Nozomu Yoshioka

Role of the lesion scar in the response to damage and repair of the central nervous system

Traumatic damage to the central nervous system (CNS) destroys the blood–brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.

LMTK1/AATYK1 Is a Novel Regulator of Axonal Outgrowth That Acts via Rab11 in a Cdk5-Dependent Manner

Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5–LMTK1–Rab11 is a novel signaling pathway involved in axonal outgrowth.

An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury.

After central nervous system (CNS) injury, meningeal fibroblasts migrate in the lesion center to form a fibrotic scar which is surrounded by end feet of reactive astrocytes. The fibrotic scar expresses various axonal growth-inhibitory molecules and creates a major impediment for axonal regeneration. We developed an in vitro model of the scar using coculture of cerebral astrocytes and meningeal fibroblasts by adding transforming growth factor-beta1 (TGF-beta1), a potent fibrogenic factor. Addition of TGF-beta1 to this coculture resulted in enhanced proliferation of fibroblasts and the formation of cell clusters which consisted of fibroblasts inside and surrounded by astrocytes. The cell cluster in culture densely accumulated the extracellular matrix molecules and axonal growth-inhibitory molecules similar to the fibrotic scar, and remarkably inhibited the neurite outgrowth of cerebellar neurons. Therefore, this culture system can be available to analyze the inhibitory property in the lesion site of CNS.

Suppression of fibrotic scar formation promotes axonal regeneration without disturbing blood-brain barrier repair and withdrawal of leukocytes after traumatic brain injury

The fibrotic scar containing type IV collagen (Col IV) formed in a lesion site is considered as an obstacle to axonal regeneration, because intracerebral injection of 2,2′‐dipyridyl (DPY), an inhibitor of Col IV triple‐helix formation, suppresses fibrotic scar formation in the lesion site and promotes axonal regeneration. To determine the role of the fibrotic scar on the healing process of injured central nervous system (CNS), the restoration of blood‐brain barrier (BBB) and withdrawal of inflammatory leukocytes were examined in mice subjected to unilateral transection of the nigrostriatal dopaminergic pathway and intracerebral DPY injection. At 5 days after injury, destruction of BBB represented by leakage of Evans blue (EB) and widespread infiltration of CD45‐immunoreactive leukocytes was observed around the lesion site, whereas reactive astrocytes increased surrounding the BBB‐destroyed area. By 2 weeks after injury, the region of EB leakage and the diffusion of leukocytes were restricted to the inside of the fibrotic scar, and reactive astrocytes gathered around the fibrotic scar. In the DPY‐treated lesion site, formation of the fibrotic scar was suppressed (84% decrease in Col IV‐deposited area), reactive astrocytes occupied the lesion center, and areas of both EB leakage and leukocyte infiltration decreased by 86%. DPY treatment increased the number of regenerated dopaminergic axons by 2.53‐fold. These results indicate that suppression of fibrotic scar formation does not disturb the healing process in damaged CNS, and suggest that this strategy is a reliable tool to promote axonal regeneration after traumatic injury in the CNS. J. Comp. Neurol. 518:3867–3881, 2010.

Small molecule inhibitor of type I transforming growth factor-β receptor kinase ameliorates the inhibitory milieu in injured brain and promotes regeneration of nigrostriatal dopaminergic axons

Transforming growth factor‐β (TGF‐β), a multifunctional cytokine, plays a crucial role in wound healing in the damaged central nervous system. To examine effects of the TGF‐β signaling inhibition on formation of scar tissue and axonal regeneration, the small molecule inhibitor of type I TGF‐β receptor kinase LY‐364947 was continuously infused in the lesion site of mouse brain after a unilateral transection of the nigrostriatal dopaminergic pathway. At 2 weeks after injury, the fibrotic scar comprising extracellular matrix molecules including fibronectin, type IV collagen, and chondroitin sulfate proteoglycans was formed in the lesion center, and reactive astrocytes were increased around the fibrotic scar. In the brain injured and infused with LY‐364947, fibrotic scar formation was suppressed and decreased numbers of reactive astrocytes occupied the lesion site. Although leukocytes and serum IgG were observed within the fibrotic scar in the injured brain, they were almost absent in the injured and LY‐364947‐treated brain. At 2 weeks after injury, tyrosine hydroxylase (TH)‐immunoreactive fibers barely extended beyond the fibrotic scar in the injured brain, but numerous TH‐immunoreactive fibers regenerated over the lesion site in the LY‐364947‐treated brain. These results indicate that inhibition of TGF‐β signaling suppresses formation of the fibrotic scar and creates a permissive environment for axonal regeneration.

LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes

It is unknown how membrane components are supplied to growing dendrites in neurons. Down-regulation of LMTK1 Ser/Thr kinase in cortical neurons increases Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, leading to dendrite growth and branching. Cdk5-LMTK1-Rab11A is a signaling pathway regulating dendrite development.

A DEAD-box RNA helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system.

We recently reported that a new monoclonal antibody, 4F2, which labels oligodendroglial lineage cells, recognizes a DEAD‐box RNA helicase Ddx54 and that Ddx54 binds to myelin basic protein (MBP) in brain and cultured oligodendrocytes. To elucidate the biological function of Ddx54, we generated a recombinant adenovirus, Ad‐shRNA:Ddx54, expressing a short hairpin RNA to silence endogenous Ddx54 protein. The virus was intraventricularly injected into the brains of mice on postnatal day (PD) 2. The brains at PD 9 were then analyzed by immunohistochemistry. In untreated normal brain sections, as well as control brains that had been injected with Ad‐β‐Gal, myelination of axons occurred in the corpus callosum with filamentous patterns of immunosignals of myelin‐associated glycoprotein (MAG) and MBP. In Ad‐shRNA:Ddx54‐injected brain, substantial amounts of MAG and MBP immunosignals were present, but MBP immunosignals accumulated in the subplate layer and did not intrude into the emerging white matter. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of Olig2; 2′,3′‐cyclic nucleotide 3′ phosphodiesterase; MAG; three MBP isoforms (14, 17.5, and 18 kDa); and QKI‐5, QKI‐6, and QKI‐7 proteins remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter‐introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Immunoprecipitation of Ddx54 protein in MBP‐transfected HEK293 cells indicated that Ddx54 may directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes.

The astrocytic lineage marker calmodulin-regulated spectrin-associated protein 1 (Camsap1): phenotypic heterogeneity of newly born Camsap1-expressing cells in injured mouse brain.

Calmodulin‐regulated spectrin‐associated protein 1 (Camsap1) has been recognized as a new marker for astrocytic lineage cells and is expressed on mature astrocytes in the adult brain (Yamamoto et al. [ 2009 ] J. Neurosci. Res. 87:503–513). In the present study, we found that newly born Camsap1‐expressing cells exhibited regional heterogeneity in an early phase after stab injury of the mouse brain. In the surrounding area of the lesion site, Camsap1 was expressed on quiescent astrocytes. At 3 days after injury, Camsap1 immunoreactivity was upregulated on glial fibrillary acidic protein‐immunoreactive (GFAP‐ir) astrocytes. Some of these astrocytes incorporated bromodeoxyuridine (BrdU) together with re‐expression of the embryonic cytoskeleton protein nestin. In the neighboring region of the lesion cavity, Camsap1 was expressed on GFAP‐negative cells. At 3 days after injury, GFAP‐ir astrocytes were absent around the lesion cavity. At this stage, NG2‐ir cells immunopositive for Camsap1 and immunonegative for GFAP were distributed in border of the lesion cavity. By 10 days, Camsap1 immunoreactivity was exclusively detected on GFAP‐ir reactive astrocytes devoid of NG2 immunoreactivity. BrdU pulse‐chase labeling assay suggested the differentiation of Camsap1+/NG2+ cells into Camsap1+/GFAP+ astrocytes. In the subependymal zone of the lateral ventricle, Camsap1‐ir cells increased after injury. Camsap1 immunoreactivity was distributed on ependymal and subependymal cells bearing various astrocyte markers, and BrdU incorporation was enhanced on such Camsap1‐ir cells after injury. These results suggest that newly born reactive astrocytes are derived from heterogeneous Camsap1‐expressing cells in the injured brain. J. Comp. Neurol. 520:1301–1317, 2012.

An Extract of Chinpi, the Dried Peel of the Citrus Fruit Unshiu, Enhances Axonal Remyelination via Promoting the Proliferation of Oligodendrocyte Progenitor Cells

The aging-induced decrease in axonal myelination/remyelination is due to impaired recruitment and differentiation of oligodendrocyte progenitor cells (OPCs). Our previous studies have shown that a monoclonal antibody to DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases, (1) specifically labels oligodendrocyte lineages, (2) binds to mRNA and protein isoforms of myelin basic proteins (MBP), and (3) regulates migration of OPCs from ventricular zone to corpus callosum in mice. It has also been demonstrated that specific loss of a 21.5 kDa MBP isoform (MBP21.5) reflects demyelination status, and oral administration of an extract of Chinpi, citrus unshiu peel, reversed the aging-induced demyelination. Here, we report that Chinpi treatment induced a specific increase in the MBP21.5, led to the reappearance of Ddx54-expressing cells in ventricular-subventricular zone and corpus callosum of aged mice, and promoted remyelination. Treatment of in vitro OPC cultures with Chinpi constituents, hesperidin plus narirutin, led to an increase in 5-bromo-2′-deoxyuridine incorporation in Ddx54-expressing OPCs, but not in NG2- or Olig2-expressing cell populations. The present study suggests that Ddx54 plays crucial role in remyelination. Furthermore, Chinpi and Chinpi-containing herbal medicines may be a therapeutic option for the aging-induced demyelination diseases.

Fibrotic scar formed in the lesion site of the central nervous system. I. Impediment for axonal regeneration

Myelin components that inhibit axonal regeneration are believed to significantly contribute tothe lack of axonal regeneration noted in the adult CNS. Three proteins found inmyelin-Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelinglycoprotein-inhibit neurite outgrowth in vitro. All these proteins interact with the samereceptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B(PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced inNgR-knockout mice after spinal cord injury. Therefore, we assessed CST regeneration inPIR-B-knockout mice. We found that hindlimb motor function, as assessed using the BassoMouse Scale, foot print test, inclined plane test, and beam walking test, did not differ betweenthe PIR-B-knockout and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sproutingin the CST of the PIR-B-knockout mice. Systemic administration of NEP1-40, NgRantagonist, to PIR-B knockout mice did not enhance the regenerative response. These resultsindicate that PIR-B knockout is not sufficient to induce extensive axonal regeneration afterspinal cord injury.