Osamu Imamura

Extracellular Signal-Regulated Kinase 2 (ERK2) Knockdown Mice Show Deficits in Long-Term Memory; ERK2 Has a Specific Function in Learning and Memory

The extracellular signal-regulated kinase (ERK) 1 and 2 are important signaling components implicated in learning and memory. These isoforms display a high degree of sequence homology and share a similar substrate profile. However, recent findings suggest that these isoforms may have distinct roles: whereas ERK1 seems to be not so important for associative learning, ERK2 might be critically involved in learning and memory. Thus, the individual role of ERK2 has received considerable attention, although it is yet to be understood. Here, we have generated a series of mice in which ERK2 expression decreased in an allele dose-dependent manner. Null ERK2 knock-out mice were embryonic lethal, and the heterozygous mice were anatomically impaired. To gain a better understanding of the influence of ERK2 on learning and memory, we also generated knockdown mice in which ERK2 expression was partially (20–40%) reduced. These mutant mice were viable and fertile with normal appearance. The mutant mice showed a deficit in long-term memory in classical fear conditioning, whereas short-term memory was normal. The mice also showed learning deficit in the water maze and the eight-arm radial maze. The ERK1 expression level of the knockdown mice was comparable with the wild-type control. Together, our results indicate a noncompensable role of ERK2-dependent signal transduction in learning and memory.

Analysis of Extracellular Signal‐Regulated Kinase 2 Function in Neural Stem/Progenitor Cells via Nervous System‐Specific Gene Disruption

Extracellular signal‐regulated kinase 2 (ERK2) is involved in a variety of cell fate decisions during development, but its exact role in this process remains to be determined. To specifically focus on the role of ERK2 in the brain, and to avoid early lethalities, we used a conditional gene‐targeting approach to preferentially inactivate Erk2 in the embryonic mouse brain. The resulting mutant mice were viable and were relatively normal in overall appearance. However, the loss of Erk2 resulted in a diminished proliferation of neural stem cells in the embryonic ventricular zone (VZ), although the survival and differentiation of these cells was unaffected. The multipotent neural progenitor cells (NPCs) isolated from ERK2‐deficient brains also showed impaired proliferation, reduced self‐renewal ability, and increased apoptosis. By neurosphere differentiation analysis we further observed that lineage‐restricted glial progenitors were increased in ERK2‐deficient mice. The decline in the self‐renewal ability and multipotency of NPCs resulting from the loss of ERK2 was found to be caused at least in part by upregulation of the JAK‐STAT signaling pathway and reduced G1/S cell cycle progression. Furthermore, by global expression analysis we found that neural stem cell markers, including Tenascin C NR2E1 (Tlx), and Lgals1 (Galectin‐1), were significantly downregulated, whereas several glial lineage markers were upregulated in neurospheres derived from ERK2‐deficient mice. Our results thus suggest that ERK2 is required both for the proliferation of neural stem cells in the VZ during embryonic development and in the maintenance of NPC multipotency by suppressing the commitment of these cells to a glial lineage.

ERK1 and ERK2 are required for radial glial maintenance and cortical lamination

ERK1/2 is involved in a variety of cellular processes during development, but the functions of these isoforms in brain development remain to be determined. Here, we generated double knockout (DKO) mice to study the individual and combined roles of ERK1 and ERK2 during cortical development. Mice deficient in Erk2, and more dramatically in the DKOs, displayed proliferation defects in late radial glial progenitors within the ventricular zone, and a severe disruption of lamination in the cerebral cortex. Immunohistochemical analyses revealed that late‐generated cortical neurons were misplaced and failed to migrate the upper cortical layers in DKO mice. Moreover, these mice displayed fewer radial glial fibers, which provide architectural guides for radially migrating neurons. These results suggest that extracellular signal‐regulated kinase signaling is essential for the expansion of the radial glial population and for the maintenance of radial glial scaffolding. Tangential migration of interneurons and oligodendrocytes from the ganglionic eminences (GE) to the dorsal cortex was more severely impaired in DKO mice than in mice deficient for Erk2 alone, because of reduced progenitor proliferation in the GE of the ventral telencephalon. These data demonstrate functional overlaps between ERK1 and ERK2 and indicate that extracellular signal‐regulated kinase signaling plays a crucial role in cortical development.

Nicotinic acetylcholine receptors mediate donepezil-induced oligodendrocyte differentiation.

Oligodendrocytes are the myelin‐forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimers disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell‐derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin‐associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil‐induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil‐induced myelin‐related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin‐related gene expression via nAChRs in neural stem cell‐derived oligodendrocyte progenitor cells.

The crucial role of Erk2 in demyelinating inflammation in the central nervous system

BackgroundBrain inflammation is a crucial component of demyelinating diseases such as multiple sclerosis. Although the initiation of inflammatory processes by the production of cytokines and chemokines by immune cells is well characterized, the processes of inflammatory aggravation of demyelinating diseases remain obscure. Here, we examined the contribution of Erk2, one of the isoforms of the extracellular signal-regulated kinase, to demyelinating inflammation.MethodsWe used the cuprizone-induced demyelinating mouse model. To examine the role of Erk2, we used Nestin-cre-driven Erk2-deficient mice. We also established primary culture of microglia or astrocytes in order to reveal the crosstalk between two cell types and to determine the downstream cascades of Erk2 in astrocytes.ResultsFirst, we found that Erk is especially activated in astrocytes within the corpus callosum before the peak of demyelination (at 4xa0weeks after the start of cuprizone feeding). Then, we found that in our model, genetic ablation of Erk2 from neural cells markedly preserved myelin structure and motor function as measured by the rota-rod test. While the initial activation of microglia was not altered in Erk2-deficient mice, these mice showed reduced expression of inflammatory mediators at 3–4 model weeks. Furthermore, the subsequent inflammatory glial responses, characterized by accumulation of microglia and reactive astrocytes, were significantly attenuated in Erk2-deficient mice. These data indicate that Erk2 in astrocytes is involved in augmentation of inflammation and gliosis. We also found that activated, cultured microglia could induce Erk2 activation in cultured astrocytes and subsequent production of inflammatory mediators such as Ccl-2.ConclusionsOur results suggest that Erk2 activation in astrocytes plays a crucial role in aggravating demyelinating inflammation by inducing inflammatory mediators and gliosis. Thus, therapies targeting Erk2 function in glial cells may be a promising approach to the treatment of distinct demyelinating diseases.

Donepezil promotes differentiation of neural stem cells into mature oligodendrocytes at the expense of astrogenesis

Oligodendrocytes are the myelin‐forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimers disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self‐renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin‐associated glycoprotein, 2′, 3′‐cyclic‐nucleotide 3′‐phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker β‐tubulin type III (Tuj1) were up‐regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down‐regulated by donepezil in a dose‐ and time‐dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte‐inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.

A novel strategy for selective gene delivery by using the inhibitory effect of blue light on jetPRIME-mediated transfection.

Photodynamic control of gene delivery is a new technology with growing applications in gene therapy and basic cell research. Main approaches of light‐selective gene delivery rely on the light‐dependent enhancement of transfection efficiency. Studies focused on light‐stimulated inhibitory regulation of transfection have rarely been reported. Here, we tried to establish a novel procedure of light‐dependent inhibition of transfection. Our experiments, conducted with several types of commercial transfection reagents, revealed that jetPRIME‐mediated transfection was strongly inhibited by blue light. Although the uptake of reagent–DNA complex was drastically reduced, preliminary exposure of cells or reagent–DNA complex to blue light had no inhibitory effect on the transfection efficiency. The inhibitory effect was wavelength‐dependent and mediated by reactive oxygen species. Partial exposure of a culture vessel to blue light resulted in selective gene delivery into cells grown on the unexposed area of the vessel. By using this approach, different types of plasmid DNA were delivered into different areas in the culture vessel. This novel approach to the inhibitory control of transfection provides practical options for research and therapeutics. Biotechnol. Bioeng. 2016;113: 1560–1567.

Role of the ERK signaling in the developing cerebral cortex

tralaterally ascending tracts followed two major, the dorsal and the ventral, paths and ipsilaterally ascending tracts followed the single dorsal path. A part of dorsally ascending axons of both sides entered the developing cerebellum to constitute chick counterparts of mammalian ventral spinocerebellar tract (VSCT, contralateral side) and dorsal spinocerebellar tracts (DSCT, ipsilateral side). Others headed to superior medullary velum located between the cerebellum and the midbrain. The time courses of axonal elongation and destinations in cerebellum were characteristically varied. Interestingly, ascending axons derived from T4 segment were caught up with axons from LS2 segment in the course of passing brachial region, suggesting that the spinal cord may provide cues for adjusting their pace of elongation. Selective visualization of spinocerebellar tract using cell-type specific enhancers will also be presented and discussed.

Neural-specific inactivation of ERK2 in mice causes abnormal laminar formation in the cerebral cortex

dyskinesia. However, little is known about recovery and regeneration mechanism of optimal neural circuits developing by precise neural activities and how neural activities form the appropriate local neural circuits with transplanted cells correctly processing the information with endogenous neurons. Therefore we investigated whether Neurogenin2 and Mash-1 can differentiate transplanted neural stem cells, which are thought to regulate differentiation of excitatory and inhibitory neuron respectively. This approach will allow us to reveal the formation mechanism of local excitatory and inhibitory circuits in the cerebral cortex. Additionally this method could be helpful to care the patients suffered from neurological disorders.