migration and recruitment of talin and focal adhesion kinase to puncta beneath the plasma membrane. The same inhibitory effect on neuronal migration was observed by overexpression of a catalytically inactive mutant of PIP5KIγ_v2 but not PIP5KIγ_v1 or PIP5KIγ_v3. These findings suggest an essential role of PIP5KIγ, particularly PIP5KIγ_i2, in neuronal migration, possibly through recruitment of adhesion components to the plasma membrane.